Enhanced Ig Production Research Articles

Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

The nervous system as ectopic germinal center: CXCL13 and IgG in lyme neuroborreliosis

Lyme neuroborreliosis (LNB) is a chronic infection in which B-cell activation, plasma cell infiltration, and enhanced Ig production in infected tissue are prominent feature. However, little is known about how B cells and plasma cells invade and persist in target organs. To assess this issue, we developed real-time PCR measurements of IgG and CXCL13 production. We used these RNA assays and specific enzyme-linked immunosorbent assays for protein and demonstrated that human peripheral blood mononuclear cells (PBMCs), stimulated by Borrelia burgdorferi sonicate, produced CXCL13 and IgG. Magnetic separation of PBMC populations and flow cytometry showed that CXCL13 is produced by dendritic cells. We then measure the expression of CXCL13 and IgG in tissues and correlated the expression of these host genes with spirochetal load. We also measured expression of dbpA and BBK32, two spirochetal genes important in chronic infection. There was a strong correlation between host immune response gene expression (CXCL13 and IgG) and spirochetal load. Immunohistochemistry of infected nonhuman primates tissue confirmed that CXCL13 is expressed in the nervous system. We conclude that persistent production of CXCL13 and IgG within infected tissue, two characteristics of ectopic germinal centers, are definitive features of LNB.

Bi-directional modulation of T cell-dependent antibody production by prostaglandin E(2).

T cell-dependent Ig production involves interaction between T cells and B cells. This study evaluated the effects of prostaglandin (PG) E(2) on Ig production in a system in which B cells were co-cultured with autologous CD4(+) T cell clones non-specifically activated by anti-CD3. The effects of PGE(2) on T cell-dependent Ig production differed substantially, depending on the T cells employed. We selected six T cell clones that were able to enhance Ig production (resistant T cell clones) and six T cell clones that inhibited Ig production in the presence of PGE(2) (sensitive T cell clones) for comparison. The resistant T cells produced high levels (>1000 pg/ml) of IL-2 and/or IL-4, and expressed high CD40L, OX40 and CD45RA, and low CD45RO. In contrast, sensitive T cells secreted low IL-2 (<500 pg/ml) and IL-4 (<200 pg/ml), and expressed low CD40, OX40 and CD45RA, and high CD45RO. Adding supernatant derived from resistant T cell clones restored Ig production inhibited by PGE(2), while removing IL-2, IL-4 or IL-10 using specific antibodies inhibited Ig production. In addition, we demonstrated a direct effect of PGE(2) on B cells to enhance Ig production. Consistently, in the presence of resistant T cells, PGE(2) increased B cell proliferation and differentiation. In conclusion, the effects of PGE(2) on Ig production consist of its indirect effects through T cells and its direct effects on B cells. The outcome of the effects can be up-regulatory or down-regulatory, depending whether resistant or sensitive T cells are involved.

In Vitro Exposure of Human Lymphocytes to Trichothecenes: Individual Variation in Sensitivity and Effects of Combined Exposure on Lymphocyte Function

The trichothecenes are mycotoxins produced by fungi of the genus Fusarium, which are commonly present in foods and feed of cereal origin. Owing to the lack of sufficient toxicological data for most of the trichothecenes, in vitro studies may contribute to risk assessments of these toxins. In the present report, human lymphocyte cultures were used to study the individual variation in sensitivity among humans and the effects on in vitro Ig production. Furthermore, proliferative responses of cells exposed to combinations of two of the toxins were studied. Four toxins, T-2 toxin, diacetoxyscirpenol (DAS), nivalenol (NIV) and deoxynivalenol (DON) were included in the study. All four of the tested trichothecenes effectively inhibited mitogen-induced lymphocyte proliferation. There were no statistically significant differences in sensitivity to the toxins between lymphocytes from female and male blood donors. The individual variation in sensitivity, evaluated as the range of IC 50 values, was rather limited (within a factor of 3 to 4). Immunoglobulin production by pokeweed-stimulated human lymphocytes was also effectively inhibited with IC 50 values similar to the IC 50 values in the proliferation tests for DON and NIV. However, IC 50 values for Ig synthesis in cultures exposed to T2 were approximately two to three times higher than the corresponding IC 50 values found in the proliferation tests. At low levels of exposure, elevated Ig production was observed in lymphocyte cultures from four out of the five blood donors tested. This effect was most pronounced on IgA synthesis. Combinations of NIV with T2, DAS or DON resulted in additive toxicity in the lymphocyte proliferation test, while combinations of DON with T2 or DAS resulted in an inhibition that was slightly lower than what could have been expected from the inhibition produced by the individual toxins. In conclusion, the tested trichothecenes inhibited both proliferation and Ig production in human lymphocytes in a dose-dependent manner with limited variation in sensitivity between individuals. Enhanced Ig production was observed in cell cultures exposed to the lower doses of the toxins. Combined exposure to two of the toxins resulted mainly in additive or antagonistic effects, although synergistic effects cannot be excluded and should be further investigated. These findings indicate that the total intake of type A and B trichothecenes should be taken into account in risk assessments.

