Human recombinant erythropoietin directly stimulates B cell immunoglobulin production and proliferation in serum-free medium.

The effect of human recombinant erythropoietin (Epo) on B cell responses was studied in a serum-free medium. Epo enhanced IgM production and thymidine uptake by a human IgM-producing lymphoblastoid cell line, CBL. This effect was specific to Epo since enhancement was blocked by anti-Epo antibody but not by control antibody. Among the various cytokines, interleukin-4 (IL-4) enhanced IgM production and thymidine uptake while IL-6 enhanced IgM production without affecting thymidine uptake. In contrast, other cytokines including IL-1 beta, IL-2, IL-5, interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), or granulocyte/macrophage colony-stimulating factor (GM-CSF) were without effect. However, the enhancing effect of Epo is different from that of IL-4 or IL-6, since Epo effect was not blocked by anti-IL-4 antibody or anti-IL-6 antibody. Moreover, specific binding of Epo was detected on CBL cells. Epo also enhanced immunoglobulin (IgG, IgM and IgA) production and thymidine uptake by purified tonsil small resting B cells stimulated by Staphylococcus aureus Cowan strain I (SAC) or by large activated B cells. In contrast, Epo had no effect on unstimulated small resting B cells. These results indicate that Epo could directly stimulate activated and differentiated B cells and could enhance B cell immunoglobulin production and proliferation.     

GM1, a ganglioside that specifically enhances immunoglobulin production and proliferation in human plasma cells

The effects of gangliosides on human plasma cell responses were studied. Among the various gangliosides tested, only GM1 enhanced immunoglobulin (Ig) production and proliferation in the human plasma cell lines, IM-9 and AF-10, while other gangliosides (GM2, GM3, GD1a, GD1b, GD3, GT1b, and GQ1b) had no effect. Among the various cytokines tested, including interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, interferon (IFN)-α and IFN-γ, only IL-6 enhanced Ig production and proliferation in IM-9 and AF-10 cells. However, the enhancement of plasma cell responses by GM1 was specific and was not mediated by IL-6, since GM1 activity was blocked by anti-GM1 monoclonal antibody (mAb), but not by control IgM, anti-IL-6 Ab or the anti-IL-6 receptor mAb, PM1. Conversely, the enhancement by IL-6 was blocked by anti-IL-6 Ab and PM1, but not by anti-GM1 mAb. GM1, but not other gangliosides, also enhanced Ig production and proliferation in freshly separated plasma cells from patients with plasma cell leukemia and in plasma cells generated in vitro. These actions of GM1 were specifically blocked by anti-GM1 mAb, but not by anti-IL-6 Ab or PM1. These results indicate that GM1 may be an important regulator of plasma cell responses.     

Nerve growth factor inhibits immunoglobulin production by but not proliferation of human plasma cell lines

The effects of nerve growth factor (NGF) on human plasma cells were studied. NGF inhibited immunoglobulin (Ig) production but not thymidine uptake by human plasma cell lines IM-9 and AF-10 in a dose-dependent fashion. This NGF-induced inhibition of Ig production was specific, since inhibition was blocked by anti-NGF serum but not by control serum. Interleukin (IL)-6 did not affect Ig production by IM-9 and AF-10; however, IL-6 restored NGF-induced inhibition of Ig production. NGF also inhibited Ig production (IgG, IgM, and IgA) without affecting thymidine uptake by PCA-1+ plasma cells generated in vitro. This inhibition was also blocked by anti-NGF serum but not by control serum and was restored by IL-6. These results suggest that NGF may interact with IL-6 in control of Ig production by plasma cells.     

Parathyroid hormone inhibits immunoglobulin production without affecting cell growth in human B cells.

