Inhibit Ig Secretion Research Articles

Mechanism of immunosuppression in zebrafish (Danio rerio) spleen induced by environmentally relevant concentrations of perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) has been identified as a new persistent organic pollutant. This pollutant is ubiquitous in water and environments. Although PFOA is toxic to fishes, the precise immunotoxicological mechanism remains unclear. In this study, HPLC-MS analysis proved that PFOA can accumulate in the spleen of zebrafish. As comparison of 7-day and 14-day data, the cumulative content in the spleen significantly increased by 26% even in the 0.1 mg/L PFOA-treated group. Morphological observations revealed that PFOA can damage immune cells in zebrafish spleen by inducing vacuolization, lipofuscin granule production, and mitochondrial swelling. The Toll-like receptor 2 (TLR2)/myeloid differentiation factor 88 (myd88)/NF-κB (P65) pathway can mediate the mRNA expression levels of interferon (IFN) and B cell-activating factor (BAFF); immunoglobulin (Ig) secretion is further regulated. RT-PCR results indicated that the expression levels of P65 and IFN in the 1 mg/L group after PFOA exposure for 7 d increased by 4.03- and 3.28-fold, respectively, in a dose-dependent manner compared with those of the control group. The linear correlation coefficient (r2) was analyzed, and the results indicated that the Ig-mediated pathway can be affected by PFOA. For example, the r2 between IgD and P65 decreased from 0.641 (7 d) to 0.295 (14 d) after the cells were exposed to PFOA for a prolonged time; the r2 between IgD and IFN increased from 0.562 (7 d) to 0.808 (14 d). The triangle plot method strongly demonstrated that increased PFOA concentration and prolonged exposure to PFOA can inhibit Ig secretion. Therefore, immune organs, particularly the spleen, of zebrafish are vulnerable to PFOA. These results can help to improve the understanding of the possible noncarcinogenic risk mechanisms induced by PFOA.

Regulation of Immunoglobulin Secretion During Adaptive Immunity (84.17)

Abstract Regulating BCR-mediated adaptive immune responses and subsequent immunoglobulin (Ig) secretion is critical for normal immune function. Defective regulation could lead to chronic inflammation, autoimmunity, or cancer. Several regulatory mechanisms control the threshold for B cell activation and Ig secretion. One mechanism negatively regulates the threshold for BCR activation through coligation of FcγRIIb and subsequent recruitment of SHIP. It was thought that FcγRIIB was the primary mechanism by which humoral responses were terminated. However, mice deficient in FcγRIIb do not exhibit excessive Ig secretion indicating other regulatory mechanisms must exist. We recently identified two additional mechanisms by which immunoglobulin secretion is attenuated during adaptive immunity. First, upon engagement of FcγRIII on dendritic cells by antibody/antigen immune complexes (ICs), dendritic cells secrete soluble mediators that repress BCR-mediated Ig secretion. Second, an autocrine mechanism functions to inhibit Ig secretion wherein B cells stimulated via the BCR with anti-μ or antigen secrete soluble mediators that repress Ig secretion. These data suggest that in addition to FcγRIIb, soluble factors secreted by both dendritic cells and B cells have a critical role in regulating immunoglobulin secretion during BCR-mediated adaptive immune responses. Studies are underway to define the relative importance of these pathways in terminating Ig secretion during adaptive immunity.

Enhancement of humoral immunity by interleukin-12.

We have found that IL-12 treatment of mice leads to long-lasting enhancement in production of most antibody isotypes in conventional B-cell responses. Initial recruitment of new B-cell clones into the response is mediated by IFN-gamma, but subsequent enhancement of Ig secretion appears to be IFN-gamma-independent. We have further found that activated B cells can directly bind IL-12. Taken together, our results suggest a two-step model for the role of IL-12 in enhancement of humoral immunity. Initially, IL-12 induces production of IFN-gamma from Th1 and NK cells. Enough cytokine can be produced from either cell type to then mediate gamma 2a heavy chain isotype switching as well as temporary suppression of IgG1 production. IL-12 further stimulates post-switched cells, including cells producing IgG1, to secrete greatly increased amounts of antibody. This step is not mediated by IFN-gamma but might be due to direct IL-12 binding to activated B lymphocytes. Depletion of B1 cells by IL-12 may further enhance antibody responsiveness since B1 cells are known to competitively inhibit Ig secretion by conventional B cells. The end result is that IL-12 causes a generalized upregulation in production of all antibodies and therefore acts as a strong adjuvant for humoral as well as cellular immunity.

