Number Of IgM-secreting Cells Research Articles

Effect of β-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells

β-glucans are carbohydrates present in the cell wall of many fungi, which are often used as immunostimulants in feeds for farmed species. Their capacity to activate innate immune responses directly acting on innate cell populations has been widely documented in fish. However, whether they can affect the functionality of adaptive immune cells has been scarcely explored. In this context, in the current work, we have determined the effects of β-glucans on rainbow trout blood IgM+ B cells in the presence or absence of 2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide (TNP-LPS), a model antigen. For this, rainbow trout peripheral blood leukocytes were incubated with different doses of β-glucans or media alone in the presence or absence of TNP-LPS for 48 h. The size, levels of expression of surface MHC II, antigen processing and phagocytic capacities and proliferation of IgM+ B cells were then studied by flow cytometry. The number of IgM-secreting cells in the cultures was also estimated by ELISpot. β-glucans significantly decreased the levels of surface MHC II expression and the antigen processing capacities of these cells, especially in the presence of TNP-LPS, while they increased their phagocytic activity. On their own, β-glucans slightly activated the proliferation of IgM+ B cells but reduced that induced by TNP-LPS. In contrast, β-glucans significantly increased the number of cells secreting IgM in the cultures. This effect of β-glucans on the IgM-secreting capacity of B cells was also confirmed through a feeding experiment, in which the IgM-secreting capacity of blood leukocytes obtained from fish fed a β-glucan-supplemented diet for one month was compared to that of leukocytes obtained from fish fed a control diet. Altogether, these findings contribute to increase our knowledge regarding the effects of β-glucans on fish adaptive responses.

L-methionine supplementation modulates IgM+ B cell responses in rainbow trout.

The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.

Bacillus subtilis Expressing the Infectious Pancreatic Necrosis Virus VP2 Protein Retains Its Immunostimulatory Properties and Induces a Specific Antibody Response

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

B1 B cell-derived bone marrow plasmacytes are the primary source of natural IgM (109.11)

Abstract Natural IgM is a unique and important component of humoral immunity. It provides early protection against microbial pathogens and helps to maintain tissue homeostasis. Though B1 cells are known to be the primary source of natural IgM, the developmental stage and anatomical location of the cells that secrete natural IgM antibody remain controversial. In order to answer this question, we first confirmed earlier observations that the spleen and BM are the primary tissues in which IgM secreting cells reside. We next examined two lines of VDJ knockin mice and found that both exhibited 10-fold reductions in serum IgM levels despite the presence of normal IgG concentrations. Whereas knockin animals had normal numbers of IgM secreting cells in the spleen and numbers of splenic and BM IgG plasma cells that were indistinguishable from normal controls, the numbers of IgM plasmacytes in the BM of knockin mice was ≤20% of normal animals. In addition, the vast majority of peritoneal B1 cells were missing in both knockin strains whereas all B2 populations remained unaffected. By sorting BM cells, we determined that only those with the CD138high IgM+ phenotype secreted IgM in vitro; analyses of the two knockin mouse strains revealed this BM population to be significantly reduced in concordance with the peripheral B1 B-cell populations. We conclude that natural IgM antibody is produced by differentiated BM plasmacytes that are derived from peripheral, B1 B-cell populations.

Enhancement of Immunoglobulin M Production in B Cells by the Extract of Red Bell Pepper

Under immunocompromised conditions such as aging, cancer, diabetes, burns, and sepsis, external agents may be useful for upregulation of the immune system to prevent infection. Dietary products have the advantage of being safe and easy to take. An extract of red bell pepper (Capsicum annuum L.) flesh markedly enhanced immunoglobulin M (IgM) production by murine B cells and their proliferation. The effect of the extract on IgM production was not abrogated by treatment with polymyxin B and was also observed in spleen cells of lipopolysaccharideunresponsive C3H/HeJ mice. Treatment of B cells with the extract resulted in an increase in the number of IgMsecreting cells and cluster of differentiation (CD)138-positive cells. The IgM production-enhancing activity was resistant to trypsin treatment but was heat-labile. The activity was eluted in a peak with a molecular weight of 6000 on gel filtration. These results demonstrated that the extract of red bell pepper enhanced IgM production in B cells and suggest that the active substance(s) in the extract is a non-proteinous heat-labile macromolecule.