The effect of parathyroid hormone (PTH) on immunoglobulin (Ig) production and proliferation in the human B-cell lines CBL, SKW, and CESS was studied. PTH inhibited Ig production from all the B-cell lines in a dose-dependent manner during 5 days of culture. As little as 0.1 ng/ml was inhibitory. PTH also inhibited Ig production from cell lines stimulated by vasoactive intestinal peptide (VIP), interleukin 2 (IL-2), and IL-6. This inhibition was not due to decreased cell growth since proliferation was not affected and cell viability was always greater than 98%. In contrast to PTH, inactivated PTH or triiodothyronine failed to affect Ig production. Inhibition by PTH was blocked by anti-PTH serum, but not by control serum. Of the various cytokines tested, IL-4 reduced the PTH-induced inhibition of Ig production, whereas other cytokines, including IL-1 beta, IL-3, IL-5, interferon alpha (IFN-alpha), IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF), failed to do so. The reducing effect of IL-4 was blocked by anti-IL-4 antibody but not by control antibody. Moreover, IFN-alpha and IFN-gamma, but not GM-CSF, overcame the reducing effect of IL-4. PTH also inhibited IgG, IgM, and IgA production by tonsillar B cells stimulated with Staphylococcus aureus Cowan strain I (SAC) and IL-6 without affecting proliferation. This inhibition was blocked by anti-IL-4 antibody but not by control antibody. These results indicate that, in addition to its regulatory effect on calcium metabolism, PTH also acts as an immunoregulatory factor, and that it interacts with the cytokine, IL-4.     

Disodium cromoglycate enhances ongoing immunoglobulin production in vitro in human B cells

The effect of disodium cromoglycate (DSCG) upon human immunoglobulin (Ig) isotypes and IgG subclasses production by purified B cells was studied. DSCG enhanced IgM, IgG1, IgG2, IgG3, IgG4 and IgA production in a dose-dependent fashion, while DSCG failed to induce IgE production at any concentrations tested by purified B cells. When B cells were separated into small resting and large activated B cells, DSCG failed to induce Ig production from small resting B cells in the presence or absence of Staphylococcus aureus Cowan strain I (SAC). In contrast, in large activated B cells DSCG significantly enhanced all types of Ig production (two-to threefold), especially IgG4 production (seven-to 11-fold), except IgE, which large B cells did not produce. The enhancement of IgG subclass production was not subclass switching, since DSCG failed to enhance IgG1 production in B cells depleted of surface IgG1+ cells (sIgG1+ cells). Similarly, DSCG did not enhance IgG2, IgG3 or IgG4 production from sIgG2-, sIgG3- or sIgG4- B cells, respectively, Interleukin-4 (IL-4) or interleukin-6 (IL-6) also enhanced Ig production except IgG4 from large activated B cells. The enhancing effect of DSCG was not mediated by IL-4 or IL-6 since anti-IL-4 or anti-IL-6 antibody failed to block the DSCG-induced enhancement. DSCG also enhanced IgG2 and IgM production from human B-cell lines GM-1500 and CBL, respectively. These results suggest that DSCG directly and preferentially stimulates activated B cells which are producing Ig and, in addition, enhances their Ig production.     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-μ antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-α (anti-TNF-α) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-α, but not other cytokines including IL-1β, IL-3, IL-5, IL-6, interferon-α (IFN-α) or IFN-γ, inhibited IL-4-mediated growth, and inhibition by TNF-α was blocked by anti-TNF-α Ab but not by control IgG. IL-4 had no effect on TNF-α binding by B cells while it decreased TNF-α production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-α by B cells, however, it enhanced TNF-α production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-α production.     

Effect of recombinant human erythropoietin on human IgE production in vitro.

Recombinant human erythropoietin enhanced spontaneous IgE production (200-300% enhancement) in cultures of peripheral blood mononuclear cells (MNC) from atopic patients. In contrast, IgG and IgA production were only slightly enhanced (30-50% enhancement), and IgM production was not affected by erythropoietin. The enhancement of IgE production by erythropoietin was indirect since it required T cells and monocytes. However, erythropoietin effect was specific since enhancement was blocked by anti-erythropoietin antibody but not by control antibody. Interleukin-4 (IL-4) also enhanced spontaneous IgE production from atopic MNC. However, the enhancing effect by erythropoietin is different from that by IL-4, since the erythropoietin effect was not blocked by anti-IL-4 antibody, and conversely IL-4 effect was not blocked by anti-erythropoietin antibody. In contrast to the enhancing effect on atopic MNC, erythropoietin failed to induce IgE production in cultures of MNC from normal donors while IL-4 induced IgE production from normal MNC. However, when normal MNC were pre-incubated with IL-4, erythropoietin enhanced IgE production from IL-4-pre-incubated MNC. Moreover, B cells separated from IL-4-pre-incubated MNC produced IgE which was enhanced by erythropoietin. However, this effect required T cells and monocytes. These results indicate that erythropoietin could regulate ongoing IgE production in vitro by T cell- and monocyte-dependent mechanisms.     