Ganglioside GQ1b enhances Ig production by human PBMCs

Background: Gangliosides are glycosphingolipids that contain sialic acid and have various immunomodulatory effects. Objective: We studied in vitro effects of gangliosides on human humoral immune responses. Methods: PBMCs from healthy volunteers were cultured with gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, and GQ1b. The amounts of IgG, IgM, and IgA and cytokine activity in the culture supernatants were measured with use of an ELISA. Proliferation was determined by [ 3 H] thymidine uptake. Results: Among the various gangliosides tested, GQ1b most strongly enhanced spontaneous IgG, IgM, and IgA production by human PBMCs. The effect of GQ1b was revealed at 0.1 μmol/L, increased dose dependently, and was maximized at 10 μmol/L. Weaker but significant stimulatory effects on the Ig production were manifested by GM2 and GD1a, whereas GT1b and GD1b were inhibitory. None of the gangliosides examined affected the proliferation of PBMCs. GQ1b did not enhance Ig production of B cells alone. Anti-IL-6 and anti-IL-10 antibody each partially blocked GQ1b-induced enhancement of the Ig production by PBMCs, and the addition of both antibodies completely blocked the enhancement. GQ1b enhanced both IL-6 and IL-10 production of T cells without affecting those of monocytes or B cells. GQ1b enhanced Ig production of T and B cell culture without monocytes. When T cells were preincubated with GQ1b, washed and added to B cells and monocytes, and then cultured together, the Ig production was also enhanced, although to a lesser extent than the whole time incubation. Exogenous IL-6 and IL-10 each enhanced Ig production of B cells alone, and the addition of both gave additive effects. Conclusion: These results suggest that GQ1b may indirectly enhance Ig production of B cells by promoting IL-6 and IL-10 production of T cells in whole PBMC culture. That GQ1b may act as an important immunostimulator is also indicated. (J Allergy Clin Immunol 1998;102:813-820.)

CD154/CD40 and CD70/CD27 interactions have different and sequential functions in T cell-dependent B cell responses: enhancement of plasma cell differentiation by CD27 signaling.

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.

B cell stimulating activity of seaweed extracts

The activity of seaweed extracts on murine and human lymphocytes was studied in vitro. The extracts of some kinds of seaweed, such as Hizikia fusiformis and Meristotheca papulosa, stimulated normal mouse spleen cells to proliferate. The responder cells are B cells, because the response was depleted by the treatment of spleen cells with anti-immunoglobulin (Ig) antibody and complement and being passed through a nylon wool column. This response is not due to lipopolysaccharide (LPS) contamination, because seaweed extracts can stimulate spleen cells of C3H/HeJ mice which are LPS low responders. Seaweed extracts also enhanced Ig production by B cells and tumor necrosis factor (TNF) production by macrophages. Furthermore, seaweed extracts stimulated human lymphocytes to proliferate. All these B cell stimulating activities of seaweed extracts associated with glycoproteins whose molecular weights resided in 100 kD. These results suggest that seaweed extracts have stimulating activity on B cells and macrophages and this ability could be used clinically for the modulation of immune responses.

Vasoactive intestinal peptide enhances immunoglobulin production and growth in human plasma cells via mechanisms that may involve protein kinase C.