IFN-gamma is a potent inducer of Ig secretion by sort-purified murine B cells activated through the mIg, but not the CD40, signaling pathway.

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were >99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.

B Cell Differentiation Factor-Induced Human B Cell Maturation: Stimulation of Intracellular Calcium Release

We have recently identified a novel human B cell differentiation factor, 446-BCDF, derived from anti-CD3-stimulated peripheral blood (PB) T cells. This novel cytokine, which may act through a pertussis toxin-sensitive G i-linked receptor, induces a 5- to 100-fold increase in immunoglobulin (Ig) secretion by SAC (0.001%, v/v)-activated PB B cells. Coculture of B cells with 446-BCDF induces a decrease in intracellular cAMP which is necessary but not sufficient to drive terminal B cell differentiation. A second signal appears to be required. We therefore measured Ca 2+ flux in indo-1 AM-loaded PB B cells. Stimulation with 446-BCDF resulted in an immediate rise in intracellular Ca 2+ comparable to that seen with the anti-IgM mAb HB57. Ca 2+ appeared to be mobilized from internal stores as pretreatment with BAPTA but not EGTA inhibited the response. Ca 2+ mobilization was critical for the induction of differentiation as BAPTA pretreatment of PB B cells completely inhibited Ig secretion without affecting cell viability. In contrast, neither SAC, rIL6, IL2, IFN-γ, nor IL4 could mobilize Ca 2+. Pertussis toxin, a G i and G o protein inhibitor, was able to inhibit 446-BCDF-induced Ca 2+ flux as well as Ig secretion. To determine whether the Ca 2+ flux was generated in the course of inositol phosphate turnover, we measured IP 3 turnover and the translocation of PKC from cytosol to membrane. An increase in IP 3 comparable to that seen with a monoclonal anti-human IgM antibody was noted and was specifically inhibited by the 446-BCDF-specific mAb 929. Interestingly, no membrane PKC was demonstrable in either SAC- or BCDF-stimulated B cells, although PMA (50 ng/ml) could directly activate PKC. To confirm these findings functionally, B cells were stimulated with SAC and 446-BCDF in the presence of two known PKC inhibitors, staurosporin and calphostin. No inhibition of Ig secretion was detected at any concentration tested (0.39-100 n M staurosporin and 0.0625-1 μ M calphostin C). These data suggest that induction of B cell differentiation is a Ca 2+-dependent and PTX-sensitive event.

Interferon-gamma in multiple myeloma.

Biological heterogeneity is a characteristic of multiple myeloma. A dysregulated cytokine network underlies the various phases of the disease. Numerous cytokines, either promoting or inhibiting plasma cell growth, are involved in tumor control. Interferon-gamma (IFN-gamma) showed the most powerful inhibiting activity on myeloma cell proliferation. This effect was demonstrated on IL-6 dependent myeloma cell lines, but not on IL-6 independent ones. It was also evident on fresh explanted bone marrow myeloma cells. The antiproliferative effect of IFN-gamma seems mainly due to the inhibition of IL-6, the central myeloma growth factor. IL-6 inhibition may occur at various levels: a downregulation of IL-6 receptor has been reported, and also a block of the IL-6 signal transduction pathway via interaction with cytoplasmic proteins such as p91 has been suggested. Our findings showed that IFN-gamma strongly inhibited myeloma cell proliferation to the same extent as dexamethasone (DEX), whereas interferon-alpha (IFN-alpha) inhibited Ig secretion. The combined use of interferons (IFNs) showed inhibitory activities both on proliferation and Ig synthesis that paralleled the effects of DEX. In some cases, IFN-gamma was also shown to augment monoclonal immunoglobulin secretion suggesting a possible differentiating activity on plasma cells. The in vitro data encouraged pilot studies to evaluate the in vivo antitumor effects of IFN-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 inhibits T cell-dependent Ig secretion by neonatal but not adult lymphocytes.