Ganglioside GQ1b enhances anti-double-stranded DNA antibody and IgG production of PBMCs from patients with systemic lupus erythematosus

BACKGROUND: Previously, we reported that ganglioside GQ1b greatly enhanced spontaneous immunoglobulin production in vitro by PBMCs from normal human subjects. OBJECTIVE: We examined in vitro effects of GQ1b on anti-double-stranded DNA (anti-dsDNA) antibody production by PBMCs from patients with systemic lupus erythematosus (SLE). METHODS: PBMCs from patients with SLE were cultured with GQ1b. IgG anti-dsDNA antibody, total IgG, and cytokine amounts in the culture supernatants and protein kinase C (PKC) activity of T cells were measured by using ELISA. RESULTS: GQ1b enhanced both anti-dsDNA and total IgG production of PBMCs from patients with SLE who were seropositive for anti-dsDNA. Among the seropositive patients, the active patients were more responsive to GQ1b in anti-dsDNA production than the inactive patients. GQ1b also enhanced total IgG production of PBMCs from patients with SLE who were seronegative for anti-dsDNA but did not induce their anti-dsDNA production. In contrast to PBMCs, GQ1b did not affect the antibody production either of purified CD5(+) or of CD5(-) B cells. Anti-IL-6 or anti-IL-10 antibody each partially blocked the GQ1b-induced enhancement of antibody production in PBMCs, and the addition of both antibodies completely blocked the enhancement. GQ1b increased IL-6 and IL-10 production of T cells. The supernatant from GQ1b-treated T cells enhanced antibody production both of CD5(+) and of CD5(-) B cells to a greater extent than that from medium-treated T cells. Exogenous IL-6 and IL-10 additively increased the antibody production both of CD5(+) and CD5(-) B cells. GQ1b-induced increases in IL-6 and IL-10 production of T cells were both blocked by PKC inhibitors, calphostin C and staurosporine. GQ1b enhanced PKC activity of T cells. CONCLUSION: These results suggest that GQ1b may polyclonally increase the production of IgG, including IgG anti-dsDNA antibody, in PBMCs from patients with SLE by promoting IL-6 and IL-10 production of T cells through the enhancement of their PKC activity.     

Vasoactive intestinal peptide stimulates immunoglobulin production and growth of human B cells.

The effect of vasoactive intestinal peptide (VIP) on human lymphoblastoid B cell lines and tonsil B cells was studied. VIP increased immunoglobulin production and proliferation by lymphoblastoid B cell line, GM-1056, in a dose-dependent manner. As little as 10(-12) M of VIP was effective, and higher concentrations of VIP induced an approximately five-fold increase in IgA production. Moreover, this enhancement was blocked by VIP antagonist. Similarly, VIP enhanced IgM and IgG production by other lymphoblastoid B cell lines, CBL and IM-9, respectively. In contrast to VIP, another neuropeptide substance P (SP) or somatostatin failed to enhance immunoglobulin production and thymidine uptake. VIP also enhanced IgA production and thymidine uptake by purified tonsil B cells. However, in contrast to B cell lines, VIP failed to enhance IgM and IgG production by tonsil B cells. SP or somatostatin failed to enhance immunoglobulin production or thymidine uptake by tonsil B cells. These results indicate that VIP acts as B cell stimulatory factor and that VIP may also have preferential effect on IgA production on tonsil B cells.     

Disodium cromoglycate inhibits immunoglobulin production in vitro without affecting cell growth in human B cell lines

The effect of disodium cromoglycate (DSCG) on human B cell lines (IM-9, GM-1056, and AF-10) was studied. DSCG inhibited immunoglobulin production by these B cell lines without affecting thymidine uptake or cell number. Thus, in addition to its antiallergic function, DSCG may also act as a B cell-modulating reagent in vitro.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

Regulation of murine in vivo IgG and IgE responses by a monoclonal anti-IL-4 receptor antibody