Promotion of IL-4- and IL-5-dependent differentiation of anti-μ-primed B cells by ascorbic acid 2-glucoside

The stable ascorbic acid derivative 2- O-α- d-glucopyranosyl- l-ascorbic acid (AA-2G) was used to investigate the role of ascorbic acid (AA) in B cell differentiation in vitro. AA-2G is stable in a solution unlike AA but is hydrolyzed by cellular α-glucosidase to release AA. Mouse spleen B cells were primed for 2 days with an anti-μ antibody in the presence of interleukin (IL)-4 and IL-5 and then washed and recultured with AA-2G in the presence of IL-4 and IL-5. AA-2G, but not AA, dose-dependently increased IgM production, the greatest enhancement being 150% at concentrations of more than 0.5 mM. In the absence of IL-4 and IL-5, primed B cells produced a negligible amount of IgM, and AA-2G had no effect. AA-2G-induced IgM production in the presence of IL-4 and IL-5 was inhibited by the α-glucosidase inhibitor castanospermine. Intracellular AA content, depleted during the priming period, increased by adding AA-2G at the start of reculture. Treatment of B cells with AA-2G resulted in an increase in the number of IgM-secreting cells, CD138-positive cells and CD45R/B220-negative cells. The number of viable cells in untreated cultures decreased gradually, but the decrease was significantly attenuated by AA-2G, resulting in about 70% more viable cells in AA-2G-treated cultures. AA-2G caused a slight but reproducible enhancement of DNA synthesis and a slight decrease in the number of cells with a sub-G1 DNA content. These results demonstrated that AA released from AA-2G enhanced cytokine-dependent IgM production in anti-μ-primed B cells and suggest that its effect is caused through promoting the differentiation of B cells to plasma cells and attenuating the gradual decrease in the number of viable cells.

Differentiation of murine B cells induced by chondroitin sulfate B

A two-step culture system was used to investigate the role of chondroitin sulfate (CS) B, which is mitogenic to B cells, in differentiation of B cells. Mouse spleen B cells were incubated for 3 days with CSB in the presence of interleukin (IL)-4 and IL-5. After washing, the cells were replated at 10 5 viable cells/well and recultured without CSB in the presence of IL-4 and IL-5. CSB dose-dependently increased IgM production, the greatest enhancement being 450%. Dextran sulfate had a similar effect, whereas other glycosaminoglycans, CSA, CSC, heparin and hyaluronic acid, were marginally effective. Treatment of B cells with CSB resulted in increases in the number of IgM-secreting cells and numbers of CD138-positive cells and CD45R/B220-negative cells. CSB-induced IgM production was inhibited by the protein kinase C (PKC) inhibitor GF109203X but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. These results demonstrated that CSB promoted differentiation of B cells in the presence of IL-4 and IL-5 and suggested that PKC but not PI3K is crucial for CSB-induced IgM production.

Activation of humoral immunity and eosinophils in neuromyelitis optica

To study immunologic alterations in patients with neuromyelitis optica (NMO). The authors studied 8 patients with NMO together with 16 healthy subjects, 16 patients with relapsing remitting multiple sclerosis (RRMS), and 16 patients with secondary progressive MS (SPMS), matched for age and sex, as controls. Because recent histopathologic studies have demonstrated that active NMO lesions consist of perivascular immunoglobulin (Ig) deposition and eosinophil infiltration, IL-5, IL-6, IL-12, IgG, and IgM production by anti-myelin oligodendrocyte glycoprotein (MOG) mononuclear cells in peripheral blood and CSF were selected for study using ELISPOT. Eotaxin-2 (Eo-2) and eotaxin-3 (Eo-3) levels were also assessed using ELISA and eosinophil cationic protein (ECP) levels by radioimmunoassay. MOG-specific responses in CSF showed significant increase in IL-5, IL-6, IgG, and IgM secreting cells in NMO patients compared with patients with RRMS, SPMS and healthy subjects. Interestingly, numbers of IgM secreting cells were significantly higher than identical specificity IgG secreting ones. Moreover, CSF Eo-2, Eo-3, and ECP levels were also significantly higher in NMO patients compared to all three control populations. Anti-MOG IL-12 secreting cells were increased in CSF and peripheral blood from NMO, RRMS, and SPMS patients when compared to healthy subjects. These observations suggest that neuromyelitis optica is associated with a major humoral immune response (particularly anti-MOG IgM production) and eosinophil activation present exclusively in CSF.

The effects of transforming growth factor-beta neutralization on postburn humoral immunity.