Prostaglandin E2 (PGE2) inhibits Ig production by adult B cells stimulated with Staphylococcus aureus and IL-2, and inhibits the production of IL-2 by anti-CD3 stimulated T cells. By contrast, PGE2 did not inhibit T cell-dependent Ig production by adult B cells. However, T cell-dependent Ig secretion by neonatal B cells was markedly inhibited by PGE2 even in the presence of supplemental IL-2. Forskolin, a direct activator of adenylate cyclase, and dibutyryl cAMP caused similar effects. PGE2 inhibited T cell-dependent Ig secretion by both neonatal CD5+ and CD5- B cells but did not inhibit Ig secretion by either CD5+ or CD5- adult peripheral blood B cells. PGE2 did not inhibit IL-2-dependent anti-CD3 stimulated neonatal T cell proliferation or T cell-dependent neonatal B cell proliferation. PGE2 inhibited neonatal B cell Ig production when addition was delayed, suggesting that initial B cell activation and proliferation were not blocked, but that PGE2 prevented subsequent differentiation. In addition, PGE2 inhibited neonatal T cell help independent of cytokine production. PGE2-induced cAMP levels in neonatal B and T cells were not different from adult levels. These results indicate that production of Ig by neonatal lymphocytes is uniquely sensitive to the inhibitory effects of agents such as PGE2 that elevate cAMP levels. This sensitivity may contribute to the suppressed in vivo responses of human neonatal B cells.

Soluble anti-mu monoclonal antibodies prime resting B cells to secrete immunoglobulins in response to interleukins-4 and -5.

Soluble anti-immunoglobulin (Ig) antibodies have been generally found to inhibit Ig secretion in B cells, via largely unknown mechanisms. To investigate this phenomenon further a two-step culture system was used in which B cells are primed for 24-72 h with various soluble monoclonal or polyclonal anti-Ig antibodies: after washing the cells were placed in readout cultures with a combination of interleukin (IL)-5 and IL-4. Using this protocol B cells primed with (mitogenic or nonmitogenic) anti-mu monoclonal antibodies differentiated into large numbers of IgM-secreting cells, comparable to responses to lipopolysaccharide. In contrast, priming with polyclonal rabbit anti-Ig or monoclonal anti-kappa antibodies, markedly inhibited Ig secretion induced by IL-4 + IL-5. In addition, anti-mu was markedly inhibitory if left in the readout cultures with the two lymphokines. These results, therefore, indicate that appropriate cross-linking of surface IgM receptors on B cells can prime the cells to secrete Ig when they are restimulated by T cell-derived lymphokines in the absence of anti-mu. In contrast co-ligation of both surface IgM and surface IgD receptors apparently results in powerful inhibition of Ig secretion, which is not reversed by stimulation with IL-4 plus IL-5.

VLA-4-fibronectin interaction is required for the terminal differentiation of human bone marrow cells capable of spontaneous and high rate immunoglobulin secretion.