Although the cytokine interleukin 4 (IL-4) stimulates LPS-activated mouse B lymphocytes to secrete both IgG1 and IgE, an anti-IL-4 antibody completely inhibits IgE responses but has little or no effect on several in vivo IgG responses. IL-4 might, therefore, have a restricted role in the generation of in vivo humoral immune responses. Alternatively, IgG1 responses might be stimulated by IL-4 secreted by T cells that are interacting directly with B cells, so that anti-IL-4 antibody cannot neutralize IL-4 before it binds to a B cell IL-4 receptor. In contrast, an antibody that blocks the IL-4 receptor (IL-4R) should equally inhibit responses to IL-4 produced proximal to or distant from a B cell. This reasoning led us to determine the ability of an anti-IL-4R mAb to affect antibody production in mice injected with a goat antibody to mouse IgD (GaM delta) or inoculated with the nematode parasite Heligmosomoides polygyrus. Anti-IL-4R mAb, like anti-IL-4 mAb, blocked IgE responses by greater than 95% and enhanced IgG2a responses to a variable extent. Anti-IL-4R mAb, however, had only a modest and variable inhibitory effect on the induction of IgG1 responses, although it caused these responses to terminate more rapidly. A combination of anti-IL-4 and anti-IL-4R mAbs totally blocked goat anti-mouse IgD antibody (GaM delta)-induced IgE production but had no additive inhibitory effect on IgG1 production. These observations are most consistent with the view that IL-4 is required for a primary IgE response, but has relatively little role in the induction of IgG1 responses in the in vivo systems studied.     

Enhancement of human lymphocyte proliferative response to purified protein derivative by an anti-interleukin-2 receptor alpha chain antibody (CD25).

While it is clear that the beta subunit of interleukin-2 receptor (IL-2R) plays a pivotal role in IL-2-induced signal transduction, the function of the alpha subunit, other than modulating the association rate of IL-2, is still unknown. It has been reported that the interaction between IL-2 and the IL-2R alpha subunit of several IL-2-dependent murine T-cell lines may result in a negative regulatory signal. To confirm this finding, we investigated the effect of an anti-IL-2R alpha antibody, CD25-8D8, on the proliferative response of human peripheral blood lymphocytes. Lymphocytes from purified protein derivative (PPD)-positive donors were cultured with PPD and various concentrations of CD25-8D8 for up to 9 days, and [3H]thymidine uptake was measured. Whereas the proliferative response of human lymphocytes to PPD was suppressed by high concentrations of CD25-8D8, subinhibitory amounts of CD25-8D8 enhanced lymphocyte proliferation by 3.5-fold (range 2.2-6.2-fold) on the second day after maximal [3H]thymidine uptake had occurred. By itself, CD25-8D8 could not induce proliferation of washed 5-day PPD-activated lymphocytes during reculturing; instead, growth enhancement by CD25-8D8 was dependent on the presence of PPD-activated culture supernatant or moderate levels of exogenous IL-2. The enhancing effect of anti-IL-2R alpha antibody, observed in both murine and human systems, reinforces the possibility that binding of IL-2 to the IL-2R alpha chain plays a negative regulatory role in signal transduction.     

Immunomodulation Induced by Ascaris suum Extract in Mice: Effect of Anti-Interleukin-4 and Anti-Interleukin-10 Antibodies

Simultaneous immunization of mice with an Ascaris suum extract (Asc) and ovalbumin (OA) markedly affects the immune response to OA. The role of interleukin (IL)-4 and IL-10 induced by Asc immunization on the modulation of antigen-specific and mitogen-induced responses was investigated following single or combined cytokine-specific monoclonal antibody (MoAb) treatment of mice before immunization with OA + Asc. Immediate hypersensitivity reactions to aggregated OA and OA-specific immunoglobulin (Ig)G2a antibody production were completely restored only when both IL-4 and IL-10 were neutralized. These findings were associated with enhanced interferon (IFN)-γ secretion by OA-stimulated lymph node (LN) cells. In addition, the Asc-specific cytokine response in anti-IL-4 plus anti-IL-10 MoAb treated mice was shifted towards a Th1 phenotype, with an increase in IFN-γ and IL-2 levels and a decrease in IL-4, but not in IL-10, levels. Consequently, Asc-specific IgG2a antibody production increased, whereas IgE titres diminished in these animals. These results indicate that IL-4 and IL-10 act together in the Asc-induced mechanism of antigen-specific pansuppression. In contrast, modulation of Concanavalin A (Con A)-induced cytokine responses in Asc-immunized mice appears to be essentially mediated by an IL-4-dependent mechanism, since the neutralization of just IL-4 (and not of IL-10), either in vivo or in vitro , changed the cytokine profile from a Th2 towards a Th1 type. However, OA and Asc-specific cell responses were not modified by either anti-IL-4 or by anti-IL-4 + anti-IL-10 MoAbs in vitro treatments, suggesting that the induction of a Th2 response to Asc components concomitant to OA immunization has a strong suppressive effect on the priming stage of OA-specific Th1 type response.     

