Intravenous Ig Regulates Anti-Desmoglein 3 IgG Production in B220- Antibody-Producing Cells in Mice with Pemphigus Vulgaris.

Intravenous Ig (IVIG) is a treatment option for intractable cases of pemphigus vulgaris (PV), an autoimmune blistering disease caused by autoantibodies against desmoglein 3 (DSG3). To investigate the efficacy of IVIG on autoantibody secretion, we produced PV model mice by adoptive transfer of immunized Dsg3-/- splenocytes to Rag2-/- mice. We found that circulating anti-DSG3 IgG ELISA titer decreased in PV model mice after 5 days of treatment with IVIG compared with PBS-treated mice, whereas the F(ab')2 fragment did not suppress the anti-DSG3 IgG titer. enzyme-linked immunospot assay revealed that IVIG treatment reduced the frequency of anti-DSG3 antibody-secreting cells in the spleen but not in lymph nodes and bone marrow. Moreover, this reduction was observed only in the splenic B220- fraction but not in the B220+ fraction. Furthermore, IVIG decreased the serum levels of anti-DSG3 IgG, even after a significant reduction of its titer, owing to antibody-mediated CD20+ B cell depletion. In addition, IVIG suppressed anti-DSG3 IgG production in B220-CD138+ plasma cells derived from PV model mice ex vivo. These results indicate that IVIG reduced autoantibody production in B220- cells containing plasma cells in PV model mice, and this function may indicate one of the mechanisms of action of IVIG on PV.

Augmented ILT3/LILRB4 Expression of Peripheral Blood Antibody Secreting Cells in the Acute Phase of Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD) is an acute, systemic vasculitis syndrome that occurs in children. The clinical symptoms and epidemiologic features of KD strongly suggest that KD is triggered by unidentified infectious agents in genetically predisposed patients. In addition, a number of studies have described the role of B cells in the development of KD. To obtain a mechanistic insight into the humoral immune response of B-lineage cells in KD patients, we examined peripheral blood antibody secreting cells (ASCs) and inhibitory immunoreceptors, immunoglobulin-like transcript (ILT)/leukocyte immunoglobulin-like receptor (LILR), on each B cell subpopulation. METHODS: Eighteen Japanese KD patients and thirteen healthy control subjects were recruited for this study. Their peripheral blood mononuclear cells were examined by flow cytometry for the number of CD19 B cells, the size of each B cell subset and the expression of the inhibitory isoforms of ILT/LILR on the B cell subset. RESULTS: The frequency of CD19CD27 ASCs was significantly increased in the acute phase of KD and reduced after high-dose intravenous immunoglobulin (IVIG) treatment. Interestingly, while ILT2/LILRB1 expression was ubiquitously observed on every B cell/ASCs subset and the level was not significantly different after IVIG, ILT3/LILRB4 (B4) was uniquely expressed on only ASCs, and its expression was significantly decreased after IVIG. CONCLUSIONS: In the acute phase of KD, the frequency of ASCs is high with augmented B4 expression, whereas it is lower with decreased B4 expression after IVIG. Further studies of B4 expression on ASCs in autoimmune and infectious diseases will be needed to confirm the significance of our findings.

Regulatory role of Peyer's patches for the inhibition of OVA-induced allergic diarrhea.

Intestinal allergic diseases are initiated by aberrant Th2-type immune responses, including elevation of IgE antibodies (Abs) and infiltration of eosinophils. However, little is known about the role of Peyer's patches (PP) in the control of allergic diseases. Using a mouse model for food allergy, we here show that mice lacking PP are more susceptible to disease development and show higher levels of antigen-specific IgE Abs than do PP-intact mice. In our study, we noted that high numbers of eosinophils infiltrated into the small intestine of PP-null mice. In contrast, the PP of intact mice contained regulatory CD4+CD25+ Foxp3+ T cells (Treg) that are known to produce high levels of IL-10, and inhibited the development of allergic diarrhea. PP-intact mice thus developed allergic diarrhea when treated with anti-CD25 or anti-IL-10 monoclonal antibody (mAb) in vivo. These studies demonstrate that PP, as the site where IL-10-producing Treg cells are created, mediate the mucosal regulatory network for the control of undesired allergic responses in the intestine.

ACSD labelling and magnetic cell separation: a rapid method of separating antibody secreting cells from non-secreting cells.

Several new ways of selecting cells have recently been developed. These include magnetic separation of cells by labelling with magnetic beads against the recombinant product, gel microdrop technology which encapsulates the cells in gelatine beads and matrix-based secretion assays. Affinity capture surface display (ACSD) is a matrix-based assay for the enrichment of high producing cells and relies on the strong affinity between biotin and avidin derivatives. Matrix-based assays have previously only been used for the enrichment of recombinant cells. Here, we have optimised this assay and developed a method of separating antibody producing cells from non-producing cells in a recombinant myeloma cell line using ACSD combined with MACS magnetic separation. The method is rapid, simple enough to become routine and adaptable to many different secreted products from recombinant mammalian cells.

Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age.

BACKGROUND: According to data from animal and in vitro studies, transforming growth factor-beta (TGF-beta) has a crucial effect on 2 essential parts of the mucosal immune system: IgA production and oral tolerance induction. OBJECTIVE: We sought to ascertain whether TGF-beta in breast milk is associated with specific IgA production and atopic disease in human subjects. METHODS: Forty-seven infants with several atopic family members were followed during their first year of life. The concentrations of TGF-beta1 and TGF-beta2 in maternal colostrum, mature milk, and the infants' sera were determined. The enzyme-linked immunospot assay was used to assess the infants' specific IgA production in response to beta-lactoglobulin, casein, gliadin, and ovalbumin. RESULTS: At 12 months, atopic dermatitis was confirmed in 29 of 47 infants; in 11, atopic disease had begun during exclusive breast-feeding (preweaning onset), whereas in 18 the disease manifested itself after weaning (postweaning onset). The concentrations of both TGF-beta1 and TGF-beta2 were higher in maternal colostrum, but not in mature milk and infants' serum, in infants with postweaning-onset atopic disease compared with those with preweaning-onset disease (P =.0008 and P =. 015, respectively). The concentration of TGF-beta2 was, and that of TGF-beta1 tended to be, higher in the colostrum of mothers whose infants had specific IgA-secreting cells at 3 months in response to at least one of the dietary antigens tested compared with those who did not have such cells (P =.048 and P =.076, respectively). CONCLUSION: TGF-beta in colostrum may prevent the development of atopic disease during exclusive breast-feeding and promote specific IgA production in human subjects.

Differential effect of estradiol on antibody secretion of murine hybridomas.

The need for increased antibody production by hybridomas has been approached by the addition to cell cultures of different growth factors; in vitro addition of estradiol-17beta (E2) to human blood lymphocytes increases the accumulation of plasma-blasts and Ig-secreting cells. Four different murine-murine hybridomas secreting different monoclonal antibodies (MAbs) were treated with E2. Specific antibody concentration was measured by enzyme-linked immunoadsorbent assay (ELISA) in culture supernatants whereas expression of E2-receptor in the hybridoma cells was determined by polymerase chain reaction (PCR). When E2 was added as a growth supplement to alpha-estrogen receptor positive murine-murine hybridomas it enhanced MAb secretion by as much as 255%, in a dose-dependant manner. This effect lasted for as long as the alpha-estrogen receptor was detected in the hybridoma cells, was inhibited by tamoxifen and was not observed in alpha-estrogen receptor negative hybridomas. The synthetic estrogen analogue diethylstilbestrol had no effect. Estradiol-17beta should be added to the list of hybridoma-inducing growth factors.

Functional analysis of rheumatoid factor-producing B cells from the synovial fluid of rheumatoid arthritis patients.

OBJECTIVE: To understand the regulation of rheumatoid factor (RF) production in rheumatoid arthritis (RA), we studied IgM-RF production by B cells isolated from the synovial fluid (SF). METHODS: Highly purified SF and peripheral blood (PB) B cells were isolated by negative selection in a fluorescence-activated cell sorter (FACS) and then cultured with either L cells, CD40 ligand (CD40L)-transfected L cells, or type B synoviocytes in the presence or absence of interleukin-2 (IL-2), IL-4, or IL-10. Total IgM and IgM-RF were detected after 14 days by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to detect cells that spontaneously produced immunoglobulin. SF B cells were also phenotypically characterized by FACS analysis. RESULTS: Terminally differentiated CD20-,CD38+ synovial plasma cells (PC) present in the SF of RA patients secreted IgM-RF in the absence of a stimulus. IgM-RF production markedly increased when SF B cells were cultured in the presence of type B RA synoviocytes together with IL-10, but independently of CD40-CD40L interaction. Although CD20-,CD38+ PC could also be demonstrated in SF B cells from patients with other forms of arthritis, IgM-RF production was restricted to the SF B cell cultures of patients with seropositive RA. The frequency of IgM-RF-producing cells among IgM-producing PC in patients with seropositive RA was estimated to be as much as 50%. CONCLUSION: These data demonstrate that terminally differentiated CD20-,CD38+ IgM-RF-producing B cells are specifically present in the inflamed joints of patients with seropositive RA. There is evidence that the local environment in the rheumatoid joint favors RF production. The relatively high frequency of IgM-RF PC in the SF B cell population provides evidence of a dominant RA-specific antigen-driven response in the development of the synovial PC repertoire.

Increased PGE2 production mediates the in vitro inhibitory effect of the human immunodeficiency virus P24 immunosuppressive heptapeptide Ch7.