Human bone marrow (BM) is a relevant site for immunoglobulin (Ig) generation in vivo. The occurrence of BM cells capable of spontaneous and high rate Ig secretion for 14 d in vitro has been described previously. Accordingly, these cells provide a suitable model for studying terminal B cell maturation within the BM. We have reported recently that these BM cells are not totally differentiated when isolated from the body, as they require inductive signals from adherent stromal BM cells to complete their maturation. Interleukin (IL)-6 produced by these adherent BM cells was identified as one such signal. The present work shows that IL-6 was necessary, but not sufficient, for the induction of BM Ig-secreting cells, since the cytokine was unable to restore missing IgG in nonadherent BM cell cultures. Supernatants (SN) obtained from cultures of stromal adherent BM cells, either freshly isolated or derived from long-term BM culture (LTBMC), restored Ig secretion by nonadherent BM cells, suggesting that additional soluble factors from BM stromal cells were required. Fibronectin (FN) was identified as that factor, as can be deduced from the following findings: (a) stromal, but not nonadherent, BM cells constitutively produced FN; (b) anti-FN antibodies markedly reduced the IgG secretion in cultures of BM mononuclear cells (BMMC), and blocked the inductive effect of stromal cell SN on nonadherent BM cells, and such a blockade could be reversed by exogenous FN; and (c) finally, although neither IL-6 nor FN alone exerted any effect, the combination of both factors induced optimal Ig secretion by nonadherent BM cells. Furthermore, VLA-4 molecules seemed to be the FN receptor that was active in this culture system, as indicated by: (a) BM Ig-secreting cells exhibited the phenotype VLA-4+ VLA-5-; (b) mAbs directed to VLA-4 (anti-CD29 and anti-CD49d), but not those directed to other adhesion molecules, inhibited Ig secretion by BMMC cultures, and this effect was reversed by FN; (c) the inductive role of the entire FN molecule could be replaced by a fragment containing the CS-1 region, but not by a fragment containing the RGDS sequence; and (d) only mAbs anti-CD49d capable of blocking VLA-4-FN interaction inhibited induction by either the FN or the CS-1-containing fragment of FN.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytokine regulation of immunoglobulin secretion by neonatal lymphocytes.

In contrast to adult lymphocytes, neonatal lymphocytes secrete minimal amounts of Ig in response to stimulation with immobilized MAb to CD3. This deficiency could be overcome by the addition of supplemental IL-2, IL-4, or IL-6, resulting in the secretion of all Ig isotypes. There were no major differences in the distribution of Ig isotypes secreted in response to the cytokines alone or in combination. The Ig secreted in response to IL-4 or IL-6 was inhibited by MAb to CD25, suggesting that the effects of IL-4 and IL-6 were dependent on IL-2. Stimulation of neonatal lymphocytes with anti-CD3 was sufficient to induce expression of IL-2 receptors (CD25) on both T and B cells. IL-4 exerted direct effects on neonatal T cells by increasing IL-2 production and promoting IL-6 production by anti-CD3-stimulated neonatal lymphocytes. Antibody to IL-4 or IL-6 did not inhibit Ig secretion in response to IL-2 and antibody to IL-6 did not consistently inhibit Ig secretion in response to IL-4. Finally, in the presence of cyclosporin, anti-CD3-stimulated neonatal lymphocytes secreted Ig only with the combination of IL-2 and IL-4. These results have delineated unique, but not Ig isotype-specific, effects of cytokines in supporting Ig secretion by anti-CD3-stimulated neonatal lymphocytes. Deficient production of these cytokines is likely to contribute to the decreased capacity of neonatal lymphocytes to generate an Ig response.

Requirement for noncognate interaction with T cells for the activation of B cell immunoglobulin secretion by IL-2

The mechanism whereby noncognate contact with activated IL-2-producing Type 1 helper T cells (TH1) induces B cell activation was examined. Small resting B cells from C57B1/6 mice were cultured, in the absence of any ligand for surface Ig, with irradiated cells of the hapten-specific, CBA-derived, F23.1 + TH1 clone E9.D4 in F23.1 (anti-T cell receptor V β8)-coated microwells. This induced polyclonal B cell activation to enter cell cycle (thymidine incorporation) at 2 days and to secrete immunoglobulin at 5 days. An anti-IL-2 mAb (S4B6) inhibited antibody production completely. Anti-IL-2 did not inhibit either LPS-induced B cell responses, or T cell activation (measured as IL-3 secretion). Anti-IL-2 receptor (anti-Tac) mAbs also inhibited T-dependent B cell responses, without affecting LPS responses. An anti-IFN-γ mAb partially inhibited Ig secretion, without affecting entry into cycle, LPS responses or T cell activation. Other antibodies (anti-IL-3, IL-4, IL-5, Thy-1.2, CD5) were not inhibitory. After 2 days of culture with F23.1-activated T cells, B cells appeared to have become responsive to IL-2, in that they could be driven to immunoglobulin production by the addition of IL-2. Flow cytometry showed no expression by these B cells of 55-kDa (Tac) IL-2 receptors. Also, rigorous removal of T cells from 2-day cocultures prevented the response to IL-2, and readdition of T cells restored it. Because the reconstituted responses were inhibited both by anti-IL-2 and by anti-Tac, IL-2 must have acted indirectly, via the T cells that were present in these cultures. Continued contact with T cells was therefore necessary for the progression of B cells to antibody secretion.

