Annett Jacobi

Disturbed Peripheral B Lymphocyte Homeostasis in Systemic Lupus Erythematosus

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19+/CD27− naive B cells more than CD19+/CD27+ memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27high/CD38+/CD19dim/surface Iglow/CD20−/CD138+ plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27high plasma cells and naive CD27− B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27+ B cells coexpressing IgD were memory B cells preferentially using VH3 family members with multiple somatic mutations. CD27high plasma cells showed a similar degree of somatic hypermutation, but preferentially employed VH4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.

Differential effects of epratuzumab on peripheral blood B cells of patients with systemic lupus erythematosus versus normal controls

Objective: B lymphocytes have been implicated in the pathogenesis of lupus and other autoimmune diseases, resulting in the introduction of B cell-directed therapies. Epratuzumab, a humanised anti-CD22 monoclonal antibody, is currently in clinical trials, although its effects on patients’ B cells are not completely understood. Methods: This study analysed the in vivo effect of epratuzumab on peripheral B cell subsets in 12 patients with systemic lupus erythematosus, and also addressed the in vitro effects of the drug by analysing anti-immunoglobulin-induced proliferation of isolated B cells obtained from the peripheral blood of 11 additional patients with lupus and seven normal subjects. Results: Upon treatment, a pronounced reduction of CD27– B cells and CD22 surface expression on CD27– B cells was observed, suggesting that these cells, which mainly comprise naïve and transitional B cells, are preferentially targeted by epratuzumab in vivo. The results of in vitro studies indicate additional regulatory effects of the drug by reducing the enhanced activation and proliferation of anti-immunoglobulin-stimulated lupus B cells after co-incubation with CD40L or CpG. Epratuzumab inhibited the proliferation of B cells from patients with systemic lupus erythematosus but not normal B cells under all culture conditions. Conclusions: Epratuzumab preferentially modulates the exaggerated activation and proliferation of B cells from patients with lupus in contrast to normal subjects, thus suggesting that epratuzumab might offer a new therapeutic option for patients with systemic lupus erythematosus, as enhanced B cell activation is a hallmark of this disease.

B cells in autoimmunity.

B-cell development is tightly regulated, including the induction of B-cell memory and antibody-secreting plasmablasts and plasma cells. In the last decade, we have expanded our understanding of effector functions of B cells as well as their roles in human autoimmune diseases. The current review addresses the role of certain stages of B-cell development as well as plasmablasts/plasma cells in immune regulation under normal and autoimmune conditions with particular emphasis on systemic lupus erythematosus. Based on preclinical and clinical data, B cells have emerged increasingly as both effector cells as well as cells with immunoregulatory potential.

HLA-DRhigh/CD27high plasmablasts indicate active disease in patients with systemic lupus erythematosus

Objectives: Monitoring of peripheral B-cell subsets in patients with systemic lupus erythematosus (SLE) revealed an activity-related expansion of CD27++CD20−CD19dim Ig-secreting cells. A similar subset has also been identified 6–8 days after tetanus/diphtheria vaccination in normal individuals and in patients with infectious disease. Methods: This subset was analysed further focussing on the HLA-DR surface expression in a cohort of 25 patients with SLE. Results: This study revealed that 86% (range 59–97%) of CD27++CD20−CD19dim cells express high levels of HLA-DR, are also expanded in the bone marrow, and represent plasmablasts enriched with anti-dsDNA secreting cells. The remaining CD27++CD20−CD19dim cells were HLA-DRlow and represent mature plasma cells. Importantly, HLA-DRhigh plasmablasts showed a closer correlation with lupus activity and anti-dsDNA levels than the previously identified CD27++CD20−CD19dim cells. Conclusion: HLA-DRhighCD27++CD20−CD19dim plasmablasts represent a more precise indicator of lupus activity and suggest that there is an overproduction or lack of negative selection of these cells in SLE.

