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Key Role of Macrophages in the Pathogenesis of CD18 Hypomorphic Murine Model of Psoriasis

      Psoriasis is a chronic skin disorder of unsolved pathogenesis affecting skin in 2–3% of the general population. Research into the pathogenesis of psoriasis has profited from suitable animal models. Previously, we reported on the CD18 hypomorphic (CD18hypo) PL/J mouse model clinically resembling human psoriasis, which is characterized by reduced expression of the common chain of β2-integrins (CD11/CD18) to only 2–16% of wild-type levels. Aside from common clinical and pathophysiological features shared with human psoriasis, the psoriasiform skin disease in CD18hypo PL/J mice also depends on the presence of CD4+ T-cells. This review focuses on the role of activated macrophages in the pathogenesis of CD18hypo T-cell-mediated mouse model of psoriasis, and extends our understanding in unrestrained pathogenic T-cells whose activation may be crucial for the recruitment and activation of macrophages within skin. The findings in the CD18hypo PL/J model are discussed in the context of current literatures of human and other autoimmune disorders.

      Abbreviations

      CD18hypo
      CD18 hypomorphic mutation
      DC
      dendritic cell
      DLN
      draining lymph node
      iNOS
      inducible nitric oxide synthase
      LT
      lymphotoxin
      MCP
      monocyte chemotactic protein
      PASI
      psoriasis activity and severity index
      Th
      T-helper cell
      TipDC
      TNF-iNOS-producing DC
      TNF-α
      tumor necrosis factor-α
      TNFR
      TNF receptor
      Treg-cells
      regulatory T-cells

      Introduction

      Skin from psoriatic patients is characterized by a dense dermal infiltrate that predominantly consists of T-cells, dendritic cells (DCs), natural killer T-cells, and macrophages (
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ). The epidermis of psoriatic skin is hyperproliferative and fails to undergo normal differentiation, resulting in a marked thickening of the epidermis and increased scale formation. The dispute in the field is whether psoriasis is a disease of autoreactive T-cells or whether it reflects an intrinsic defect within the epidermis, or both. There is more recent evidence from genetic mouse models that macrophages can contribute to T-cell-mediated and epidermis-mediated psoriasiform skin inflammation (
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ).
      Activated macrophages are major producers of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 (
      • Salkowski C.A.
      • Neta R.
      • Wynn T.A.
      • Strassmann G.
      • van Rooijen N.
      • Vogel S.N.
      Effect of liposome-mediated macrophage depletion on LPS-induced cytokine gene expression and radioprotection.
      ;
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ), and play a crucial role in modulating immune responses (
      • Holt P.G.
      • Oliver J.
      • Bilyk N.
      • McMenamin C.
      • McMenamin P.G.
      • Kraal G.
      • et al.
      Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages.
      ;
      • Thepen T.
      • Kraal G.
      • Holt P.G.
      The role of alveolar macrophages in regulation of lung inflammation.
      ;
      • Gordon S.
      The macrophage.
      ). Macrophages are heterogeneous and versatile bone marrow-derived cells that produce a wide range of mediators and exert a multitude of biological functions (
      • Ganz T.
      Macrophage function.
      ). They can not only serve as antigen-presenting cells, but also directly inhibit antigen presentation by DCs (
      • Holt P.G.
      • Oliver J.
      • Bilyk N.
      • McMenamin C.
      • McMenamin P.G.
      • Kraal G.
      • et al.
      Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages.
      ). T-cell proliferation, phenotype, and ultimately the type of immune response induced, can distinctly be influenced by macrophages (
      • Thepen T.
      • Kraal G.
      • Holt P.G.
      The role of alveolar macrophages in regulation of lung inflammation.
      ,
      • Thepen T.
      • Langeveld-Wildschut E.G.
      • Bihari I.C.
      • van Wichen D.F.
      • van Reijsen F.C.
      • Mudde G.C.
      • et al.
      Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial TH2 response to a TH1 response in situ: an immunocytochemical study.
      ;
      • Strickland D.
      • Kees U.R.
      • Holt P.G.
      Regulation of T-cell activation in the lung: alveolar macrophages induce reversible T-cell anergy in vitro associated with inhibition of interleukin-2 receptor signal transduction.
      ;
      • Grewe M.
      • Bruijnzeel-Koomen C.A.
      • Schopf E.
      • Thepen T.
      • Langeveld-Wildschut A.G.
      • Ruzicka T.
      • et al.
      A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis.
      ). Macrophages are specifically polarized by the microenvironment to mount different functional programs. Initial signals from microbes, through their pathogen-associated molecular patterns (
      • Schnare M.
      • Holt A.C.
      • Takeda K.
      • Akira S.
      • Medzhitov R.
      Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88.
      ), followed by a second signal, such as IFN-γ, gives rise to “classically activated macrophages”. In early immune responses, IFN-γ is produced by natural killer and natural killer T-cells; later the main source of IFN-γ are antigen-specific T-helper-1 (Th1) cells (
      • Young H.A.
      Unraveling the pros and cons of interferon-gamma gene regulation.
      ). These stimuli generally activate macrophages to produce TNF-α, monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), IFN-γ, and to promote strong IL-12-mediated Th1 responses. When macrophages are activated in the presence of IL-4, IL-10, transforming growth factor-β, or glucocorticoids, they become “alternatively activated macrophages” being characterized by dectin-1 expression and supporting Th2-associated immune responses. However, activation of dectin-1 with the fungal β-glucan triggers a severe autoimmune arthritis in genetically susceptible mice (
      • Yoshitomi H.
      • Sakaguchi N.
      • Kobayashi K.
      • Brown G.D.
      • Tagami T.
      • Sakihama T.
      • et al.
      A role for fungal {beta}-glucans and their receptor dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice.
      ). Thus, in inflammatory processes macrophages contribute to both initiation of inflammation and to its resolution. However, during chronic inflammatory responses in the skin and most likely other tissues, interaction between macrophages and T-cells may lead to a vicious cycle, which, by itself, is capable of maintaining local inflammation without the necessity of external stimuli (
      • Avice M.N.
      • Demeure C.E.
      • Delespesse G.
      • Rubio M.
      • Armant M.
      • Sarfati M.
      IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation.
      ).
      To characterize mouse macrophages in various biological processes, a panel of antibody-defined markers, expressed during different stages of mouse macrophage development, was previously reported. These markers included (1) macrophage precursors and immature macrophages (ER-MP12, ER-MP20, ER-MP54, ER-MP58); (2) mature macrophages in general (F4/80, BM8, Mac-1, Mac-2, ER-BMDM1); (3) macrophage subsets (ER-HR3, ER-MP23, ER-TR9, Forssman antigen, MOMA-1, MOMA-2, Monts-4, SER-4), and (4) IFN-γ-stimulated macrophages (H-2Ia, LFA-1, intercellular adhesion molecule-1, 158.2, MBR-2, TM-2, TM-4, TM-5) (
      • Leenen P.J.
      • de Bruijn M.F.
      • Voerman J.S.
      • Campbell P.A.
      • van Ewijk W.
      Markers of mouse macrophage development detected by monoclonal antibodies.
      ).
      In the past, some surface markers, including CD14, CD68, HLA-DR, RM3/1, and CD11c, have been used to describe the phenotypic diversity of the dermal macrophage population in humans (
      • Weber-Matthiesen K.
      • Sterry W.
      Organization of the monocyte/macrophage system of normal human skin.
      ;
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ). Besides these markers, a recent study reported that a population of dermal macrophage/macrophage-like cells expresses CD163 and factor XIIIa in normal human skin (
      • Nestle F.O.
      • Nickoloff B.J.
      Deepening our understanding of immune sentinels in the skin.
      ;
      • Zaba L.C.
      • Fuentes-Duculan J.
      • Steinman R.M.
      • Krueger J.G.
      • Lowes M.A.
      Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.
      ). CD163+ cells phagocytose large particles in a tattoo and have the structural features of macrophages (
      • Zaba L.C.
      • Fuentes-Duculan J.
      • Steinman R.M.
      • Krueger J.G.
      • Lowes M.A.
      Normal human dermis contains distinct populations of CD11c+BDCA-1+ dendritic cells and CD163+FXIIIA+ macrophages.
      ). CD163 is a hemoglobin/haptoglobin complex-binding macrophage scavenger receptor expressed on the majority of tissue macrophages (
      • Fabriek B.O.
      • Dijkstra C.D.
      • van den Berg T.K.
      The macrophage scavenger receptor CD163.
      ). Expression of factor XIIIa is related to cell activation and is inducible via IL-4 in alternatively activated macrophages (
      • Torocsik D.
      • Bardos H.
      • Nagy L.
      • Adany R.
      Identification of factor XIII-A as a marker of alternative macrophage activation.
      ).
      In human psoriasis, the number of epithelium-lining macrophages was reported to increase in lesional skin. These macrophages, which line dermal–epidermal junctions, may play a role in the regulation of epidermal proliferation and differentiation (
      • van den Oord J.J.
      • de Wolf-Peeters C.
      Epithelium-lining macrophages in psoriasis.
      ;
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ) or vigorous interactions between macrophages and keratinocytes (
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ), and may be involved in the pathogenesis of psoriasis (
      • van den Oord J.J.
      • de Wolf-Peeters C.
      Epithelium-lining macrophages in psoriasis.
      ;
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ). Macrophages secrete a variety of proinflammatory cytokines, such as TNF-α, IL-1β, IFN α/β, IL-6, IL-10, IL-12, and IL-18 cytokines, under different conditions (
      • Willment J.A.
      • Lin H.H.
      • Reid D.M.
      • Taylor P.R.
      • Williams D.L.
      • Wong S.Y.
      • et al.
      Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide.
      ).
      Research into the pathogenesis of human psoriasis has profited, at least in part, from suitable animal models. Most of these, however, reveal only a single or a few aspects resembling human psoriasis (
      • Carroll J.M.
      • Romero M.R.
      • Watt F.M.
      Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.
      ;
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Pasparakis M.
      • Courtois G.
      • Hafner M.
      • Schmidt-Supprian M.
      • Nenci A.
      • Toksoy A.
      • et al.
      TNF-mediated inflammatory skin disease in mice with epidermis-specific deletion of IKK2.
      ;
      • Sano S.
      • Chan K.S.
      • Carbajal S.
      • Clifford J.
      • Peavey M.
      • Kiguchi K.
      • et al.
      Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model.
      ;
      • Zenz R.
      • Eferl R.
      • Kenner L.
      • Florin L.
      • Hummerich L.
      • Mehic D.
      • et al.
      Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins.
      ).
      Previously, introduction of an insertion mutation in the murine CD18 gene, resulting in duplication of exons 2 and 3, yielded a mouse model with severe reduction of CD18 expression, with only 2–16% of wild-type (wt) levels (
      • Wilson R.W.
      • Ballantyne C.M.
      • Smith C.W.
      • Montgomery C.
      • Bradley A.
      • O'Brien W.E.
      • et al.
      Gene targeting yields a CD18-mutant mouse for study of inflammation.
      ). Due to this hypomorphic CD18 mutation (CD18hypo), a chronic inflammatory skin disease develops in PL/J mice, which closely resembles human psoriasis clinically, histologically, in its polygenic nature and in its response to therapy (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ;
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ). Affected mice present with erythema, crusts, and scaling, as well as abnormal keratinocyte proliferation/differentiation, subcorneal microabscesses, and increased inflammatory infiltrate. In severely affected mice, reversible alopecia was observed, a feature, which may only rarely, if at all, occur in human psoriasis (
      • Shuster S.
      Psoriatic alopecia.
      ). The psoriasiform skin disease was only observed when the CD18hypo mutation was backcrossed on the PL/J, but not on the C57BL/6J or 129/SvEv inbred mouse strains. Homozygous mutant mice on a PL/J × C57BL/6J F1 background did not develop the disease, despite the CD18hypo mutation. Backcross analysis suggests that, in addition to the CD18hypo mutation, a small number of other genes determine susceptibility to the disease (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ;
      • Kess D.
      • Lindqvist A.K.
      • Peters T.
      • Wang H.
      • Zamek J.
      • Nischt R.
      • et al.
      Identification of susceptibility loci for skin disease in a murine psoriasis model.
      ). Hence, the CD18hypo PL/J model qualifies as a polygenic model for chronic skin inflammation. Such a polygenic nature has been claimed for human psoriasis (
      • Schon M.P.
      • Boehncke W.H.
      Psoriasis.
      ).
      As in patients treated for severe psoriasis (
      • van de Kerkhof P.C.
      • Weemaes C.M.
      Skin manifestations in congenital deficiency of leucocyte-adherence glycoproteins (CDLG).
      ;
      • Feldman S.R.
      • Ravis S.M.
      • Fleischer Jr, A.B.
      • McMichael A.
      • Jones E.
      • Kaplan R.
      • et al.
      Betamethasone valerate in foam vehicle is effective with both daily and twice a day dosing: a single-blind, open-label study in the treatment of scalp psoriasis.
      ), this chronic psoriasiform skin disease of the CD18hypo PL/J mouse model can be suppressed by corticosteroids (dexamethasone), suggesting involvement of an inflammatory process. T-cells are important in the generation of the inflammatory skin disease in this CD18hypo PL/J model (
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ;
      • Barlow S.C.
      • Xu H.
      • Weaver C.T.
      • Lindsey J.R.
      • Schoeb T.R.
      • Bullard D.C.
      Development of dermatitis in CD18-deficient PL/J mice is not dependent on bacterial flora, and requires both CD4+ and CD8+ T lymphocytes.
      ). This is analogous to the affected skin of psoriatic patients in which CD4+ T-cells prevail (
      • Morel P.
      • Revillard J.P.
      • Nicolas J.F.
      • Wijdenes J.
      • Rizova H.
      • Thivolet J.
      Anti-CD4 monoclonal antibody therapy in severe psoriasis.
      ;
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ;
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ;
      • Conrad C.
      • Boyman O.
      • Tonel G.
      • Tun-Kyi A.
      • Laggner U.
      • de Fougerolles A.
      • et al.
      Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.
      ).
      CD18 represents the common β2-chain of the β2-integrin family. β2-Integrins (CD11/CD18) are leukocyte adhesion molecules exclusively expressed on hemopoietic cells and are responsible for cell–cell contacts in a variety of inflammatory interactions (
      • Hynes R.O.
      Integrins: a family of cell surface receptors.
      ). At present, four different β2-integrins have been characterized, all of which are heterodimeric cell-surface molecules consisting of the CD18 molecule and one of the CD11 molecules: CD11a, CD11b, CD11c, or CD11d. These heterodimeric molecules interact with more than 20 known ligands, of which the most prominent belong to the intercellular adhesion molecule family (
      • Carlos T.M.
      • Harlan J.M.
      Leukocyte-endothelial adhesion molecules.
      ).
      The pathogenic role of β2-integrins in human psoriasis and other inflammatory skin diseases is less well understood. Circumstantial evidence indicating that reduced CD18 expression may causally be involved in the development of this psoriasiform dermatitis, comes from the clinical observation that some patients suffering from leukocyte adhesion deficiency syndrome-1, with only moderately reduced CD18 expression levels, develop a psoriasiform skin disease (
      • van Pelt J.P.
      • Kuijpers S.H.
      • van de Kerkhof P.C.
      • de Jong E.M.
      The CD11b/CD18–integrin in the pathogenesis of psoriasis.
      ). Linkage analysis of psoriasis families has identified a region on chromosome-17, including among other loci the intercellular adhesion molecule-2 locus, an important ligand of the CD11/CD18 heterodimers (
      • Tomfohrde J.
      • Silverman A.
      • Barnes R.
      • Fernandez-Vina M.A.
      • Young M.
      • Lory D.
      • et al.
      Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q.
      ). Furthermore, polymorphisms in the CD18 gene have been found that predispose to autoimmune diseases (
      • Gencik M.
      • Meller S.
      • Borgmann S.
      • Sitter T.
      • Menezes Saecker A.M.
      • Fricke H.
      • et al.
      The association of CD18 alleles with anti-myeloperoxidase subtypes of ANCA-associated systemic vasculitides.
      ;
      • Meller S.
      • Jagiello P.
      • Borgmann S.
      • Fricke H.
      • Epplen J.T.
      • Gencik M.
      Novel SNPs in the CD18 gene validate the association with MPO-ANCA+ vasculitis.
      ).
      Until today, the cause of psoriasis remains unknown. Here, we describe the causal contribution of activated macrophages in the initiation and maintenance of the psoriasiform skin disease, and summarize recent data on the impaired function of regulatory T-cells (Treg-cells) being responsible for the accelerated proliferation of pathogenic T-cells, which contribute to the recruitment and activation of macrophages in CD18hypo mouse model of psoriasis. Understanding the role of activated macrophages in the pathogenesis of psoriasiform skin disease in CD18hypo PL/J mouse model will help to develop new approaches for design of therapeutics in chronic inflammatory skin diseases, such as human psoriasis and other inflammatory diseases.