Functional studies examining the subpopulation of human B lymphocytes responding to high molecular weight B cell growth factor.

Mature human B lymphocytes perform many functions including antibody secretion, Ag presentation, preservation of memory for Ag, and lymphokine secretion. Individual resting B cells receive multiple sequential signals that determine the function(s) that will be performed by those cells. Activation signals such as Ag or Staphylococcus aureus Cowan I (Sac) stimulate overlapping but different subpopulations of B cells. After activation, B cells may be induced to proliferate by a variety of B cell growth factors (BCGF) including IL-2, IL-4, TNF-alpha, low molecular weight BCGF (LMW-BCGF), and high molecular weight BCGF (HMW-BCGF). Little information exists to explain why so many different BCGFs are involved with human B cell proliferation. The current studies were designed to examine the role HMW-BCGF plays in selecting B cells for particular functions. HMW-BCGF but not LMW-BCGF was found to inhibit Ig secretion when it was included in culture with Sac-activated B cells and B cell differentiation factors (BCDFs) including IL-6. Sorting resting B lymphocytes into surface IgD+ and IgD- populations and then stimulating each population with anti-mu revealed that the cells most responsive to HMW-BCGF resided in the surface IgD- sorted population. Sorting activated B lymphocytes into BA5 (HMW-BCGFR)+ and BA5- populations revealed that BA5+ B cells stimulated with BCDF (in the absence of HMW-BCGF) produced predominantly IgG, whereas the BA5- population produced both IgG and IgM. Finally, expansion of peripheral B cells from tetanus toxoid-immunized donors with either HMW-BCGF or LMW-BCGF revealed that the HMW-BCGF-expanded population produced predominantly IgG tetanus-specific antibody in the presence of BCDF (in the absence of HMW-BCGF), whereas the LMW-BCGF-expanded population produced IgM much greater than IgG tetanus-specific antibody. Thus, HMW-BCGF may function to expand a subpopulation of B cells for memory B cell functions.

Analysis of cell proliferation and μ-RNA processing during activation of mouse B-cells by anti-μ and T lymphokines

Anti-immunoglobulin (anti-Ig, anti-μ is commonly used) activates resting mouse B-cells to proliferate but not to differentiate and secrete Ig. Differentiation requires additional help from T-cells including soluble factors such as lymphokines. The capability of lymphokines, alone and in combination, to promote the differentiation of anti-μ activated B-cells has been investigated. Some lymphokines, like interleukin (IL) 2 and 3, as well as human-interferon β-2 (IL-6), have no significant effect on differentiation. IL-4 and 5 maintain cell growth but do not lead to differentiation, which requires multiple factors present in ConA supernatant or partially purified TRF. Anti-μ and interferon-γ (IFN-γ) exert both positive and negative effects on B-cell maturation. Anti-ji induces cell proliferation. IFN-γ enhances Ig transcription, but it has no apparent proliferation or differentiation activity. Anti-μ and IFN-γ inhibit Ig secretion by causing the accumulation of nuclear μ-RNA precursors. Although phorbol ester plus ionomycin induce cell proliferation, the negative effect of anti-μ in RNA processing could not be mimicked by these reagents. I show that anti-μ and IFN-μ interfere with the splicing of nuclear hnRNA. This phenomenon is independent of known 2'–5'(A)n synthetase activity. The data suggest that post-transcriptional regulation of μ-RNA processing might be a critical event in controlling the generation of the plasma cells (which secrete IgM), memory precursor cells or abortive cells (both of which do not secrete IgM).