Blood dendritic cells in systemic lupus erythematosus exhibit altered activation state and chemokine receptor function

Background Dendritic cells (DCs) have a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Reduced numbers of blood DCs and the accumulation of DCs at inflammatory sites have been observed in SLE. One crucial feature of DCs is their ability to migrate. Objective To analyse the maturation/activation state and the migratory capacity of different DC precursor subsets in SLE to further elucidate their role in autoimmunity. Methods Plasmacytoid DCs (pDCs), myeloid DCs (mDCs) and monocytes from patients with SLE, healthy volunteers and healthy volunteers immunised with tetanus/diphtheria were examined by flow cytometry for expression of subset-specific antigens (BDCA-2, CD11c, CD14, HLA-DR), activation/maturation markers (CD83, CD86, CD40, BLyS) and chemokine receptors (CCR1, CCR5, CCR7, ChemR23). Additionally, migratory capacity to chemokine receptors was investigated in vitro using the chemokines RANTES, CCL19 and chemerin. Results SLE monocytes and mDCs had higher CD86 and B-lymphocyte stimulatory factor (BLyS) expression levels. ChemR23 expression was lower in SLE pDCs and mDCs. Basal and CCL19-specific migration levels were higher in SLE pDCs. Altered DC function in SLE had no correlative changes in chemokine receptor expression, whereas immunisation-induced blood DC migration patterns in healthy donors were accompanied by changes in chemokine receptor expression. Conclusions The phenotypic and migratory disturbances observed in SLE blood DCs could result in altered distribution of DCs in peripheral tissues, contributing to dysregulated immune responses and autoimmunity.

Prolactin enhances the in vitro production of IgG in peripheral blood mononuclear cells from patients with systemic lupus erythematosus but not from healthy controls

OBJECTIVES Recent evidence suggests that prolactin (PRL) plays a part in the pathogenesis of systemic lupus erythematosus (SLE). Because B cell hyperreactivity and autoantibodies are characteristic hallmarks of SLE, this study aimed at assessing the impact of this pituitary hormone on IgG production by stimulating peripheral blood mononuclear cells (PBMC) with PRL. METHODS PBMC from 11 patients with SLE assessed by the ECLAM score and eight healthy controls were incubated with PRL and cultured for seven days. IgG production was measured by enzyme linked immunosorbent assay (ELISA). RESULTS Spontaneous IgG production of SLE PBMC was significantly enhanced compared with that found in healthy controls. After PRL stimulation, the IgG concentrations of supernatants from SLE PBMC were significantly higher than those of unstimulated PBMC (median 394 ng/ml). Of note, the physiological concentration of PRL (20 ng/ml) induced IgG production more effectively (median 1139 ng/ml) than PRL at 100 ng/ml (median 1029 ng/ml). In contrast, preincubation with PRL did not stimulate IgG production in normal PBMC. A significant correlation between PRL induced IgG production and the disease activity (ECLAM) of the patients with SLE was seen. Moreover, the maximum amount of PRL induced IgG depended on the serum PRL concentrations of the patients with SLE. CONCLUSIONS The results suggest that PBMC from patients with SLE have an extraordinarily high susceptibility to PRL, showing the most striking effect at a concentration usually found in vivo. This indicates a potential role for mild hyperprolactinaemia in the pathogenesis of SLE, influencing both IgG production and disease activity.

Immunglobulin repertoire analysis provides new insights into the immunopathogenesis of Sjögren's syndrome

This review focuses on the use of immunglobulin (Ig) variable region genes by B cells from patients with primary Sjögrens syndrome (pSS) and the biologic insights that this provides. Comparison of the Ig repertoire from the blood and parotid gland of pSS patients with that of normal donors suggests that there are typical disturbances of B cell homeostasis with depletion of memory B cells from the peripheral blood and accumulation/retention of these antigen-experienced B cells in the inflamed tissue. Although there are clonally expanded B cells in the parotid gland, generalized abnormalities in the B cell repertoire are also found in pSS patients. The vast majority of the current data indicate that there is no major molecular abnormality in generating the IgV chain repertoire in patients with pSS. In contrast, disordered selection leads to considerable differences in the V(L) gene usage and V(H) CDR3 length of the B cell Ig repertoire in pSS patients. The nature of the influences that lead to disordered selection in pSS remains to be determined, but should provide important clues to the etiology of this autoimmune inflammatory disorder.