Burn injury induces immunosuppression, which is associated with an increased susceptibility to infection. Our laboratory has demonstrated that burn injury also impairs humoral immunity. We reported that burn injury enhanced expression of transforming growth factor-beta (TGF-beta) mRNA and that exogenous TGF-beta further impaired humoral immunity. The objective of this study was to clarify the role of TGF-beta on humoral immunity after burn injury with a neutralizing experiment. Twelve BALB/c mice were randomly divided into two groups: sham and burn. Anesthetized mice received a 20% full-thickness burn or sham injury. The murine splenocytes containing 1.5 x 10 cells/mL were cultured with 2.5 microg/mL of lipopolysaccharide with or without 0.5 ng/mL of TGF-beta or 1 microg/mL of anti-TGF-beta neutralizing antibody, if necessary. Concentrations of immunoglobulin (Ig) M in the cell culture supernatant were determined by enzyme-linked immunosorbent assay and the number of IgM-secreting cells in the culture was measured by enzyme-linked immunospot assay. After 2-day culture, neutralization of TGF-beta dramatically restored IgM synthesis after burn injury. After 5-day culture, however, it restored IgM concentration but failed to restore a number of IgM-secreting cells. This neutralizing experiment demonstrated that TGF-beta is one of the inhibitors of IgM synthesis after burn injury. However, neutralization of TGF-beta was not enough to completely restore humoral immunity after burn injury. Investigation of the mechanism of impaired IgM synthesis after burn injury should be continued.

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). Part III: Lymphocytes.

The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a T-cell-specific mitogen, concanavalin A (ConA), or a B-cell-specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVB-exposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine gamma-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG-specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyte-related functions in fish.

Transforming growth factor-beta impairs postburn immunoglobulin production by limiting B-cell proliferation, but not cellular synthesis.

Transforming growth factor-beta (TGF-beta) has been shown to be an inhibitor of immunoglobulin (Ig) synthesis and may contribute to decreased Ig synthesis after burn injury. This study investigated the relationship between TGF-beta and Ig synthesis after burn injury. Twenty-four BALB/c mice received either a 30% body surface area full-thickness contact burn or no burn. Splenocytes were isolated 8 days after burn and were cultured with 0, 0.05 or 0.5 ng/mL TGF-beta. After culture, total IgG and total IgM were measured by enzyme-linked immunosorbent assay. The number of IgM-secreting cells per 10(5) cells was measured by enzyme-linked immunoabsorbent spot forming assay. Total IgM per IgM-secreting cell (pg/cell) was calculated. Total IgG, IgM, IgM-secreting cells, and B-cell number after culture were decreased by burn injury, and the decrease was exacerbated by the presence of TGF-beta. The total IgM per IgM-secreting cells, however, was significantly increased by TGF-beta at 0.5 ng/mL. These data demonstrates that TGF-beta does not specifically impair IgM secretion by committed IgM B cells but appears to decrease B-cell proliferation or clonal expansion.

Normal Uterine Cervix: Characterization of Isolated Lymphocyte Phenotypes and Immunoglobulin Secretion

Isolation of viable cervical lymphocyte populations and characterization of their function in healthy tissue is necessary to understand immunity in the genital tract. Normal, cervical tissue was digested using a multi-enzymatic digestion procedure. Lymphocytes were characterized using FACS analysis and ELISPOT analysis for immunoglobulin secreting cells. Following the digestion procedure, 0.16 x 10(6) +/- 0.8 cells/g of tissue with a viability of 90-98% were isolated from normal cervical tissue. FACS analysis determined that B lymphocytes were the predominant cell type in normal cervical tissue representing a significantly higher percentage than that found in peripheral blood (P = 0.015). T lymphocytes and NK cells represented a significantly lower percentage than that found in peripheral blood (P = 0.0001 and 0.026, respectively). The largest percentage of immunoglobulin secreting cells isolated were secreting IgG followed by IgA. A limited number of IgM secreting cells were detected. IgA2 secreting cells represented 34.46 +/- 4.6% of the total number of IgA plasma cells. These studies represent the first analysis of viable mononuclear cells isolated from normal cervical tissue. The results form a baseline from which it will now be possible to compare changes that occur at the cervical squamocolumnar junction in response to infection or neoplasia.