      Macrophage infiltration is a hallmark in the inflamed skin of CD18hypo PL/J mice

      Subsets of both human and mouse monocytes can develop into macrophages in various disease states (
      • Grage-Griebenow E.
      • Flad H.D.
      • Ernst M.
      Heterogeneity of human peripheral blood monocyte subsets.
      ;
      • Sunderkotter C.
      • Nikolic T.
      • Dillon M.J.
      • van Rooijen N.
      • Stehling M.
      • Drevets D.A.
      • et al.
      Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.
      ). A subset of monocytes patrols tissues through long-range crawling on the resting endothelium (
      • Auffray C.
      • Fogg D.
      • Garfa M.
      • Elain G.
      • Join-Lambert O.
      • Kayal S.
      • et al.
      Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior.
      ). This patrolling behavior was required for rapid tissue invasion at the site of an infection by this “resident” monocyte population, which initiated an early immune response and differentiated into tissue “resident” macrophages (
      • Auffray C.
      • Fogg D.
      • Garfa M.
      • Elain G.
      • Join-Lambert O.
      • Kayal S.
      • et al.
      Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior.
      ). In peripheral inflammation, Ly-6Cmed/high monocytes are recruited to the affected sites to become inflammatory exudate macrophages (
      • Sunderkotter C.
      • Nikolic T.
      • Dillon M.J.
      • van Rooijen N.
      • Stehling M.
      • Drevets D.A.
      • et al.
      Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response.
      ). Interestingly, monocytes are abundant in the lesions of T-cell-mediated diseases (
      • Iwahashi M.
      • Yamamura M.
      • Aita T.
      • Okamoto A.
      • Ueno A.
      • Ogawa N.
      • et al.
      Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis.
      ;
      • Schlitt A.
      • Heine G.H.
      • Blankenberg S.
      • Espinola-Klein C.
      • Dopheide J.F.
      • Bickel C.
      • et al.
      CD14+CD16+ monocytes in coronary artery disease and their relationship to serum TNF-alpha levels.
      ). These findings suggest that tissue macrophages derived from patrolling monocytes may contribute to the pathogenesis of autoimmune diseases and may represent a target for treatment.
      Psoriasis is regarded as a T-cell-mediated disease (
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Nickoloff B.J.
      • Wrone-Smith T.
      Injection of pre-psoriatic skin with CD4+ T cells induces psoriasis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Conrad C.
      • Boyman O.
      • Tonel G.
      • Tun-Kyi A.
      • Laggner U.
      • de Fougerolles A.
      • et al.
      Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.
      ). However, there is an increasing body of correlative evidence that macrophages may also be relevant for its pathophysiology (
      • Gillitzer R.
      • Wolff K.
      • Tong D.
      • Muller C.
      • Yoshimura T.
      • Hartmann A.A.
      • et al.
      MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.
      ;
      • Nickoloff B.J.
      Characterization of lymphocyte-dependent angiogenesis using a SCID mouse: human skin model of psoriasis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ). Macrophages lining the epidermal–dermal junction have repetitively been described in human psoriasis (
      • Gillitzer R.
      • Wolff K.
      • Tong D.
      • Muller C.
      • Yoshimura T.
      • Hartmann A.A.
      • et al.
      MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.
      ;
      • van den Oord J.J.
      • de Wolf-Peeters C.
      Epithelium-lining macrophages in psoriasis.
      ;
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ;
      • Nickoloff B.J.
      • Wrone-Smith T.
      Injection of pre-psoriatic skin with CD4+ T cells induces psoriasis.
      ;
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ).
      As in psoriasis, activated macrophages were found to play major roles in other T-cell-mediated autoimmune diseases such as rheumatoid arthritis (
      • Huang Q.
      • Ma Y.
      • Adebayo A.
      • Pope R.M.
      Increased macrophage activation mediated through toll-like receptors in rheumatoid arthritis.
      ), multiple sclerosis (
      • Greter M.
      • Heppner F.L.
      • Lemos M.P.
      • Odermatt B.M.
      • Goebels N.
      • Laufer T.
      • et al.
      Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis.
      ), and the experimental autoimmune encephalitis (
      • Drew P.D.
      • Storer P.D.
      • Xu J.
      • Chavis J.A.
      Hormone regulation of microglial cell activation: relevance to multiple sclerosis.
      ), or in type-I diabetes in the non-obese diabetic mouse model (
      • Rosmalen J.G.
      • Martin T.
      • Dobbs C.
      • Voerman J.S.
      • Drexhage H.A.
      • Haskins K.
      • et al.
      Subsets of macrophages and dendritic cells in nonobese diabetic mouse pancreatic inflammatory infiltrates: correlation with the development of diabetes.
      ).
      In this review, we will focus on the role of macrophages in CD18hypo PL/J mouse model for psoriasis and other T-cell-mediated diseases (
      • Jun H.S.
      • Yoon C.S.
      • Zbytnuik L.
      • van Rooijen N.
      • Yoon J.W.
      The role of macrophages in T cell-mediated autoimmune diabetes in nonobese diabetic mice.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ).
      To define the contribution of macrophages, we investigated the composition of the inflammatory cellular infiltrate in the psoriasiform skin of the CD18hypo mice and the respective skin draining lymph nodes (DLNs). Immunohistological analysis of skin sections taken from affected CD18hypo and CD18wt mice showed remarkably increased number of F4/80+ macrophages in diseased skin of CD18hypo PL/J mice compared with skin of CD18wt animals. In skin DLNs, an increased number of MOMA-2+ monocyte/macrophages, which had apparently entered via the medullar and subcapsular sinuses, were detected in histology and confirmed by FACS analysis (Figure 1). Interestingly, similar as in human psoriasis, these macrophages belong to the classical activation type, as they strongly stained for iNOS and TNF-α, as well as of the alternative activation type, as they expressed dectin-1 on their surface. Our finding is of particular interest as the infiltrating macrophages in the murine psoriasis model were similarly described for human psoriasis (
      • Nickoloff B.J.
      Characterization of lymphocyte-dependent angiogenesis using a SCID mouse: human skin model of psoriasis.
      ;
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ). The pathogenic involvement of these sub-epidermally located macrophages designated as “epithelium lining macrophages” is largely unknown in human psoriasis (
      • Gillitzer R.
      • Wolff K.
      • Tong D.
      • Muller C.
      • Yoshimura T.
      • Hartmann A.A.
      • et al.
      MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.
      ;
      • van den Oord J.J.
      • de Wolf-Peeters C.
      Epithelium-lining macrophages in psoriasis.
      ;
      • Djemadji-Oudjiel N.
      • Goerdt S.
      • Kodelja V.
      • Schmuth M.
      • Orfanos C.E.
      Immunohistochemical identification of type II alternatively activated dendritic macrophages (RM 3/1+3, MS-1+/-, 25F9-) in psoriatic dermis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Vestergaard C.
      • Just H.
      • Baumgartner Nielsen J.
      • Thestrup-Pedersen K.
      • Deleuran M.
      Expression of CCR2 on monocytes and macrophages in chronically inflamed skin in atopic dermatitis and psoriasis.
      ). Here, we show that the number of macrophages was greatly increased in the lesional skin of the chronic inflammatory skin disease in CD18hypo mice corresponding to human psoriasis. Therefore, the CD18hypo PL/J mouse model is a valuable tool to study the potential role of macrophages in the pathogenesis psoriasis.
      Figure thumbnail gr1
      Figure 1Increase in macrophages numbers in lesional skin and skin DLNs of affected CD18hypo mice. Skin cryosections from CD18wt (a) and affected CD18hypo mice (b) were stained with F4/80-Alexa-488 for detecting infiltrating macrophages (green) into the skin. Cell nuclei (blue) were counterstained with 4′,6-diamidino-2-phenylindole (original magnification × 40; inset original magnification × 100). e, epidermis; d, dermis; h, hair follicle. The dotted lines indicate the border between the epidermis and dermis. (c) To quantify macrophages in the skin of affected CD18hypo and CD18wt mice, the positively stained cells were calculated. For all measurements, the median of macrophages counted in 12 high-power fields (HPFs) (n=4) is presented (**P<0.0001 by Student's t-test). Immunostaining with macrophage/monocyte–FITC (clone MOMA-2) was performed on cryosections of skin DLNs from CD18wt (d) and affected CD18hypo mice (e). Infiltrated macrophages (green) were found in the medullar and subcapsular sinuses, as indicated by arrows. Cell nuclei (red) were counterstained with propidium iodide (original magnification × 20). To quantify macrophages in the skin DLNs of CD18wt (f) and affected CD18hypo mice (g), skin DLNs cells were labeled with MOMA-2–FITC mAb and analyzed by flow cytometry. Dotted line, isotype control; solid histogram, MOMA-2 staining. (h) Total number of macrophages in skin DLNs of CD18hypo and CD18wt mice (n=6). One representative experiment out of three is shown (**P<0.01 by Student's t-test).
      (This figure was reproduced from
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      , with permission from the American Society for Clinical Investigation).