Differential effects of cyclophosphamide and mycophenolate mofetil on cellular and serological parameters in patients with systemic lupus erythematosus

IntroductionDisease activity and therapy show an impact on cellular and serological parameters in patients with systemic lupus erythematosus (SLE). This study was performed to compare the influence of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) therapy on these parameters in patients with flaring, organ-threatening disease.MethodsSLE patients currently receiving CYC (n = 20), MMF (n = 25) or no immunosuppressive drugs (n = 22) were compared using a cross-sectional design. Median disease activity and daily corticosteroid dose were similar in these treatment groups. Concurrent medication, organ manifestations, and disease activity were recorded, and cellular and serological parameters were determined by routine diagnostic tests or flow cytometric analysis. In addition follow-up data were obtained from different sets of patients (CYC n = 24; MMF n = 23).ResultsAlthough both drugs showed a significant effect on disease activity and circulating B cell subsets, only MMF reduced circulating plasmablasts and plasma cells as well as circulating free light chains within three months of induction therapy. Neither MMF nor CYC were able to reduce circulating memory B cells. MMF lowered IgA levels more markedly than CYC. We did not observe a significant difference in the reduction of IgG levels or anti-dsDNA antibodies comparing patients receiving MMF or CYC. In contrast to MMF, induction therapy with CYC was associated with a significant increase of circulating CD8+ effector T cells and plasmacytoid dendritic cells (PDCs) after three months.ConclusionsThe results indicate differences between MMF and CYC with regard to the mechanism of action. MMF, but not CYC, treatment leads to a fast and enduring reduction of surrogate markers of B cell activation, such as circulating plasmablasts, plasma cells and free light chains but a comparable rate of hypogammaglobulinemia.

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome.

Patients with Sjögrens syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related VL chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of VL chain genes caused by selection and clonal expansion of B cells expressing particular VL genes. In addition, the data document an accumulation of B cells bearing mutated VL gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.

Enhanced Mutational Activity and Disturbed Selection of Mutations in VH Gene Rearrangements in a Patient with Systemic Lupus Erythematosus

To determine the impact of somatic hypermutation and selective influences on the VH gene repertoire in SLE, the mutational frequency and pattern of mutations in nonproductively and productively rearranged VH genes obtained from genomic DNA of individual CD 19+ B cells were analyzed in a patient with SLE. The mutational frequencies of nonproductive (6.54×10−2) as well as of productive (4.38×10−2) VH rearrangements were significantly higher in the SLE patient than in normal controls (3.8×10−2 p<0.001 and 3.3×10−2; p<0.001, respectively). Analysis of nonproductive rearrangements documented only minor abnormalities of the targeting of the mutator in the SLE patient. The majority of “mutational hot spots”, although different than in normals, appeared in the CDRs and an increased frequency of mutations in RGYW/WRCY sequences was observed. Moreover, no biases in base pair changes were found in the nonproductive repertoire. In contrast, there was a selection against A and T mutations and towards G mutations within the productive repertoire. Importantly, there were no significant differences in the R/S ratios of mutations within the FRs between the nonproductive and productive repertoire consistent with abnormalities in elimination of B cells expressing VH genes with these mutations. The result of this abnormality was a significantly higher R/S ratio of the VHgenes in the productive repertoire of the SLE patient compared to normals (p<0.05). These data indicate that the mutational machinery was markedly enhanced in this SLE patient but exhibited nearly normal targeting, whereas selective influences were abnormal. These findings suggest that both enhanced mutational activity and disturbances in selection may have played a role in the emergence of autoreactivity in this SLE patient