Inhibition of pokeweed mitogen-induced Ig secretion by IgG monoclonal antibodies to MHC class I and Class II molecules requires binding of the intact antibody

Seven purified IgG monoclonal antibodies reactive with different epitopes on DQw1, DR, HLA-A3 or p85 glycoprotein of human lymphocytes have each been shown to inhibit pokeweed mitogen (PWM)-induced IgG and IgM secretion in a dose-dependent manner. Binding of these antibodies to their target antigens was required for the suppression. Antibodies of IgG1, IgG2a, and IgG2b subclasses were able to inhibit both IgG and IgM secretion in the PWM system. The mechanisms by which two of the monoclonal antibodies (MoAbs)—77.34, specific for the class II antigen DQw1, and GAP A3, specific for the class I antigen HLA-A3-caused inhibition—were analyzed. The suppressive effects of 77.34 and GAP A3 were maximal when added at the initiation of the culture period. No inhibition of IL-2 production or cellular proliferation was detected. Supernatants obtained from inhibited cultures were not themselves suppressive. The F(ab′) 2 fragments of either 77.34 or GAP A3 failed to influence PWM-Ig secretion, indicating that intact IgG molecules were required. This suggests that the observed inhibition might be mediated via Fc receptors. Together F(ab′) 2 fragments of either 77.34 or GAP A3 and a control IgG2a protein did not reconstitute the inhibitory effects of intact 77.34 or GAP A3. Suppression, therefore, required intact Fc portions on the same IgG molecules as those that bound to DQw1 or HLA-A3. These studies suggest that populations of IgG molecules that crosslink sufficient numbers of Fc receptors with other cell surface antigens on peripheral blood mononuclear cells (PBMs) during the early stages of B-cell activation can inhibit Ig secretion. Crosslinking of B-cell Fc receptors with SIg has been proposed by others to act as a negative signal for Ig production; our data raise the possibility that crosslinking of FcR with B-cell plasma membrane components other than SIg can also suppress Ig secretion.

Selective effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and corticosteroid on in vitro lymphocyte maturation.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the corticosteroid dexamethasone have potent effects on lymphocyte function, although the effects of the former have not been well characterized. In the present studies murine B cell maturation was used as a model system to examine and compare the effects of TCDD and dexamethasone on cell function. Immunosuppression by TCDD and dexamethasone is mediated by binding to specific intracellular R referred to as the Ah and glucocorticoid R, respectively. Although both compounds were comparable in their ability to inhibit antibody responses to the T-independent antigen TNP-LPS, the events responsible for suppression were found to be distinct. Dexamethasone, although affecting multiple stages of B cell maturation, had its primary effect very early, manifested by inhibition of the phosphoinositide signal transduction pathway. This was evidenced by a decrease in accumulation of inositol phosphate and surface Ia antigen expression as well as an inability to enter the cell cycle after stimulation with anti-Ig. In contrast, neither early signaling events nor proliferation were affected in B cells treated with TCDD. However, TCDD inhibited Ig secretion after stimulation of B cells with T cell-replacing factor, suggesting that TCDD modulates the differentiation of B cells into plasma cells. These differential results were confirmed by monitoring the expression of surface antigens that occur on B cells, including Ia, 7D4, and PC.2, during this maturational process. Whereas dexamethasone inhibited the expression of surface antigens that occur early in maturation (Ia and 7D4), TCDD blocked only the expression of the plasma cell marker PC.2. Although TCDD altered later stages of the B cell cycle, the presence of TCDD was required at the time of initial activation to be effective, suggesting that TCDD may interfere with early cell programming.

In vitro immunoglobulin synthesis by human intestinal lamina propria lymphocytes.