      Macrophages represent an important source of TNF-α in lesional skin of CD18hypo PL/J mice

      TNF-α is a multifunctional cytokine that mediates inflammation, immune response, and apoptosis (
      • Locksley R.M.
      • Killeen N.
      • Lenardo M.J.
      The TNF and TNF receptor superfamilies: integrating mammalian biology.
      ). Inappropriate production or persistent activation of TNF-α participates in a wide spectrum of diseases, including septic shock, diabetes, cancer, graft rejection, rheumatoid arthritis, and Crohn's disease (
      • Banno T.
      • Gazel A.
      • Blumenberg M.
      Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.
      ). Accumulating evidence indicates that TNF-α also has a significant role in normal development and homeostasis of several organs. Mice deficient in TNF-α lack germinal center and show increased susceptibility to microbial pathogens due to incomplete inflammatory responses (
      • Pasparakis M.
      • Alexopoulou L.
      • Episkopou V.
      • Kollias G.
      Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.
      ). TNF receptor-1 (TNFR1)-mutant mice show similar abnormalities, in addition to defective formation of Peyer's patches (
      • Fu Y.X.
      • Chaplin D.D.
      Development and maturation of secondary lymphoid tissues.
      ). In skin, TNF-α is the master cytokine promoting inflammatory diseases, such as psoriasis, contact dermatitis, drug eruptions, cutaneous T-cell lymphoma, and others (
      • Teraki Y.
      • Moriya N.
      • Shiohara T.
      Drug-induced expression of intercellular adhesion molecule-1 on lesional keratinocytes in fixed drug eruption.
      ). The concentration of TNF-α is distinctly increased in skin after injury (
      • Kock A.
      • Schwarz T.
      • Kirnbauer R.
      • Urbanski A.
      • Perry P.
      • Ansel J.C.
      • et al.
      Human keratinocytes are a source for tumor necrosis factor alpha: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.
      ) and is essential for angiogenesis during wound healing (
      • Leibovich S.J.
      • Polverini P.J.
      • Shepard H.M.
      • Wiseman D.M.
      • Shively V.
      • Nuseir N.
      Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha.
      ). There is evidence that the levels of TNF-α receptors are highly upregulated in the involved skin areas of psoriatic patients (
      • Kristensen M.
      • Chu C.Q.
      • Eedy D.J.
      • Feldmann M.
      • Brennan F.M.
      • Breathnach S.M.
      Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kDa TNF receptor.
      ), and that the biological activity of TNF-α is elevated in psoriatic skin lesions (
      • Ettehadi P.
      • Greaves M.W.
      • Wallach D.
      • Aderka D.
      • Camp R.D.
      Elevated tumour necrosis factor-alpha (TNF-alpha) biological activity in psoriatic skin lesions.
      ).
      TNF-α is biologically active as trimer (
      • Pfeffer K.
      Biological functions of tumor necrosis factor cytokines and their receptors.
      ). It is produced primarily by activated macrophages and other cell types, including epidermal keratinocytes (
      • Chen G.
      • Goeddel D.V.
      TNF-R1 signaling: a beautiful pathway.
      ), DCs (
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ), and mast cells (
      • Echtenacher B.
      • Mannel D.N.
      • Hultner L.
      Critical protective role of mast cells in a model of acute septic peritonitis.
      ;
      • Malaviya R.
      • Ikeda T.
      • Ross E.
      • Abraham S.N.
      Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-[alpha].
      ;
      • Ackermann L.
      • Harvima I.T.
      Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-alpha and their degranulation is associated with expression of ICAM-1 in the epidermis.
      ). A low level of TNF-α is present in the upper layer of the healthy epidermis, but its synthesis and release from keratinocytes are greatly augmented by injury, infection, UV irradiation, and contact allergy (
      • Kock A.
      • Schwarz T.
      • Kirnbauer R.
      • Urbanski A.
      • Perry P.
      • Ansel J.C.
      • et al.
      Human keratinocytes are a source for tumor necrosis factor alpha: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.
      ). Of the two distinct cell-surface receptors for TNF-α, TNFR1, and TNFR2, keratinocytes mainly express TNFR1 (
      • Banno T.
      • Gazel A.
      • Blumenberg M.
      Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.
      ). The binding of TNF-α to TNFR1 triggers a series of intracellular events resulting in activation of transcription factors, including nuclear factor-κB, activator protein-1, CCAAT enhancer-binding protein β, and others (
      • Baud V.
      • Karin M.
      Signal transduction by tumor necrosis factor and its relatives.
      ), which are responsible for induction of genes important for diverse biological processes, including cell growth and death, and immune, inflammatory, and stress responses (
      • Chen G.
      • Goeddel D.V.
      TNF-R1 signaling: a beautiful pathway.
      ). TNF-α activates the immune responses through induction of signals, such as IL-1 and IL-8, transforming growth factor-β, intercellular adhesion molecule-1, and so on (
      • Banno T.
      • Gazel A.
      • Blumenberg M.
      Effects of tumor necrosis factor-alpha (TNF alpha) in epidermal keratinocytes revealed using global transcriptional profiling.
      ).
      To study the cellular sources of TNF-α in the lesional skin of affected CD18hypo mice, double immunofluorescence stainings performed on cryosections derived from lesional skin of CD18hypo mice and healthy skin from CD18wt mice revealed that CD117+ mast cells, CD31+ endothelial cells, and especially F4/80+ macrophages were important sources of TNF-α (Figure 2). This correlates with the finding that macrophages are important source of TNF-α in psoriatic lesions derived from patients (
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ).
      Figure thumbnail gr2
      Figure 2Activated macrophages are an important source of TNF-α. To investigate the cellular origin of TNF-α in lesional skin infiltrate, skin samples from CD18wt and affected CD18hypo mice were double stained with anti-mouse TNF-α–FITC (green) or TNF-α-phycoerythrin (red), together with the cell-specific markers CD117–Cy3 anti-mouse for mast cells (red) (a, b), CD31-Alexa-488 for endothelial cells (green) (d, e), keratin-14-Alexa-488 for keratinocytes (green) (g, h), and F4/80-Alexa-488 for macrophages (green) (j, k). The overlay (yellow) represents TNF-α-producing cells. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue) (original magnification × 20). e, epidermis; d, dermis; h, hair follicle. Dotted lines indicate the border between epidermis and dermis. Quantitative analysis of the TNF-α-producing cells was performed by counting the cells staining positively for both TNF-α and one of the cell markers CD117–Cy3 for mast cells (c), CD31 for endothelial cells (f), keratin-14 for keratinocytes (i), and F4/80 for macrophages (l) (yellow dots) in the lesional skin of CD18hypo mice as compared with in CD18wt mice. Data are presented as median of positive counts in 12 high-power fields (n=12) (**P<0.01 by Student's t-test).
      (This figure was reproduced from
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      , with permission from the American Society for Clinical Investigation).
      The suggested cellular sources of TNF-α release in human psoriasis include T-cells, mast cells, and endothelial cells (
      • Schlaak J.F.
      • Buslau M.
      • Jochum W.
      • Hermann E.
      • Girndt M.
      • Gallati H.
      • et al.
      T cells involved in psoriasis vulgaris belong to the Th1 subset.
      ;
      • Ackermann L.
      • Harvima I.T.
      Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-alpha and their degranulation is associated with expression of ICAM-1 in the epidermis.
      ). Notably, in a recent paper CD68+ macrophages and CD11c+ dermal DCs were identified as important TNF-α source in human psoriasis and upon treatment with adalimumab (anti-TNF-α antibody) macrophage levels decreased in the plaque psoriasis, with complete resolution of clinical psoriasis (
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ). In line with this finding (
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ), we also identified CD68+ macrophages as important TNF-α source in human psoriatic skin, which had distinctly decreased number and TNF-α concentration following bath-PUVA therapy (our unpublished data). This was also found in a T-cell-independent mouse model, with an increase in TNF-α in macrophages (
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
      ). Additionally, a distinct population of TNF-α and inducible nitric oxide synthase-expressing CD11c+ DCs, resembling murine “TipDCs” (TNF-iNOS-producing DCs), has been found to abundantly infiltrate human psoriatic plaques and to be reduced after treatment with efalizumab (anti CD11a) (
      • Lowes M.A.
      • Chamian F.
      • Abello M.V.
      • Fuentes-Duculan J.
      • Lin S.L.
      • Nussbaum R.
      • et al.
      Increase in TNF-alpha and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a).
      ). As β2-integrins are virtually undetectable on macrophages derived from CD18hypo mice, these cells most likely belong to the pool of detected classically activated macrophages.
      The most abundantly studied macrophage phenotype is the classically activated macrophage characterized by production of TNF-α and iNOS. This macrophage subset develops in response to proinflammatory stimuli such as Th1 cytokines or bacterial products (
      • Aderem A.
      • Ulevitch R.J.
      Toll-like receptors in the induction of the innate immune response.
      ). Macrophages differentiating in the presence of Th2 cytokines have been designated as alternatively activated macrophages. This subset is characterized by enhanced expression of dectin-1, eosinophil chemotactic factor (Ym1/ECF-L), and arginase-1. These markers allow to distinguish them from classically activated macrophages (
      • Stein M.
      • Keshav S.
      • Harris N.
      • Gordon S.
      Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.
      ). The infiltrating macrophages as found in the CD18hypo PL/J mouse model reveal both classical and alternative activation markers, as similarly described for human psoriasis (
      • Nickoloff B.J.
      Characterization of lymphocyte-dependent angiogenesis using a SCID mouse: human skin model of psoriasis.
      ). Interestingly, both classically activated macrophages, characterized by production of TNF-α and iNOS, and alternatively activated macrophages, characterized by enhanced dectin-1 and arginase-1 expression, are present in the lesional skin of CD18hypo PL/J mice (
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ).