Regulation of PTX3, a key component of humoral innate immunity in human dendritic cells: stimulation by IL-10 and inhibition by IFN-gamma

The protopypic long pentraxin 3 (PTX3) is a unique, humoral pattern-recognition receptor, which plays a nonredundant function in innate resistance to pathogens. Dendritic cells (DC) of myelomonocytic origin, but not plasmacytoid DC, are a major source of PTX3 in response to Toll-like receptor (TLR) engagement. The present study was designed to explore the regulation of PTX3 production in DC. PTX3 production was induced by TLR ligands, CD40 ligand, and interleukin (IL)-1beta and was suppressed by dexamethasone, 1alpha, 25-dihydroxivitamin D3, and prostaglandin E2. It was unexpected that lipopolysaccharide (LPS)-stimulated PTX3 production was enhanced by IL-10 and inhibited by IL-4 and interferon-gamma (IFN-gamma). Enhancement of PTX3 production by IL-10 was also evident when Pam3 Cys-Ser-(Lys)4.3HCl, a TLR2-TLR1 agonist, polyionisicpolycytidylic acid, a TLR3 agonist, and IL-1beta were used as stimuli. The effect of IL-10 was blocked by an anti-IL-10 monoclonal antibody (mAb) or an anti-IL-10 receptor alpha mAb, which also reduced the LPS-induced production. Thus, production of PTX3 in DC is subjected to a distinct regulatory network, with inhibition by IFN-gamma and enhancement by IL-10. The amplification by IL-10 of production of a nonredundant component of fluid-phase innate immunity mirrors the IL-10 stimulatory function on B cells in adaptive immunity. As PTX3 is also an extracellular matrix component, IL-10-enhanced PTX3 production may play a role in orchestration of tissue remodeling in chronic inflammation.     

Involvement of interleukin (IL)-13, but not IL-4, in spontaneous IgE and IgG4 production in nephrotic syndrome

Nephrotic syndrome (NS) is a renal disease characterized by proteinuria and hypoalbuminemia. In NS patients without any allergic disease, serum IgE and IgG4 levels were selectively increased, and peripheral blood mononuclear cells (MNC) spontaneously produced IgE and IgG4. T cells produced interleukin (IL)-13 spontaneously, and B cells constitutively expressed IL-13 receptors (IL-13R). In addition, T cells stimulated surface IgE-negative (sIgE^?) and sIgG4^? B cells to produce IgE and IgG4, respectively, and IgE and IgG4 production was specifically blocked by anti-IL-13 antibody (Ab). MNC from atopic dermatitis (AD) patients also produced IgE and IgG4 spontaneously. However, in AD patients, T cells spontaneously produced IL-4, but not IL-13, and B cells constitutively expressed IL-4R, but not IL-13R. T cells stimulated sIgE^? and sIgG4^? B cells to produce IgE and IgG4, respectively, and the production was specifically blocked by anti-IL-4 Ab. On the other hand, sIgE⁺ and sIgG4⁺ B cells from both NS and AD patients spontaneously produced IgE and IgG4, respectively, and this production was not affected by T cells, anti-IL-4 Ab, or anti-IL-13 Ab. These results indicate that IL-13 is involved in the enhanced production of IgE and IgG4 in NS, while IL-4 is involved in these responses in AD.     

The effects of retinoic acid on immunoglobulin synthesis: Role of interleukin 6

Retinoic acid (RA) and its parent compound, retinol (ROH, vitamin A), have been recognized as important immunopotentiating agents. Previous studies from our laboratory have demonstrated that RA can augment formalin-treatedStaphylococcus aureus (SAC)-stimulated immunoglobulin (Ig) synthesis of cord blood mononuclear cells (CBMC). To determine the mechanism(s) by which RA modulates Ig synthesis, we studied the effects of RA on B cells and cytokine production. The addition of RA (10^–5 to 10^–10 M) to Epstein-Barr virus (EBV)-transformed B-cell clones derived from either adult or cord blood B cells augmented Ig secretion twofold. In contrast, cell proliferation was inhibited as measured by³H-thymidine incorporation. We evaluated two cytokines known to be constitutively produced by EBV cell lines, IL-1 and IL-6. While RA had no effect on IL-1 production, IL-6 synthesis was greatly enhanced (20- to 45-fold), which was also reflected by an increase in steady-state mRNA levels for IL-6 but not TNF- or TGF- on Northern blot analysis. Polyclonal rabbit anti-IL-6 antibodies were used to block the augmenting effects of RA on Ig synthesis of adenoidal B cells. RA-induced augmentation in IgG and IgA synthesis was blocked 58 and 29%, respectively, by anti-IL-6 antibodies. These studies suggest that the enhancing effects of RA on Ig synthesis are mediated, at least in part, by the autocrine or paracrine effects of IL-6 on B-cell differentiation.     