The concentration of IgG and IgA was measured in the supernatants of peripheral blood mononuclear cells and of cells harvested from the intestinal lamina propria, which were cultured in vitro in the presence or absence of mitogens. The lamina propria mononuclear cells were harvested by collagenase digestion of macroscopically normal mucosa from 10 fresh surgical resections for carcinoma. Secretion of IgA in cultures of unstimulated lamina propria mononuclear cells greatly exceeded that of IgG. The addition of pokeweed mitogen increased Ig secretion by cultures of peripheral blood mononuclear cells but decreased Ig secretion by lamina propria mononuclear cells. The addition of concanavalin A suppressed Ig synthesis by pokeweed mitogen stimulated cells more in cultures of peripheral blood mononuclear cells than in lamina propria mononuclear cells. Cycloheximide inhibited Ig secretion by more than 90% in cultures of peripheral blood mononuclear cells, but there was less inhibition in cultures of lamina propria mononuclear cells. In the four unstimulated cultures of lamina propria mononuclear cells examined, over 75% of the Ig was secreted in the first three to four days of culture. The results indicate that lamina propria mononuclear cells are refractory to the inductive and suppressive signals of mitogens, and represent an activated cell population which is committed to Ig secretion before being cultured.

IMMUNOREGULATION IN AN ISOLATED 12-YEAR-OLD BOY WITH CONGENITAL SEVERE COMBINED IMMUNODEFICIENCY

We report the evaluation of in vitro immunoregulation in a 12-year-old untreated boy with severe combined immunodeficiency (SCID). Severely hypogammaglobulinemic, the patient was incapable of a specific antibody response to either natural substances or administered antigens. Ficoll-Hypaque-isolated peripheral blood mononuclear cells (MNL) from the patient failed to respond to pokeweed mitogen (PWM) with the normal increment in immunoglobulin-secreting cells (Ig-SC), as measured by a reverse hemolytic plaque assay. Since the patient was lymphopenic, his MNL were relatively enriched for monocytes (range = 51-81%). Removal of phagocytic cells or the addition of unrelated irradiated helper T lymphocytes resulted in enhanced, but still suboptimal response to PWM, suggesting some intrinsic defect in B lymphocyte function. Co-culture of patient MNL with normal MNL resulted in marked suppression (12% of predicted) of PWM-induced Ig-SC. Suppressor activity was unaffected by prior irradiation of patient MNL, but was substantially reversed (99% of predicted) by removal of his phagocytic cells; whereas the combination of the two procedures further reversed suppression (184% of predicted). The patient's MNL consistently demonstrated subnormal percentages of T3+ and T4+ cells and subnormal to low normal percentages of T8+ cells. These data suggest both an intrinsic defect in B lymphocyte function, and a relative excess of monocytes which could further inhibit Ig secretion by B lymphocytes. Natural killer cell function was severely depressed.

TPA, tumor promoter-induced suppression of immunoglobulin secretion in human blood lymphocytes.

12-O-Tetradecanoylphorbol-13-acetate (TPA) modulates DNA synthesis and differentiation of normal and malignant human lymphoid cells. Using the reverse plaque forming assay and radioimmunoassay, we showed that nontoxic concentrations of TPA (5 to 10 ng/ml) inhibited Ig secretion of peripheral blood lymphocytes. This inhibition was dependent on T lymphocytes and not monocytes; TPA treatment of the B cell-enriched fraction slightly enhanced Ig secretion. Suppression was evident when the proportion of TPA-pretreated T lymphocytes exceeded 50%. TPA-induced suppressor cells were present in both OKT8+ (suppressor/cytotoxic) and OKT4+ ("helper/inducer") subpopulations. The suppression was diminished but not abolished by the irradiation of T lymphocytes. In addition, TPA treatment modulated the expression of OKT4 antigen, whereas the expression of OKT8, 9.6 (sheep erythrocyte receptors) and surface Ig remained unchanged. Modulation of OKT4 was energy dependent and was not blocked by a maximal saturation of TPA receptors at 4 degrees C. We postulate that TPA-induced suppression of Ig secretion is T cell dependent and is likely to be associated with proliferation and activation of OKT8+ and OKT4+ lymphocytes and the induction of OKT4+ suppressor cells.