      Blocking TNF-α by etanercept results in improvement of the psoriasiform skin inflammation in CD18hypo PL/J mice

      In the past 20 years it has become clear that epidermal changes in psoriasis are secondary to robust immune activation within psoriatic plaques characterized by increased numbers and activation of both T lymphocytes and DCs (professional antigen-presenting cells) (
      • Gottlieb A.B.
      • Lifshitz B.
      • Fu S.M.
      • Staiano-Coico L.
      • Wang C.Y.
      • Carter D.M.
      Expression of HLA-DR molecules by keratinocytes, and presence of Langerhans cells in the dermal infiltrate of active psoriatic plaques.
      ;
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ;
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Krueger J.G.
      The immunologic basis for the treatment of psoriasis with new biologic agents.
      ;
      • Conrad C.
      • Boyman O.
      • Tonel G.
      • Tun-Kyi A.
      • Laggner U.
      • de Fougerolles A.
      • et al.
      Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.
      ). Some animal models demonstrate a distinct role for T-cell activation, local cutaneous T-cells, and TNF-α (
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ). Clinically, agents that specifically block T-cells or TNF-α clear human psoriasis (
      • Chaudhari U.
      • Romano P.
      • Mulcahy L.D.
      • Dooley L.T.
      • Baker D.G.
      • Gottlieb A.B.
      Efficacy and safety of infliximab monotherapy for plaque-type psoriasis: a randomised trial.
      ;
      • Gottlieb A.B.
      • Masud S.
      • Ramamurthi R.
      • Abdulghani A.
      • Romano P.
      • Chaudhari U.
      • et al.
      Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-[alpha] monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris.
      ,
      • Gottlieb A.B.
      • Matheson R.T.
      • Lowe N.
      • Krueger G.G.
      • Kang S.
      • Goffe B.S.
      • et al.
      A randomized trial of etanercept as monotherapy for psoriasis.
      ;
      • Gottlieb A.B.
      Etanercept for the treatment of psoriasis and psoriatic arthritis.
      ;
      • Gordon K.B.
      • Bonish B.K.
      • Patel T.
      • Leonardi C.L.
      • Nickoloff B.J.
      The tumour necrosis factor-alpha inhibitor adalimumab rapidly reverses the decrease in epidermal Langerhans cell density in psoriatic plaques.
      ). TNF-α blocking induced by either etanercept, a form of soluble p75 TNF-α receptor that binds both TNF-α and lymphotoxin (LT) (
      • Gottlieb A.B.
      Etanercept for the treatment of psoriasis and psoriatic arthritis.
      ), or infliximab, a chimeric, monoclonal anti-TNF-α antibody (
      • Gottlieb A.B.
      • Masud S.
      • Ramamurthi R.
      • Abdulghani A.
      • Romano P.
      • Chaudhari U.
      • et al.
      Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-alpha monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris.
      ), or adalimumab, a fully human-derived recombinant monoclonal antibody against TNF-α (
      • Chew A.L.
      • Bennett A.
      • Smith C.H.
      • Barker J.
      • Kirkham B.
      Successful treatment of severe psoriasis and psoriatic arthritis with adalimumab.
      ), results in improvement or resolution of psoriasis. Clinical remission is associated with substantially decreased numbers of intraepidermal T-cells and normalization of epidermal proliferation and differentiation, as measured by decreased epidermal thickness and normalized protein expression of keratin-16 (
      • Gottlieb A.B.
      • Masud S.
      • Ramamurthi R.
      • Abdulghani A.
      • Romano P.
      • Chaudhari U.
      • et al.
      Pharmacodynamic and pharmacokinetic response to anti-tumor necrosis factor-[alpha] monoclonal antibody (infliximab) treatment of moderate to severe psoriasis vulgaris.
      ). TNF-α, LT-α, and LT-β are NF-κB-regulated key mediators of inflammation (
      • Aggarwal B.B.
      Signalling pathways of the TNF superfamily: a double-edged sword.
      ). A recent publication shows both TNF and LTs signaling being required for development of the skin phenotype in the IKBα deficiency mouse model of psoriasis (
      • Rebholz B.
      • Haase I.
      • Eckelt B.
      • Paxian S.
      • Flaig M.J.
      • Ghoreschi K.
      • et al.
      Crosstalk between keratinocytes and adaptive immune cells in an I[kappa]B[alpha] protein-mediated inflammatory disease of the skin.
      ). It is not known whether resolution of psoriasis induced by TNF-α/LT blockade is due to effects on T-cells, DCs, macrophages, or a combination of these. So far it is shown that in humans TNF blockade by etanercept induces early inhibitory effects on IL-23 release from DCs, which is known to drive Th17-cell proliferation, as well as having direct effects on Th17 products (IL17, IL22, CCL20) and late inhibitory effects on Th1 effectors such as IFN-γ (
      • Zaba L.C.
      • Cardinale I.
      • Gilleaudeau P.
      • Sullivan-Whalen M.
      • Suarez Farinas M.
      • Fuentes-Duculan J.
      • et al.
      Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.
      ). Notably, levels of TipDCs, a new type of inflammatory myeloid CD11c+ DCs described recently in psoriasis (
      • Lee E.
      • Trepicchio W.L.
      • Oestreicher J.L.
      • Pittman D.
      • Wang F.
      • Chamian F.
      • et al.
      Increased expression of interleukin 23 p19 and p40 in lesional skin of patients with psoriasis vulgaris.
      ), were significantly reduced in the lesional skin of psoriatic patients with etanercept treatment (
      • Zaba L.C.
      • Cardinale I.
      • Gilleaudeau P.
      • Sullivan-Whalen M.
      • Suarez Farinas M.
      • Fuentes-Duculan J.
      • et al.
      Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.
      ). TipDCs produce multiple inflammatory products, including iNOS, TNF-α, IL-20, and IL-23, in psoriatic lesions (
      • Zaba L.C.
      • Cardinale I.
      • Gilleaudeau P.
      • Sullivan-Whalen M.
      • Suarez Farinas M.
      • Fuentes-Duculan J.
      • et al.
      Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.
      ). Among them, IL-23 released by TipDCs is supposed to directly drive Th17-cell proliferation (
      • Zaba L.C.
      • Cardinale I.
      • Gilleaudeau P.
      • Sullivan-Whalen M.
      • Suarez Farinas M.
      • Fuentes-Duculan J.
      • et al.
      Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.
      ). It is of interest to identify whether TNF-α-producing macrophages in CD18hypo PL/J mouse model of psoriasis share phenotypic and functional features with TipDCs in human psoriasis.
      Anti-TNF-α treatment was shown to resolve inflammation in a number of autoimmune diseases such as experimental autoimmune encephalomyelitis (
      • Schilling S.
      • Goelz S.
      • Linker R.
      • Luehder F.
      • Gold R.
      Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration.
      ), autoimmune diabetes in non-obese diabetic mice (
      • Wu A.J.
      • Hua H.
      • Munson S.H.
      • McDevitt H.O.
      Tumor necrosis factor-alpha regulation of CD4+CD25+ T cell levels in NOD mice.
      ), collagen-induced arthritis (
      • Willment J.A.
      • Lin H.H.
      • Reid D.M.
      • Taylor P.R.
      • Williams D.L.
      • Wong S.Y.
      • et al.
      Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide.
      ), or chronic ulcerative colitis (
      • Popivanova B.K.
      • Kitamura K.
      • Wu Y.
      • Kondo T.
      • Kagaya T.
      • Kaneko S.
      • et al.
      Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis.
      ; Table 1).
      Table 1The role of activated macrophages in mouse models of psoriasis and other autoimmune diseases
      ModelMacrophages in the inflammatory infiltrateDisease resolution after macrophage depletionDisease resolution after anti-TNF-α treatmentReference
      Mouse models of psoriasis
       CD18hypo PL/JYesYesYes
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
       Targeted IKK2YesYesYes
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
       Inducible epidermal deletion of JunBYesNot reportedNot reported
      • Zenz R.
      • Eferl R.
      • Kenner L.
      • Florin L.
      • Hummerich L.
      • Mehic D.
      • et al.
      Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins.
       IL-Ira KOYesNot reportedNot reported
       K5-latent TGF-β1YesNot reportedNot reported
       Flacky skin (fsn/fsn)YesNot reportedNot reported
       Xenograft prepsoriatic skin on AGR129NoNot reportedYes
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
       Involucrin–MEKlYesNot reportedNot reported
       K14–VEGFYesNot reportedNot reported
       Tie2 (Tek)YesNot reportedNot reported
       Integrin αE KOYesNot reportedNot reported
      • Schon M.P.
      • Schon M.
      • Warren H.B.
      • Donohue J.P.
      • Parker C.M.
      Cutaneous inflammatory disorder in integrin {alpha}E (CD103)-deficient mice.
       Xenograft prepsoriatic skin on SCIDYesNot reportedYes
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      Mouse models for autoimmune diseases
       Experimental autoimmune encephalomyelitisYesYesYes
      • Schilling S.
      • Goelz S.
      • Linker R.
      • Luehder F.
      • Gold R.
      Fumaric acid esters are effective in chronic experimental autoimmune encephalomyelitis and suppress macrophage infiltration.
       Autoimmune diabetes (NOD mice)YesYesAge-dependent effects
      • Wu A.J.
      • Hua H.
      • Munson S.H.
      • McDevitt H.O.
      Tumor necrosis factor-alpha regulation of CD4+CD25+ T cell levels in NOD mice.
      ;
      • Nikolic T.
      • Geutskens S.B.
      • van Rooijen N.
      • Drexhage H.A.
      • Leenen P.J.
      Dendritic cells and macrophages are essential for the retention of lymphocytes in (peri)-insulitis of the nonobese diabetic mouse: a phagocyte depletion study.
       Collagen-induced arthritisYesYesYes
      • Van Lent P.L.
      • Holthuysen A.E.
      • Van Rooijen N.
      • Van De Putte L.B.
      • Van Den Berg W.B.
      Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis.
       Chronic ulcerative colitisYesNot reportedYes
      • Popivanova B.K.
      • Kitamura K.
      • Wu Y.
      • Kondo T.
      • Kagaya T.
      • Kaneko S.
      • et al.
      Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis.
       Serum-induced arthritisYesYesNot reported
      • Solomon S.
      • Rajasekaran N.
      • Jeisy-Walder E.
      • Snapper S.B.
      • Illges H.
      A crucial role for macrophages in the pathology of K/B x N serum-induced arthritis.
       Autoimmune hemolytic anemiaYesYesNot reported
      • Jordan M.B.
      • Hildeman D.
      • Kappler J.
      • Marrack P.
      An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder.
      IKK, inhibitor of nuclear factor-κB kinase; K14, keratin-14; K5, keratin-5; KO, knockout; NOD, non-obese diabetic; SCID, severe combined immunodeficiency; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor.
      Mouse models of psoriasis and other autoimmune diseases that were confirmed for presence of macrophages and TNF-α are listed.
      Etanercept is an ideal agent to study an effect of TNF-α inhibition on cellular immune regulation in plaques, because its actions are thought to be due to neutralization of TNF-α and not due to depletion of cells bearing cell-surface TNF-α.
      Following the observation that macrophages are a major source for TNF-α, its causal relevance in the formation of the psoriasiform skin disease in the CD18hypo PL/J mouse model for psoriasis was analyzed. TNF-α was neutralized by administrating etanercept once a day at a dose of 100μg per mouse. To evaluate the severity of the psoriasiform phenotype, an adapted psoriasis activity and severity index (PASI) score was used for affected CD18hypo PL/J mice before and after treatment with etanercept or the control (
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). Neutralization of TNF-α by etanercept significantly decreased the adapted PASI score after 30 days of treatment and significantly decreased the number of macrophages in affected skin and skin DLNs. In contrast, no significant improvement was observed in the mice treated with 0.9% NaCl (control) (Figure 3).
      Figure thumbnail gr3
      Figure 3Reduction of the psoriasiform phenotype of affected CD18hypo mice by administration of etanercept. Etanercept (100μg per mouse) was administrated intraperitoneally every day for a period of 30 days to neutralize TNF-α. In parallel, injection of 0.9% NaCl was used as a control. A significant difference in the adapted PASI score appears after treatment with etanercept (a) (P=0.0079 by Student's t-test), but not after treatment with 0.9% NaCl control (b) (P=0.6514 by Student's t-test).
      (This figure was reproduced from
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      , with permission from the American Society for Clinical Investigation.)
      Our data provide evidence for a mechanistic basis, with activated TNF-α-releasing macrophages playing an essential role in chronic psoriasiform skin disease. In fact, this view was confirmed by the recent finding that macrophages and DCs constitute a major source of TNF-α in human psoriasis (
      • Marble D.J.
      • Gordon K.B.
      • Nickoloff B.J.
      Targeting TNFalpha rapidly reduces density of dendritic cells and macrophages in psoriatic plaques with restoration of epidermal keratinocyte differentiation.
      ). This provides the basis for understanding the successful therapy of human psoriasis with a variety of TNF-α-inhibitory agents. The next generation of small molecules targeting different steps of TNF-α signaling has been developed, some of which are currently being tested in clinical trials (
      • Palladino M.A.
      • Bahjat F.R.
      • Theodorakis E.A.
      • Moldawer L.L.
      Anti-TNF-alpha therapies: the next generation.
      ;
      • Mease P.J.
      • Gladman D.D.
      • Ritchlin C.T.
      • Ruderman E.M.
      • Steinfeld S.D.
      • Choy E.H.
      • et al.
      Adalimumab for the treatment of patients with moderately to severely active psoriatic arthritis: results of a double-blind, randomized, placebo-controlled trial.
      ;
      • Weinberg J.M.
      • Bottino C.J.
      • Lindholm J.
      • Buchholz R.
      Biologic therapy for psoriasis: an update on the tumor necrosis factor inhibitors infliximab, etanercept, and adalimumab, and the T-cell-targeted therapies efalizumab and alefacept.
      ;
      • Cordoro K.M.
      • Feldman S.R.
      TNF-alpha inhibitors in dermatology.
      ).

