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Generation of Increased Numbers of HLA-DRhigh IgG+ Plasma Cells in the Peripheral Blood of Patients with Bullous Pemphigoid: NC16a-Specific Cells Belong to the Short-Lived Plasma Blast Population
Bullous pemphigoid (BP) is a rare, usually severe and potentially life-threatening disease. Immunologically, this disorder is characterized by the presence of circulating IgG autoantibodies targeting distinct adhesion molecules of the dermoepidermal basement membrane zone (
). Binding of autoantibodies to adhesion structures of the skin leads to a massive loss of function, resulting in blister formation. The majority of BP patients possesses high-affinity IgG autoantibodies directed against the 180kDa BP autoantigen (BP180;
Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.
). The immunodominant site of BP180 is located within the short NC16a domain, which is recognized by more than 90% of BP patients. In the previous studies we have identified the presence of memory B cells specific for the NC16a domain, which can be induced in vitro to synthesize autoantibodies (
Memory B cells specific for the NC16a domain of the 180kDa antigen can be enriched from the peripheral blood of bullous pemphigoid patients and induced to synthesize specific antibodies.
). The bulk of antibodies measurable in the serum are synthesized by terminally differentiated B lymphocytes, that is, plasma cells (PCs), mostly residing in the bone marrow (BM). Release from secondary organs, migration to the BM, and competition for the apparently limited number of survival niches lead to the transient presence of PCs in the peripheral blood (
Owing to the rarity of PCs in the peripheral blood and the lack of appropriate methods to identify their antigen specificity, few studies exist that investigate this cell population. Recently, it has been suggested that humoral memory reactions to classic bacterial and viral antigens are characterized by the generation of long-lived PCs. In contrast, antibody-mediated autoimmune responses are characterized by short-lived PCs generated by repeated antigen stimulation of B cells (
). Therefore we asked whether an analysis of the PC population in the peripheral blood of BP patients supports this concept.
Samples of whole blood (40ml) were obtained after informed consent from healthy, aged, and gender-matched controls and BP patients. The study was approved by the ethical committee of the University Hospital, Cologne and was conducted according to the Declaration of Helsinki Principles. For immunomagnetic enrichment of circulating CD138+ PCs from peripheral blood mononuclear cells were incubated with CD138 monoclonal antibody-conjugated microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained directly on the enrichment column with monoclonal antibodies recognizing the surface markers CD138, CD19, CD27, CD38, HLA-DR, and intracellular IgG, A (icIgG, A) after permeabilization with saponin (
). No difference in the expression pattern of CD19, CD27, and CD38 between controls and patients could be detected. However, in BP patients the frequency of IgG-expressing CD138+ cells (50±8.8%) was significantly increased (P<0.0001) compared with the frequency in controls (24±7.9%; see Figure 1a and b). The significant increase of the frequency of icIgG+ CD138+ cells in BP patients occurred at the expense of IgA-expressing PCs resulting in a respective reduction of the frequency of icIgA+ CD138+ cells (data not shown). CD138+ cells were further characterized for expression of HLA-DR, a marker of recently activated plasma blasts (PBs). 72±7.0% of the CD138+ icIgG+ PCs of BP patients showed an HLA-DRhigh expression, whereas in controls HLA-DR expression was significantly less pronounced with 50±9.3% (see Figure 2) of the CD138+ cells being HLA-DRhigh (P<0.0001).
Figure 1Detection of icIgG+CD138+ in controls and bullous pemphigoid (BP) patients and identification of NC16a-specific plasma blasts in BP patients. (a, b) CD138+ cells were enriched from the peripheral blood of healthy donors and BP patients with CD138 monoclonal antibody-conjugated microbeads and stained intracellularly with an anti-IgG-FITC antibody directly on the enrichment column. A representative experiment for a control (a) and BP patient (b) is shown. Please note the increase of icIgG+ CD138+ cells in b. (c, d) CD138+ cells were stained directly on the enrichment column with HLA-DR-PE and after permeabilization stained intracellularly with NC16a-Alexa Fluor 647. Dot plots (c, control) and (d, BP patient) show the two-color analysis. Only CD138+ cells were included in the analysis. The given percentage indicates the number of NC16a-specific cells among the CD138+ HLA-DR++ cell population.