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7), corresponding to a conserved sequence of human immunodeficiency virus (HIV) core protein p24 (amino acids 232-238), was able to specifically abrogate antigen-induced responses in cultures of normal human peripheral blood lymphocytes (PBL). Addition of recombinant human interferon-gamma (IFN-gamma) to Ch7-suppressed cultures restored the capacity to mount an antigen-specific antibody response, suggesting that a cytokine imbalance may be at the basis of the Ch7 immunosuppressive activity. In the present paper we show that the Ch7-dependent in vitro immunosuppression was accompanied by a significant up-regulation of prostaglandin E2 (PGE2) production and induction of interleukin-10 (IL-10)-secreting cells. In the presence of the PGE2 inhibitor indomethacin, IL-10 up-regulation was prevented and the induction of a specific antibody response was partially restored. PGE2 is indeed an important regulator of immune responses with the ability to differentially affect cytokine production. Thus, our results demonstrate that the Ch7 immunosuppressive epitope may primarily act by up-regulating PGE2 production and, through this mediator, by causing a cytokine dysregulation, finally responsible for immune response suppression.

ELISPOT for detecting antibody-secreting cells in response to infections and vaccination.

Based on immunoassay principles, methods have been developed for the analysis of secreted products at the cellular level. This approach offers substantial methodological advantages compared to traditional immunoassays. In a number of applications cell-based methods have proved able to overcome many of the problems inherent to immunoassays of biological fluids. This review focuses on applications of ELISPOT in natural infections and vaccinations of human individuals. The studies reviewed here have contributed to our understanding of the B-cell responses in infections and the independence of mucosal and systemic immune responses. Whilst diagnostic applications are rare, enzyme immunospot assays have been extensively used in testing the immunogenicity of vaccines. In particular, B-cell responses to mucosal vaccines are better covered with this cellular assay.

Influence of fluorescent antibody probe specificity on flow cytometric analysis of antibody-producing cells.

In the flow cytometric analysis of the stability of antibody-producing cells, fluorescent antibody probes specific for immunoglobulin heavy chains have been widely utilized to quantify intracellular antibodies. To investigate the effect of the specificity of antibody probes on flow cytometric analysis, non-producing subclones of the 6-31 transfectoma were stained with fluorescein isothiocyanate (FITC)-conjugated anti-human IgGs specific for heavy chain and light chain, respectively. The use of heavy chain-specific probe identified heavy chain-only producers as producers, whereas the use of light chain-specific probe identified light chain-only producers as producers. Thus, both heavy chain-specific and light chain-specific antibody probes should be used for the accurate evaluation of heterogeneous non-producing population. Furthermore, the results of the flow cytometric analysis were confirmed by immunoblotting, suggesting that flow cytometry is a useful technique for the rapid evaluation of the stability of transfectomas producing chimeric antibody.

Coordination of immunoglobulin chain folding and immunoglobulin chain assembly is essential for the formation of functional IgG.

The first constant domain (CH1) of immunoglobulin heavy (H) chains is essential for BiP-mediated retention of unassembled H chains in the endoplasmic reticulum (ER). Here, we demonstrated that both wild-type and a mutant gamma chain lacking the CH1 domain bind BiP when they are reduced in vivo. However, only oxidized mutant H chain dimers are released from BiP interaction, whereas oxidized wild-type gamma chain dimers still bind BiP. In light (L) chain-producing cells, some of the mutant H chains accumulate with L chains in ER-derived vesicles and some are secreted as IgG. Furthermore, only half of the secreted antibodies bind antigen. We found the same with a mutant gamma chain, in which the CH1 domain was replaced by a CH3 domain. Therefore, we propose that BiP interaction with incompletely folded CH1 domains is required to mediate correct assembly of H and L chains.

Bacterial endotoxins: comparison of mitogenic, polyclonal, antibody-inducing and toxicity activities.

The mitogenic effects on mouse spleen lymphocytes were determined in a large series of commercially available and laboratory-prepared lipopolysaccharides (LPS) obtained from Escherichia, Salmonella, Serratia and Shigella species; part of these LPS preparations was chemically modified prior to testing. In order to establish whether the degree of mitogenic activity corresponds with other biological effects of these preparations, polyclonal activity, capability to induce specific antibody formation and toxicity were determined for selected LPS's with different mitogenic effects. Some of the detoxication procedures used succeeded in reducing the toxicity of LPS while preserving its high mitogenic activitione of the Fe-detoxified preparations of LPS (from the R-form of Shigella dysenteriae serovar 1) exhibited a medium-degree efficacy in all parameters studied. Generally, there was no correlation between the degree of mitogenic activity and the polyclonal and antibody-inducing activities, but in some instances polyclonal activity did correlate with the antibody-inducing activity.

[A method of immunofluorescent detection of antibody-producing cells and antigens in tissues]. / K metodike immunoliuminestsentnogo vyiavleniia v tkaniakh antiteloobrazuiushchikh kletok i antigenov.

The authors have developed a simple technique for complete and selective suppression of the granulocytes nonspecific fluorescence. The method consists of an additional treatment of the histological sections in a medium for myeloperoxidase detection. Resulting staining of the granulocyte cytoplasm completely inhibits its fluorescence.