      Mechanism of macrophage ‘suicide’ using clodronate (CL2MDP) liposomes

      As macrophages represent a major, but not the only, source of TNF-α, use of TNF-α inhibitors with subsequent resolution of psoriasis provides only circumstantial evidence that macrophages play a major role in human psoriasis.
      To gain more insight into the role of macrophages in various models of inflammation, several approaches have been used to reduce macrophage activity, including blockade of secreted cytokines (
      • Elliott M.J.
      • Maini R.N.
      • Feldmann M.
      • Kalden J.R.
      • Antoni C.
      • Smolen J.S.
      • et al.
      Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis.
      ), downregulation of macrophage activity (
      • van Roon J.A.G.
      • Bijlsma J.W.J.
      • van de Winkel J.G.J.
      • Lafeber F.P.J.G.
      Depletion of synovial macrophages in rheumatoid arthritis by an anti-Fc{gamma}RI-calicheamicin immunoconjugate.
      ) using Ricin A-coupled antibodies directed against the high-affinity IgG receptor, CD64 (
      • Thepen T.
      • van Vuuren A.J.
      • Kiekens R.C.
      • Damen C.A.
      • Vooijs W.C.
      • van De Winkel J.G.
      Resolution of cutaneous inflammation after local elimination of macrophages.
      ), or liposomes containing dichloromethylene diphosphonate (clodronate liposomes) (
      • Van Rooijen N.
      • Kors N.
      • vd Ende M.
      • Dijkstra C.D.
      Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate.
      ;
      • Kurimoto I.
      • van Rooijen N.
      • Dijkstra C.D.
      • Streilein J.W.
      Role of phagocytic macrophages in induction of contact hypersensitivity and tolerance by hapten applied to normal and ultraviolet B-irradiated skin.
      ; Figure 4). Previously, macrophage depletion with clodronate liposomes proved to be effective in several animal models of diseases where activated macrophages were involved in the pathogenic mechanism, such as autoimmune diabetes in non-obese diabetic mice (
      • Nikolic T.
      • Geutskens S.B.
      • van Rooijen N.
      • Drexhage H.A.
      • Leenen P.J.
      Dendritic cells and macrophages are essential for the retention of lymphocytes in (peri)-insulitis of the nonobese diabetic mouse: a phagocyte depletion study.
      ), experimental allergic encephalomyelitis (
      • Polfliet M.M.
      • van de Veerdonk F.
      • Dopp E.A.
      • van Kesteren-Hendrikx E.M.
      • van Rooijen N.
      • Dijkstra C.D.
      • et al.
      The role of perivascular and meningeal macrophages in experimental allergic encephalomyelitis.
      ;
      • Greter M.
      • Heppner F.L.
      • Lemos M.P.
      • Odermatt B.M.
      • Goebels N.
      • Laufer T.
      • et al.
      Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis.
      ), serum-induced arthritis
      • Solomon S.
      • Rajasekaran N.
      • Jeisy-Walder E.
      • Snapper S.B.
      • Illges H.
      A crucial role for macrophages in the pathology of K/B x N serum-induced arthritis.
      ), collagen-induced arthritis (
      • Van Lent P.L.
      • Holthuysen A.E.
      • Van Rooijen N.
      • Van De Putte L.B.
      • Van Den Berg W.B.
      Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis.
      ), or experimental autoimmune anemia (
      • Jordan M.B.
      • Hildeman D.
      • Kappler J.
      • Marrack P.
      An animal model of hemophagocytic lymphohistiocytosis (HLH): CD8+ T cells and interferon gamma are essential for the disorder.
      ; Table 1). Clodronate liposomes have been shown to efficiently and selectively deplete phagocytic macrophages by apoptosis from spleen, liver, lymph nodes, and skin, whereas other non-phagocytic immunocompetent cells, such as T-cells, B cells, and mature DCs, are not eliminated (
      • Van Rooijen N.
      • Kors N.
      • vd Ende M.
      • Dijkstra C.D.
      Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate.
      ;
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ). Additionally, intradermal injections of clodronate liposomes did not deplete mature Langerhans cells from the epidermis (
      • Kurimoto I.
      • van Rooijen N.
      • Dijkstra C.D.
      • Streilein J.W.
      Role of phagocytic macrophages in induction of contact hypersensitivity and tolerance by hapten applied to normal and ultraviolet B-irradiated skin.
      ). The large liposomes (>0.8μm) used in our study were selectively phagocytosed by macrophages. After injection of clodronate liposomes into the lesional skin, skin macrophages phagocytosed clodronate liposomes and the phospholipids bilayers of the liposomes were disrupted by lysosomal phospholipase. Clodronate was then released intracellularly, and drove macrophages into apoptosis (Figure 4).
      Figure thumbnail gr4
      Figure 4Mechanism of macrophage depletion using clodronate liposomes in vivo. To selectively deplete macrophages in vivo, clodronate (CL2MDP) liposomes have successfully been employed in a variety of experimental systems. The mechanism of macrophage depletion using clodronate liposomes is as follows. Macrophage ‘suicide’ liposomes, encapsulating the clodronate molecules are ingested by macrophages via endocytosis. After fusion with lysosomes containing phospholipases (arrowheads), the latter disrupt the bilayers of the liposomes. The more concentric bilayers are disrupted, the greater is the clodronate release into the cytoplasm of the cell. The cells are then killed by clodronate through apoptosis.
      (This figure was modified from http://www.clodronateliposomes.org.)