Figure 2The frequency of icIgG+ and HLA-DRhigh plasma blasts is significantly increased in patients with bullous pemphigoid (BP). Using the Student's t-test (GraphPad Prism, La Jolla, CA), we observed a significant difference in the frequency of IgG+ and HLA-DRhigh plasma blasts among the CD138+ cell population between BP patients (BP) (IgG n=9 (a); HLA-DR n=11 (b)) and control persons (Co) (IgG (a)/HLA-DR (b) n=11) (***P<0.0001).
To investigate whether the higher frequency of CD138+HLA-DRhigh PCs correlates with disease activity in BP patients, we divided the patients into nonactive (-), active (+), and highly active (++) groups. The activity level was defined in regard to fresh blisters, erosions, crusts, redness, and itchy rash as previously described. A significant correlation between frequency of HLA-DRhigh cells of patients with high active disease and disease activity (P<0.0001) was observed (data not shown).
To identify autoantigen-specific PCs, we stained CD138+ cells intracellulary with recombinant NC16a coupled to Alexa Fluor 647 (Invitrogen, Karlsruhe, Germany). As shown in Figure 1c and d, PBs specific for NC16a are only detectable in BP patients. On average 1.26% NC16a-specific CD138+ cells were detected in the CD138+ icIgG+ population. Autoantigen-specific PCs could be detected in BP patients mainly in the HLA-DRhigh population (d) with a frequency of 0.99±0.67%. In contrast, no NC16a-specific cells could be detected in pemphigus vulgaris patients or controls.
In this study we could identify a CD138++CD19dimCD27highCD38high Ig-secreting cell subset, most probably representing recently generated, short-lived PBs, a precursor cell population that differs clearly from mature PCs of BM and tonsils (
The heterogeneity shown by human plasma cells from tonsil, blood and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression.
were able to identify among the CD27++CD20−CD19dim cell population newly generated HLA-DR++ PBs, which intensively appeared in patients with active systemic lupus erythematosus. Consistent with our own data, this subpopulation was quite dominant in the peripheral blood and their absolute number or frequency correlated with disease activity. We also found a significant correlation between the frequency of the HLA-DRhigh cell population and disease activity. Both studies support the hypothesis that HLA-DRhigh PBs are the immediate product of an immune activation and are associated directly with disease activity (
Unexpectedly, only a low number of icIgG+ CD138+ cells were specific for the main BP antigen NC16a. This may have several reasons: (1) IgG PCs are directed to other epitopes of BP180 and BP230, (2) an autoantigen-independent bystander activation of other unrelated B cells (
), and (3) a combination of (1) and (2). The low frequency of autoantigen-specific IgG+ PCs detected in BP is different from up to 20 × higher frequencies detected 6–8 days after immunization (
). It may therefore be speculated that this difference is due to the lower amounts of autoantigen continuously presented to the immune system in BP patients (
The findings of this study suggest that BP is accompanied by a broad autoantigen-independent activation of the PC system in the peripheral blood. The detectable antigen-specific autoreactive PCs belong to the short-lived PB subset, which might contribute to the high remission rate of the disease.
REFERENCES
Diaz L.A.
Ratrie III, H.
Saunders W.S.
et al.
Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.
Memory B cells specific for the NC16a domain of the 180kDa antigen can be enriched from the peripheral blood of bullous pemphigoid patients and induced to synthesize specific antibodies.
The heterogeneity shown by human plasma cells from tonsil, blood and bone marrow reveals graded stages of increasing maturity, but local profiles of adhesion molecule expression.
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