Immunoglobulin-Producing Cells in Human Prostate

Immunohistological techniques were used to investigate the presence of immunoglobulin-producing cells in human prostate. Surgical samples from 14 patients with prostate hyperplasia were analyzed. Lymphoid infiltration was rarely seen and was in no way comparable with the classical structure of mucosae-associated lymphoid tissue (MALT). Plasma cells were observed in nine cases. They produced predominantly IgA and lambda chains, but noticeable numbers of IgM-secreting cells were also seen. These cells were most numerous in patients with antecedents of infection or prostatitis. These data suggest that the local immune system of the prostate is in relation with the MALT, and can be colonized by MALT-derived cells in response to bacterial stimuli.

Interleukin-1 stimulation of human B-lymphoblast differentiation

Transformed B-cell lines have provided useful tools for analyzing B-cell responses to growth and differentiation factors. SKW 6.4 is a human B-lymphoblastoid line that differentiates in response to B-cell differentiation factor (BCDF) but not to other T-cell-derived lymphokines. The present studies were undertaken to determine whether monocyte-derived factors such as interleukin-1 (IL-1) could also promote the maturation of SKW 6.4 cells. Cells were cultured with and without supernatants derived from adherent monocytes of normal individuals. The number of Ig-producing plaque-forming cells in control media cultures gradually increased over a period of 4 days. Monocyte supernatants caused a 3- to 10-fold further increase in the number of IgM-secreting cells over that found in control media cultures. this stimulation by monocyte supernatants was maximal on the third or fourth day of culture and was not accompanied by an increase in proliferation. Recombinant IL-1 added to cultures of SKW 6.4 also produced a dose-related increase in the number of IgM-secreting cells without stimulating proliferation. A polyclonal antiserum against IL-1 prevented the increase in IgM-secreting cells which occurred with the addition of monocyte supernatants. Finally, IL-1 activity was detected in unstimulated SKW 6.4 supernatants. We conclude from these studies that SKW 6.4 cells both secrete IL-1 and differentiate in response to exogenous IL-1. SKW 6.4 may therefore provide a useful tool for future studies on mechanisms of IL-1-induced B cell maturation. It is possible that the basal numbers of IgM-secreting cells seen in unstimulated cultures results from an autocrine mechanism of IL-1-induced differentiation. Finally, care should be taken to exclude IL-1 from supernatants that are being tested for BCDF activity using SKW 6.4 cells.

Production and secretion of immunoglobulins by in vitro-activated human B lymphocytes.

Activation induced by pokeweed mitogen in cultures of mononuclear cells from human blood was followed sequentially by simultaneous quantitation of live cells, thymidine incorporation, cells displaying cytoplasmic, IgM, IgG, IgA or IgD, cells secreting IgM, IgG or IgA and cumulated IgM secretion. Maximal cellular activity was found after 7 days of cultures, with means of 16000 IgM-, 20700 IgG- and 9900 IgA-secreting cells per 10(6) originally cultured cells. The cumulated IgM secretion after 21 days of culture averaged 10400 ng per 10(6) originally cultured cells. A close correlation was found between the number of IgM-secreting cells and the cumulated IgM secretion.

A Modified Technique for Assaying Avian Immunoglobulin-M-Secreting Cells

A localized complement-dependent hemolytic assay in modified Cunningham chambers for enumerating avian immunoglobulin-M (IgM)-secreting spleen cells is defined. The maximum number of IgM-secreting spleen cells was present six days after sheep red blood cells (SRBCs) were injected. Intra-abdominal injection of SRBCs resulted in greater but more variable numbers of IgM-secreting cells at the time of maximum response than did either intramuscular or intravenous injection. Fresh chicken serum (FCS) with 10% guinea pig serum was an equally or more effective source of complement for the assay than was FCS alone.

Induction of an IgM anti-(bovine)-IgG response in mice by bacterial lipopolysaccharide.

BACTERIAL endotoxin stimulates the appearance in mice of a large number of plaque forming cells (PFC) secreting an IgM globulin with specificity for bovine IgG (bovine gamma globulin; BGG). Reversed plaquing (antibody on the target erythrocyte) has enabled us to enumerate the total number of IgM secreting cells and show that the anti-BGG PFC comprise at least 10% of the total. Plaque inhibition with free antigen shows that anti-BGG PFC stimulated by LPS, as distinct from those specifically stimulated, secrete a product with low avidity. The evidence reported both here and in a previous study1, leads us to suggest that substances such as LPS stimulate the production of an M-anti-G response. The rheumatoid-factor-like molecule which is produced may possibly serve to amplify complement fixation by high avidity/specificity IgG antibodies.