      Improvement of the psoriasiform skin inflammation after depletion of skin macrophages in CD18hypo PL/J mice and T-cell independent epidermal inhibitor of nuclear factor-κB kinase 2-deficient mice

      To analyze whether activated macrophages are mandatory in the pathogenesis of the psoriasiform skin inflammation in CD18hypo PL/J mice, macrophages were eliminated from the skin of affected CD18hypo mice using clodronate liposomes. Depletion of macrophages in CD18hypo PL/J mice with a severe inflammatory phenotype led to remarkable improvement of the psoriasiform skin inflammation after 6 weeks of treatment (Figure 5). The reduction in severity and extent of erythema, plaque formation, and scaling after treatment with clodronate liposomes was highly significant (7.67±1.03 versus 2.33±0.52, P=0.0022) (Figure 5). In contrast, no significant changes in PASI score were observed in four mice treated with control liposomes (P=0.3429) (Figure 5). Interestingly, macrophages also play a pathogenic role in a T-cell-independent, epidermis-based psoriasis murine model (
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
      ). This model was generated by deletion of inhibitor of nuclear factor-κB kinase 2, specifically in the epidermis. Notably, also in this model, macrophage levels are increased at the interface between the epidermis and dermis, and upon treatment with clodronate liposomes or etanercept (anti-TNF-α), the skin inflammation resolved (
      • Stratis A.
      • Pasparakis M.
      • Rupec R.A.
      • Markur D.
      • Hartmann K.
      • Scharffetter-Kochanek K.
      • et al.
      Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.
      ). These interesting data derived from two independent mouse models very much suggest that macrophages play a central role in both epidermis-based and T-cell-mediated pathways of inflammation. There is clinical evidence that human psoriasis may not be a homogeneous T-cell-mediated entity. In fact, some psoriatic patients treated with biologics against T-cell emigration and/or activation do not profit from this at all. This may support the view that the therapeutic modulation of macrophage function contributes to an alternate therapeutic strategy for psoriasis patients-independent of whether their disease is mediated by T-cells or not.
      Figure thumbnail gr5
      Figure 5Improvement of the psoriasiform phenotype of affected CD18hypo mice by depletion of macrophages after local injection with clodronate liposomes. Clodronate liposomes were injected subcutaneously at a dose of 50mg/200μl weekly. Representative clinical picture of a CD18hypo mouse with severe psoriasiform dermatitis before (a) and after 40 days of treatment with clodronate liposomes (b). The severity of the psoriasiform phenotype as assessed by the adapted PASI score was significantly reduced after clodronate treatment (c), but not after treatment with control liposomes (d) (P=0.3429 by Student's t-test).
      (This figure was reproduced from
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      , with permission from the American Society for Clinical Investigation.)
      Apart from the success of directly targeting TNF-α in the treatment of human psoriasis (
      • Antoni C.
      • Manger B.
      Infliximab for psoriasis and psoriatic arthritis.
      ;
      • Gordon K.B.
      • Bonish B.K.
      • Patel T.
      • Leonardi C.L.
      • Nickoloff B.J.
      The tumour necrosis factor-alpha inhibitor adalimumab rapidly reverses the decrease in epidermal Langerhans cell density in psoriatic plaques.
      ), therapies that most likely impair macrophage function by diminishing their production and release of TNF-α, such as IL-4 (
      • Ghoreschi K.
      • Thomas P.
      • Breit S.
      • Dugas M.
      • Mailhammer R.
      • van Eden W.
      • et al.
      Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease.
      ), IL-11 (
      • Trepicchio W.L.
      • Ozawa M.
      • Walters I.B.
      • Kikuchi T.
      • Gilleaudeau P.
      • Bliss J.L.
      • et al.
      Interleukin-11 therapy selectively downregulates type I cytokine proinflammatory pathways in psoriasis lesions.
      ), and IL-12 (
      • Kauffman C.L.
      • Aria N.
      • Toichi E.
      • McCormick T.S.
      • Cooper K.D.
      • Gottlieb A.B.
      • et al.
      A phase I study evaluating the safety, pharmacokinetics, and clinical response of a human IL-12 p40 antibody in subjects with plaque psoriasis.
      ), have earlier been reported to improve this medical condition. Recently, IL-20, which is produced by macrophages (
      • Wolk K.
      • Kunz S.
      • Asadullah K.
      • Sabat R.
      Cutting edge: immune cells as sources and targets of the IL-10 family members?.
      ;
      • Pestka S.
      • Krause C.D.
      • Sarkar D.
      • Walter M.R.
      • Shi Y.
      • Fisher P.B.
      Interleukin-10 and related cytokines and receptors.
      ), has been discovered to be upregulated in psoriatic skin (
      • Romer J.
      • Hasselager E.
      • Norby P.L.
      • Steiniche T.
      • Thorn Clausen J.
      • Kragballe K.
      Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine a or calcipotriol.
      ;
      • Otkjaer K.
      • Kragballe K.
      • Funding A.T.
      • Clausen J.T.
      • Noerby P.L.
      • Steiniche T.
      • et al.
      The dynamics of gene expression of interleukin-19 and interleukin-20 and their receptors in psoriasis.
      ). It promotes hyperproliferation and abnormal differentiation of keratinocytes both in vitro and in vivo (
      • Blumberg H.
      • Conklin D.
      • Xu W.F.
      • Grossmann A.
      • Brender T.
      • Carollo S.
      • et al.
      Interleukin 20: discovery, receptor identification, and role in epidermal function.
      ;
      • Romer J.
      • Hasselager E.
      • Norby P.L.
      • Steiniche T.
      • Thorn Clausen J.
      • Kragballe K.
      Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine a or calcipotriol.
      ;
      • Otkjaer K.
      • Kragballe K.
      • Funding A.T.
      • Clausen J.T.
      • Noerby P.L.
      • Steiniche T.
      • et al.
      The dynamics of gene expression of interleukin-19 and interleukin-20 and their receptors in psoriasis.
      ;
      • Wolk K.
      • Witte E.
      • Wallace E.
      • Docke W.D.
      • Kunz S.
      • Asadullah K.
      • et al.
      IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis.
      ). Furthermore, IL-20-transgenic mice displayed skin abnormalities reminiscent of psoriasis (
      • Blumberg H.
      • Conklin D.
      • Xu W.F.
      • Grossmann A.
      • Brender T.
      • Carollo S.
      • et al.
      Interleukin 20: discovery, receptor identification, and role in epidermal function.
      ). It is suggested that IL-20 indirectly exerts its proliferative effects on keratinocytes via immune cells present in the psoriatic skin (reviewed by
      • Stenderup K.
      • Rosada C.
      • Worsaae A.
      • Clausen J.T.
      • Norman Dam T.
      Interleukin-20 as a target in psoriasis treatment.
      ). Accumulating evidences indicate that IL-20 is likely to be a target in psoriasis treatment. Thus, in addition to other beneficial effects, these agents released by activated macrophages may also exert their therapeutic efficacy by silencing macrophages.
      Most recently, a new drug-conjugated antibody (CD64–calicheamicin (CD64-CaMi)) directed to the high-affinity receptor for IgG (FcγRI) was shown to selectively eliminate activated synovial macrophages in rheumatoid arthritis (
      • van Roon J.A.G.
      • Bijlsma J.W.J.
      • van de Winkel J.G.J.
      • Lafeber F.P.J.G.
      Depletion of synovial macrophages in rheumatoid arthritis by an anti-Fc{gamma}RI-calicheamicin immunoconjugate.
      ). Based On our findings, and that of others, future studies may develop the potential therapeutic agents to directly deplete activated macrophages for treatment of autoimmune diseases such as psoriasis.

      Simultaneous injection of recombinant JE/MCP-1 and recombinant TNF-α results in induction of the psoriasiform skin inflammation in healthy skin of CD18hypo PL/J mice