Differential effects of gangliosides on human IgE and IgG4 production

The effects of gangliosides on human IgE and IgG4 production were studied. Of the various gangliosides tested, only GM2 and GM3 inhibited the IgE and IgG4 production induced by interleukin (IL)-4 plus hydrocortisone (HC), or that induced by IL-13 plus HC, in human surface IgE- and IgG4-negative (sIgE^?, sIgG4^?) B cells without affecting the production of IgG1, IgG2, IgG3, IgM, IgA1 or IgA2. In contrast, GM1, GD1a, GD1b, GD3, GT1b and GQ1b were without effects. The GM2- and GM3-mediated inhibition was specific, since each was blocked by a corresponding antibody. Of the various factors tested, IL-6, IL-10, and tumor necrosis factor (TNF)-α enhanced the IgE and IgG4 production induced by IL-4 plus HC or by IL-13 plus HC, while IL-8 and transforming growth factor (TGF)-β inhibited these responses. However, only TNF-α counteracted the GM2- and GM3-mediated inhibition of IgE and IgG4 production, while IL-6, IL-10, anti-IL-8 monoclonal antibody and anti-TGF-β antibody failed to do so. Anti-TNF-α monoclonal antibody, but not control IgG1, not only inhibited IgE and IgG4 production in the absence of TNF-α but also blocked the counteraction of inhibition by TNF-α. In cultures containing IL-4 plus HC or IL-13 plus HC. GM2 and GM3 specifically inhibited TNF-α production without affecting TNF-α receptors, IL-6 production or IL-6 receptors. These results indicate that GM2 and GM3 inhibit IgE and IgG4 production by inhibiting endogenous TNF-α production.     

Human natural killer (NK) cells produce a late-acting B-cell differentiation activity

The supernatant of unstimulated purified NKH-1 bearing human natural killer (NK) cells was found to enhance ongoing immunoglobulin synthesis. This NK-Cell supernatant (NKSN) enhanced IgE, IgG, and IgA synthesis from corresponding B-cell lines without increasing thymidine incorporation or cell number. Separation of NKH-1+ cells into CD3- or CD3+ cells showed that this activity was produced by the CD3- population. Recombinant human interleukin (IL)-1, IL-2, IL-4, interferon (INF)-beta 1, INF-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha, or partially purified low molecular weight B-cell growth factor (BCGF) failed to provide the same enhancement of Ig synthesis. While the NKSN contained small amounts of IL-6 (0.1 U/ml) and IL-6 could increase Ig synthesisin vitro, the optimal IL-6 enhancement was far less than that observed with NKSN. NKSN also enhanced ongoing Ig synthesis fromin vivo activated B cells obtained from peripheral blood or bone marrow but failed to induce Ig synthesis from resting orin vitro activated B cells. These results demonstrate that human NK (CD3-, NKH-1+) cells can produce B-cell differentiation activity capable of regulating Ig productionin vivo, which appears to be distinct from the activity of previously described cytokines.     

Enhanced gamma interferon production through activation of Valpha14(+) natural killer T cells by alpha-galactosylceramide in interleukin-18-deficient mice with systemic cryptococcosis

We showed recently that activation of Valpha14(+) natural killer T cells (NKT cells) by alpha-galactosylceramide (alpha-GalCer) resulted in increased gamma interferon (IFN-gamma) production and host resistance to intravenous infection with Cryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to alpha-GalCer-induced IFN-gamma synthesis. Here we examined the role of IL-18 in alpha-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-gamma in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-gamma correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-gamma synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-gamma in WT mice. Finally, the enhanced production of IFN-gamma in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in alpha-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-gamma synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection with C. neoformans and a ligand-specific activation of Valpha14(+) NKT cells.