      MCP-1 is an important chemoattractant for macrophage recruitment in various inflammations; an antagonist of MCP-1 has been reported to inhibit arthritis in the MRL-lpr mouse model (
      • Gong J.-H.
      • Ratkay L.G.
      • Waterfield J.D.
      • Clark-Lewis I.
      An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model.
      ). Inhibition of MCP-1 and its receptor (CCR2) pathway by another antagonist against CCR2 ameliorates development of diabetic nephropathy in animal model (
      • Kanamori H.
      • Matsubara T.
      • Mima A.
      • Sumi E.
      • Nagai K.
      • Takahashi T.
      • et al.
      Inhibition of MCP-1/CCR2 pathway ameliorates the development of diabetic nephropathy.
      ). Interestingly, 17β-estradiol (E2) was successfully used to inhibit MCP-1 production in human keratinocytes derived from psoriatic skin in vitro (
      • Kanda N.
      • Watanabe S.
      17Beta-estradiol inhibits MCP-1 production in human keratinocytes.
      ). The beneficial effects of antagonists of MCP-1 or its receptor CCR2 suggest that blocking macrophage migration by inhibiting MCP-1/CCR2 interaction could have therapeutic application in inflammatory diseases, such as psoriasis. However its causal contribution has not been elucidated in psoriasis.
      Murine MCP-1 (CCL-2), originally termed JE, is considered to be the equivalent of human MCP-1, even though it has an extra 49-amino-acid fragment at the C-terminus. Murine MCP-1 is a 125-amino-acid (25–30kDa) member of the C–C subfamily of chemokines (
      • Ernst C.A.
      • Zhang Y.J.
      • Hancock P.R.
      • Rutledge B.J.
      • Corless C.L.
      • Rollins B.J.
      Biochemical and biologic characterization of murine monocyte chemoattractant protein-1. Identification of two functional domains.
      ). It is produced by immune and non-immune cells in response to various stimuli, including TNF-α, IL-1β, IL-4, viruses, and endotoxins. MCP-1 has been shown to have chemoattractant properties for monocytes, memory T-cells, natural killer cells, mast cells, and basophils (
      • Carroll J.M.
      • Romero M.R.
      • Watt F.M.
      Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.
      ;
      • Tuaillon N.
      • Shen D.F.
      • Berger R.B.
      • Lu B.
      • Rollins B.J.
      • Chan C.C.
      MCP-1 expression in endotoxin-induced uveitis.
      ). MCP-1 is postulated to be involved in several diseases, including arteriosclerosis, rheumatoid arthritis, and multiple sclerosis (
      • Yla-Herttuala S.
      • Lipton B.A.
      • Rosenfeld M.E.
      • Sarkioja T.
      • Yoshimura T.
      • Leonard E.J.
      • et al.
      Expression of monocyte chemoattractant protein 1 in macrophage-rich areas of human and rabbit atherosclerotic lesions.
      ;
      • Huang D.R.
      • Wang J.
      • Kivisakk P.
      • Rollins B.J.
      • Ransohoff R.M.
      Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis.
      ). These data indicate that MCP-1 is a major proinflammatory cytokine. Studies using a murine model of Schistosoma mansoni-induced pulmonary granuloma, a murine model of acute septic peritonitis, and studies of murine models of lipopolysaccharide-induced sepsis indicate that MCP-1 can also act as an anti-inflammatory cytokine (
      • Chensue S.W.
      • Warmington K.S.
      • Ruth J.H.
      • Sanghi P.S.
      • Lincoln P.
      • Kunkel S.L.
      Role of monocyte chemoattractant protein-1 (MCP-1) in Th1 (mycobacterial) and Th2 (schistosomal) antigen-induced granuloma formation: relationship to local inflammation, Th cell expression, and IL-12 production.
      ;
      • Matsukawa A.
      • Hogaboam C.M.
      • Lukacs N.W.
      • Lincoln P.M.
      • Strieter R.M.
      • Kunkel S.L.
      Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4.
      ;
      • Bone-Larson C.L.
      • Hogaboam C.M.
      • Steinhauser M.L.
      • Oliveira S.H.
      • Lukacs N.W.
      • Strieter R.M.
      • et al.
      Novel protective effects of stem cell factor in a murine model of acute septic peritonitis. Dependence on MCP-1.
      ). Another role for MCP-1 has been described in the development of Th1 and Th2 (
      • Matsukawa A.
      • Lukacs N.W.
      • Standiford T.J.
      • Chensue S.W.
      • Kunkel S.L.
      Adenoviral-mediated overexpression of monocyte chemoattractant protein-1 differentially alters the development of Th1 and Th2 type responses in vivo.
      ).
      MCP-1-deficient mice (MCP-1-/-) have been generated (
      • Lu B.
      • Rutledge B.J.
      • Gu L.
      • Fiorillo J.
      • Lukacs N.W.
      • Kunkel S.L.
      • et al.
      Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.
      ). They develop normally and have normal hematological profiles, including normal number of macrophages. Data show that despite expression of other chemokines in these mice, MCP-1 is essential for monocyte recruitment (
      • Lu B.
      • Rutledge B.J.
      • Gu L.
      • Fiorillo J.
      • Lukacs N.W.
      • Kunkel S.L.
      • et al.
      Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.
      ). In addition, a role for MCP-1 in arteriosclerosis and experimental autoimmune encephalomyelitis has been demonstrated in MCP-1-/- mice. MCP-1-/- mice fed a high-cholesterol diet have less lipid deposition throughout the aorta than do wt mice (
      • Lu B.
      • Rutledge B.J.
      • Gu L.
      • Fiorillo J.
      • Lukacs N.W.
      • Kunkel S.L.
      • et al.
      Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice.
      ;
      • Aiello R.J.
      • Bourassa P.A.
      • Lindsey S.
      • Weng W.
      • Natoli E.
      • Rollins B.J.
      • et al.
      Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice.
      ;
      • Gosling J.
      • Slaymaker S.
      • Gu L.
      • Tseng S.
      • Zlot C.H.
      • Young S.G.
      • et al.
      MCP-1 deficiency reduces susceptibility to atherosclerosis in mice that overexpress human apolipoprotein B.
      ). MCP-1 plays an important role in the initiation of monocyte accumulation and lipid deposition in atherosclerosis. Similarly, MCP-1-/- mice appear to be markedly resistant to experimental autoimmune encephalomyelitis after active immunization, with drastically impaired recruitment of macrophages to the CNS (
      • Huang D.R.
      • Wang J.
      • Kivisakk P.
      • Rollins B.J.
      • Ransohoff R.M.
      Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis.
      ). MCP-1 is necessary for Th1 immune responses during experimental autoimmune encephalomyelitis, and macrophage recruitment to the inflamed CNS is essential for primed T-cells to execute a Th1-effector program in experimental autoimmune encephalomyelitis.
      Monocytes form a significant component of the inflammatory reaction occurring in the skin involved in atopic dermatitis and psoriasis, and MCP-1 and CCR2 interaction is likely of importance for the monocyte/macrophage trafficking of inflammatory skin disorders (
      • Vestergaard C.
      • Just H.
      • Baumgartner Nielsen J.
      • Thestrup-Pedersen K.
      • Deleuran M.
      Expression of CCR2 on monocytes and macrophages in chronically inflamed skin in atopic dermatitis and psoriasis.
      ). Previous studies demonstrated that the strongest MCP-1 signal in psoriatic lesions is found above the dermal–epidermal junction, and this may explain the characteristic sub-basal distribution of dermal macrophages, suggesting that MCP-1 is important in regulating the interaction between proliferating keratinocytes and dermal macrophages in psoriasis pathogenesis (
      • Gillitzer R.
      • Wolff K.
      • Tong D.
      • Muller C.
      • Yoshimura T.
      • Hartmann A.A.
      • et al.
      MCP-1 mRNA expression in basal keratinocytes of psoriatic lesions.
      ).
      To study whether the synergistic action of a factor recruiting macrophages and a macrophages-activating proinflammatory cytokine may result in induction of the psoriasiform skin inflammation, recombinant JE/MCP-1 and recombinant TNF-α were simultaneously injected. Indeed, 4 days after combined administration of recombinant JE/MCP-1 and recombinant TNF-α in unaffected skin of CD18hypo PL/J mice, skin lesions were induced around the injection sites, which were identical to those seen in the spontaneously occurring chronic psoriasiform skin inflammation in CD18hypo PL/J mice. Interestingly, clinical manifestation of the chronic psoriasiform skin inflammation was reflected histologically and by immunostaining showing an accumulation and activation of macrophages. However, virtually no recruitment of CD4+ T-cells was observed at day 10 after treatment (
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ). These data may be in line with previous data showing that CD4+ T-cells are able to activate macrophages by release of Th1 cytokines such as IFN-γ. In fact, CD4+ T-cells of the CD18hypo mice have a 40-fold increase in IFN-γ release (
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). However, once macrophages are activated directly from proinflammatory cytokines such as TNF-α, they have the capacity to induce the psoriasiform inflammatory skin disease independent of CD4+ T-cells. As human psoriasis is highly variable in its clinical picture, our finding is of particular interest. It is possible that psoriasis is also pathogenetically not a homogeneous entity, and while some cases may be caused by a T-cell-dependent activation of macrophages, others may occur via T-cell-independent mechanisms of macrophage activation (
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ). Only recently, the causal role of macrophages in other T-cell-mediated disease like diabetes mellitus (
      • Jun H.S.
      • Yoon C.S.
      • Zbytnuik L.
      • van Rooijen N.
      • Yoon J.W.
      The role of macrophages in T cell-mediated autoimmune diabetes in nonobese diabetic mice.
      ), systemic lupus erythematosus (
      • Varghese B.
      • Haase N.
      • Low P.S.
      Depletion of folate-receptor-positive macrophages leads to alleviation of symptoms and prolonged survival in two murine models of systemic lupus erythematosus.
      ), and rheumatoid arthritis (
      • van Roon J.A.G.
      • Bijlsma J.W.J.
      • van de Winkel J.G.J.
      • Lafeber F.P.J.G.
      Depletion of synovial macrophages in rheumatoid arthritis by an anti-Fc{gamma}RI-calicheamicin immunoconjugate.
      ) was reported.

      Pathogenic T-cells escaped from the control of dysfunctional regulatory T-cells are involved in activation of macrophages in CD18hypo PL/J mice

      Even though there is still a debate in the field, most researchers and clinicians regard psoriasis as a T-cell-mediated inflammatory skin disease (
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Nickoloff B.J.
      • Wrone-Smith T.
      Injection of pre-psoriatic skin with CD4+ T cells induces psoriasis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Schon M.P.
      • Boehncke W.H.
      Psoriasis.
      ;
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ;
      • Conrad C.
      • Boyman O.
      • Tonel G.
      • Tun-Kyi A.
      • Laggner U.
      • de Fougerolles A.
      • et al.
      Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.
      ). Dysfunctional CD4+CD25+ Treg-cells in peripheral blood in association with accelerated proliferation of CD4+CD25- responder T-cells may contribute to development of human psoriasis (
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ). So far, their causal role and interaction with other T-cell subtypes is poorly understood in psoriasis.
      We now have experimental evidence of the causal contribution of CD18hypo Treg-cells in the development of psoriasiform skin disease in the CD18hypo murine model of psoriasis. Our data show that CD18hypo Treg-cells are dysfunctional in suppressing pathogenic T-cell responses, whereas CD18wt Treg-cells function normally. The reduced CD18 expression on Treg-cells resulted in disruption of the immunological synapse between DCs and Treg-cells, eventually impairing generation of functional antigen-specific Treg-cells in CD18hypo PL/J mice. Moreover, CD18hypo Treg-cells have disrupted cell–cell contact with DCs, with subsequently decreased expression of transforming growth factor-β1. The reduced level of transforming growth factor-β1 is responsible for the decreased suppressor function of Treg-cells, with enhanced generation of activated pathogenic T-cells, which are causal for the onset and severity of psoriasiform skin disease (data submitted for publication).
      The role of CD18 in Treg-cell activation/education and function in the control of autoimmunity is not fully understood (
      • Marski M.
      • Kandula S.
      • Turner J.R.
      • Abraham C.
      CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells.
      ). Marski et al. injected CD18-deficient T-cells into severe combined immunodeficient mice and observed severe colitis. However, under lymphopenic conditions, homeostatic expansion of pathogenic T-cells may be responsible for the induction of the colitis; the role of Treg-cells remains unclear in this model.
      In contrast to the complete CD18 deficiency in CD18-null mice, where neither Treg-cells nor responder T-cells can migrate into the skin (
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ), T-cell emigration into skin does occur in the CD18hypo PL/J mouse model, thus allowing studies on Treg-cell/DC interaction in the lymph node and the skin. Our major finding is that CD18wt Treg-cells, when adoptively transferred into affected CD18hypo PL/J mice even at low numbers, led to resolution of the psoriasiform skin phenotype. This clearly strengthens the view that not only absent (
      • Marski M.
      • Kandula S.
      • Turner J.R.
      • Abraham C.
      CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells.
      ), but also reduced CD18 expression, results in a severe defect in regulatory function of Treg-cells in the CD18hypo PL/J mice.
      Reduced Treg-cell function in CD18hypo PL/J mice is consistent with previous results that IL-2- or IL-2R-deficient mice are deficient in CD4+CD25+ suppressor T-cells in secondary lymphoid organ (
      • Furtado G.C.
      • Curotto de Lafaille M.A.
      • Kutchukhidze N.
      • Lafaille J.J.
      Interleukin 2 signaling is required for CD4(+) regulatory T cell function.
      ). Given the known defect in the activation and IL-2 production of CD18-/- T-cells (
      • Marski M.
      • Kandula S.
      • Turner J.R.
      • Abraham C.
      CD18 is required for optimal development and function of CD4+CD25+ T regulatory cells.
      ), we suggest that low CD18 expression results in impaired immune synapse formation between DCs and Treg-cells, with subsequently impaired T-cell receptor activation, eventually leading to decreased proliferation and reduced suppressor function of Treg-cells. This view is distinctly supported by the impaired cluster formation between DCs and CD18hypo Treg-cells as compared with that between DCs and CD18wt Treg-cells (data submitted for publication). Disturbance in the immune synapse between DC–Treg-cells or in the downstream signaling pathway have been reported for other autoimmune diseases (
      • van de Kerkhof P.C.
      • Weemaes C.M.
      Skin manifestations in congenital deficiency of leucocyte-adherence glycoproteins (CDLG).
      ;
      • Gencik M.
      • Meller S.
      • Borgmann S.
      • Sitter T.
      • Menezes Saecker A.M.
      • Fricke H.
      • et al.
      The association of CD18 alleles with anti-myeloperoxidase subtypes of ANCA-associated systemic vasculitides.
      ;
      • Meller S.
      • Jagiello P.
      • Borgmann S.
      • Fricke H.
      • Epplen J.T.
      • Gencik M.
      Novel SNPs in the CD18 gene validate the association with MPO-ANCA+ vasculitis.
      ). It remains to be further studied whether polymorphisms or mutations contributing to immune synapse and/or T-cell receptor activation in DC–Treg-cell contacts may be responsible for the observed lack of Treg-cell-mediated suppression of autoimmune diseases.
      Interestingly, αE (CD103)-deficient mice also develop the cutaneous inflammatory phenotype (
      • Schon M.P.
      • Schon M.
      • Warren H.B.
      • Donohue J.P.
      • Parker C.M.
      Cutaneous inflammatory disorder in integrin {alpha}E (CD103)-deficient mice.
      ). αEβ7-Integrin is normally expressed by CD4+ and CD8+ T-cells in mucosal and other epithelial compartments such as skin or lung, and is thought to mediate intraepithelial retention of T-cells (
      • Sigmundsdottir H.
      • Johnston A.
      • Gudjonsson J.E.
      • Valdimarsson H.
      Differential effects of interleukin 12 and interleukin 10 on superantigen-induced expression of cutaneous lymphocyte-associated antigen (CLA) and [alpha]E[beta]7 integrin (CD103) by CD8+ T cells.
      ). The expression of the αEβ7-integrin is a marker for a subset of highly potent, functionally distinct Treg-cells specialized for cross talk within epithelial environments (
      • Lehmann J.
      • Huehn J.
      • de la Rosa M.
      • Maszyna F.
      • Kretschmer U.
      • Krenn V.
      • et al.
      Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells.
      ). Similar to the CD18hypo mouse model, the phenotype of (CD103)-deficient mice may indicate that αEβ7-integrin is directly involved in the function or generation of Treg-cells as loss of its expression results in such a severe inflammatory phenotype (
      • Schon M.P.
      • Schon M.
      • Warren H.B.
      • Donohue J.P.
      • Parker C.M.
      Cutaneous inflammatory disorder in integrin {alpha}E (CD103)-deficient mice.
      ;
      • Lehmann J.
      • Huehn J.
      • de la Rosa M.
      • Maszyna F.
      • Kretschmer U.
      • Krenn V.
      • et al.
      Expression of the integrin alpha Ebeta 7 identifies unique subsets of CD25+ as well as CD25- regulatory T cells.
      ).
      With the identification of macrophages to play a critical role in the psoriasiform skin disease, a new model for pathogenesis events in psoriasis is emerging (Figure 6). In this model, dermal DC activation is the key event for the development of psoriasis in predisposed individuals. Many stimuli can lead to activation of dermal DCs, including injury and infection with organisms that trigger DC pathogen–recognition receptors. Once dermal DCs are activated, they trigger the activation of autoreactive T-cells, leading to their proliferation (reviewed by
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ).
      Figure thumbnail gr6
      Figure 6An emerging model of psoriasis pathogenesis in humans. Periperal tolerance is based on the antigen-specific control of pathogenic T-cells by Treg-cells. The initiating step in the development of psoriasis in predisposed individuals is the activation of DCs. Many insults can lead to activation of DCs. Activated DCs present an antigenic peptide in the context of MHC, CD18, and other adhesion/costimulatory molecules forming DC–T-cell contacts both to Treg-cells and autoreactive T-cells. Under wt condition of CD18 expression, activation of antigen-specific autoreactive T-cells upon contact with the antigen is suppressed by Treg-cells. In CD18hypo PL/J mice, low CD18 expression on Treg-cells results in an impaired synapse being formed between DCs and Treg-cells, with decreased proliferation. Due to impaired activation/education of CD18hypo Treg-cells, Treg-cells cannot sufficiently suppress the activation of autoreactive T-cells, which recruit and activate macrophages within the dermis by production of IFN-γ or others. Activated macrophages with TNF-α release induce activation of epidermal cells, which in turn induces production of MCP-1 and other chemotactic cytokines by epidermal cells. These chemotactic agents induce influx of monocytes from the blood, which undergo differentiation into macrophages and myeloid dendritic cells to maintain and amplify the inflammation and stimulate epidermal proliferation, a key feature of psoriaisis (
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ).
      (This figure was modified from
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      .)
      In case Treg-cells are dysfunctional, as shown in CD18hypo PL/J mouse model of psoriasis and in human psoriasis (
      • Sugiyama H.
      • Gyulai R.
      • Toichi E.
      • Garaczi E.
      • Shimada S.
      • Stevens S.R.
      • et al.
      Dysfunctional blood and target tissue CD4+CD25high regulatory T cells in psoriasis: mechanism underlying unrestrained pathogenic effector T cell proliferation.
      ), we suggest a sequence of pathogenic events in the development of the psoriasiform skin disease, where a decrease in CD18 expression impairs DC-Treg-cell interaction, with diminished suppressive function and subsequent hyperactivation of pathogenic T-cells (
      • Bullard D.C.
      • Scharffetter-Kochanek K.
      • McArthur M.J.
      • Chosay J.G.
      • McBride M.E.
      • Montgomery C.A.
      • et al.
      A polygenic mouse model of psoriasiform skin disease in CD18-deficient mice.
      ;
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ). These activated pathogenic T-cells, together with activated dermal DCs, secrete inflammatory cytokines that induce production of MCP-1 and other chemotactic factors by keratinocytes, leading to an influx of macrophages and DCs (reviewed by
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ). The recruitment and activation of macrophages subsequently over-produce the proinflammatory cytokine TNF-α, leading to overall amplification and maintenance of the inflammatory process and thus the psoriasiform skin disease (Figure 6).
      The polygenic CD18hypo PL/J murine psoriasis model described here may help to define modifier genes, which in addition to the CD18hypo mutation, contribute to the psoriasiform phenotype. The independent genetic approach may help to define novel gene/genes interacting with the function of β2-integrins, genes, which may also provide important insight into the pathogenetic sequence of human psoriasis.

      Perspectives and Conclusion

      Several other T-cell-mediated mouse models of psoriasis have a phenotype similar to psoriasis. Xenograft models, in which symptomless (PN) skin derived from patients with psoriasis are engrafted onto severe combined immunodeficient mice or AGR129 mice, are the only models that closely reflect the complete genetic, immunological, and phenotypic changes of human psoriasis (
      • Wrone-Smith T.
      • Nickoloff B.J.
      Dermal injection of immunocytes induces psoriasis.
      ;
      • Schon M.P.
      • Detmar M.
      • Parker C.M.
      Murine psoriasis-like disorder induced by naive CD4+ T cells.
      ;
      • Nickoloff B.J.
      • Wrone-Smith T.
      Injection of pre-psoriatic skin with CD4+ T cells induces psoriasis.
      ;
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Conrad C.
      • Boyman O.
      • Tonel G.
      • Tun-Kyi A.
      • Laggner U.
      • de Fougerolles A.
      • et al.
      Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.
      ;
      • Gudjonsson J.E.
      • Johnston A.
      • Dyson M.
      • Valdimarsson H.
      • Elder J.T.
      Mouse models of psoriasis.
      ). These models have conclusively shown that psoriasis is a T-cell-mediated disease. The psoriatic skin lesions described in xenograft models demonstrate many features resembling the CD18hypo PL/J mouse model of psoriasis, including thickening and hyperkeratosis of the epidermal layer, infiltration of immunocytes (CD4+ T-cells, CD8+ T-cells, dermal DCs, macrophages, mast cells), and proliferation of blood vessels. In these xenograft models, a number of cytokines characteristic of psoriasis were upregulated, including IFN-γ, TNF-α, IL-1, IL-6, and IL-12. Such cytokines were markedly increased in the CD18hypo PL/J mouse model of psoriasis. Notably, similar to the CD18hypo PL/J mouse model, neutralization of TNF-α using an mAb against TNF-α (infliximab) or a soluble TNF-receptor fusion protein (etanercept) significantly decreased the psoriatic phenotype in xenograft models (
      • Boyman O.
      • Hefti H.P.
      • Conrad C.
      • Nickoloff B.J.
      • Suter M.
      • Nestle F.O.
      spontaneous development of psoriasis in a new animal model shows an essential role for resident T cells and tumor necrosis factor-{alpha}.
      ;
      • Gordon K.B.
      • Bonish B.K.
      • Patel T.
      • Leonardi C.L.
      • Nickoloff B.J.
      The tumour necrosis factor-alpha inhibitor adalimumab rapidly reverses the decrease in epidermal Langerhans cell density in psoriatic plaques.
      ). However, studies on the role of activated macrophages in these animals are still lacking, and whether activation of macrophages is involved in the pathogenesis of psoriatic inflammation in xenograft models, remains to be clarified.
      In conclusion, the herein summarized data from the CD18hypo PL/J psoriasis mouse model in the context of the current literature demonstrates that the psoriasiform inflammatory skin disorder critically depends on an appropriate activation of macrophages, with sufficient release of TNF-α. In this model, T-cells are required for macrophage recruitment and activation, as previously shown by the fact that depletion of CD4+ T-cells from these mice induced complete resolution of skin disease (
      • Kess D.
      • Peters T.
      • Zamek J.
      • Wickenhauser C.
      • Tawadros S.
      • Loser K.
      • et al.
      CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.
      ;
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ;
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ). Our recent data shed light on a mechanistic basis of the involvement of a shifted immune balance between regulatory T-cells and autoreactive T-cells. Dysfunctional Treg-cells caused by reduced expression of CD18 in CD18hypo PL/J mice are responsible for unrestrained activation of pathogenic T-cells. Based on our findings (
      • Wang H.
      • Peters T.
      • Kess D.
      • Sindrilaru A.
      • Oreshkova T.
      • Van Rooijen N.
      • et al.
      Activated macrophages are essential in a murine model for T cell-mediated chronic psoriasiform skin inflammation.
      ), an emerging model of psoriasis pathogenesis in humans suggests that dermal macrophages, once activated by T-cell cytokines, then produce large amounts of TNF-α, leading to skin changes (
      • Clark R.A.
      • Kupper T.S.
      Misbehaving macrophages in the pathogenesis of psoriasis.
      ).
      These features shared with human psoriasis make this model a valuable tool for future investigations into the pathogenesis of chronic inflammatory skin diseases such as human psoriasis—including their polygenic base—and for future preclinical studies.
      The CD18hypo PL/J mouse model, thus, allowed us to provide evidence for the important role of macrophages in the pathogenesis of a T-cell-mediated animal model for psoriasis, as being also suggested for other autoimmune diseases (Table 1). The causal role of macrophages in a variety of inflammatory diseases just starts to emerge.

      Conflict of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      We are grateful to Heidi Hainzl for technical assistance with immunohistochemistry. This work was supported by the German Research Foundation (DFG) within the SFB 497 “Signals and Signal Processing during Cellular Differentiation”, C7, and individual research grants from the German Research Foundation (DFG) SCHA 411/12-1, SCHA 411/12-2, and SCHA 411/12-3.

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