The effects of various neuropeptides on human plasma cells were studied. Of the various neuropeptides tested, vasoactive intestinal peptide (VIP) enhanced Ig production and growth in human plasma cell lines, IM-9 and AF-10, and in plasma cells generated in vivo (four out of four patients with plasma cell leukemia) and in vitro. In contrast, other neuropeptides (neuropeptide Y, somatostatin, substance P, peptide YY, neurokinin A, calcitonin gene-related peptide, chole-cystokinin octapeptide, and beta-endorphin) were ineffective. Moreover, VIP-induced enhancement was specifically blocked by VIP receptor antagonist. Among the various cytokines, IL-6, GH, and insulin-like growth factor I (IGF-I) also enhanced Ig production and thymidine uptake in plasma cells. However, VIP-induced enhancement was not mediated by IL-6, GH, or IGF-I because antibodies to these cytokines failed to block VIP-induced enhancement. Phorbol 12,13 dibutyrate enhanced Ig production and thymidine uptake in plasma cells, and the Phorbol 12,13 dibutyrate-induced enhancement was blocked by H7 (a protein kinase C inhibitor) but not by H8 (a protein kinase A inhibitor). Similarly, VIP-induced enhancement was blocked by H7 but not by H8. Collectively, VIP enhances plasma cell responses via mechanisms that may involve protein kinase C.

Vasoactive intestinal peptide differentially modulates human immunoglobulin production

The effects of vasoactive intestinal peptide (VIP) on human immunoglobulin (Ig) production were studied in (1) B cell lines; (2) anti-CD40 mAb-stimulated B cells from non-atopic donors; and (3) unstimulated mononuclear cells from atopic patients. In B cell lines, GM-1056, IM-9, and CBL, VIP enhanced IgA1, IgG1 and IgM production, respectively, in a dose-dependent fashion, while the other neuropeptides somatostatin (SOM) or substance P (SP) failed to do so. Among the various cytokines examined including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, and G-CSF, IL-6 and IL-10 also enhanced Ig production. However, VIP-induced enhancement of Ig production was specific, and was not mediated via these cytokines, since enhancement was blocked by the VIP antagonist, while SOM and SP antagonists, anti-IL-6 mAb, or anti-IL-10 Ab failed to do so. In anti-CD40 mAb-stimulated B cells from non-atopic donors, VIP selectively induced IgA1 and IgA2 production without affecting IgG1, IgG2, IgG3, IgG4, IgM, or IgE production. This stimulatory effect was specifically blocked by the VIP antagonist, but not by SOM or SP antagonists, anti-IL-5 mAb, anti-IL-10 Ab, or anti-TGF-β Ab. VIP induced IgA1 and IgA2 production by surface IgA1 − (sIgA1−) and sIgA2 − B cells, respectively, while this agent had no effect on sIgA1 + and sIgA2 + B cells. In contrast, in unstimulated mononuclear cells from atopic patients, VIP selectively inhibited spontaneous IgE and IgG4 production without affecting IgG1, IgG2, IgG3, IgM, IgA1, or IgA2 production. This inhibitory effect was specifically blocked by the VIP antagonist, but not by anti-IFN-α Ab, anti-IFN-γ mAb, anti-IL-12 Ab, or anti-TGF-β Ab. VIP did not inhibit IgE or IgG4 production in B cells or in B cells cultured with either T cells or monocytes. However, VIP inhibited IgE and IgG4 production when B cells were cultured with both T cells and monocytes.

Expression of CD45RB and CD27 identifies subsets of CD4+ memory T cells with different capacities to induce B cell differentiation.

The capacity of four subsets of CD4+ memory T cells, defined by expression of CD45RB and CD27, to provide help for B cells was examined. Larger amounts of Ig were induced by CD45RBdimCD27- cells compared with the CD45RBdimCD27+ population, whereas CD45RBbrightCD27+ or CD27- cells were poor inducers of Ig synthesis. Mitomycin C treatment, which prevents suppressive activity, markedly enhanced Ig production supported by each subset except for CD45RBbrightCD27- cells. Mitomycin C-treated CD45RBdim cells remained the most efficient inducers of Ig production, but no difference was detected between CD27+ and CD27- cells. The subsets also differed in their ability to proliferate and secrete cytokines, but these differences did not explain variations in the capacity to provide help for B cells. Both CD27- subsets exhibited decreased proliferation and uniquely secreted IL-4, with the CD45RBdimCD27- subset producing the greatest quantities of IL-4. No differences in IL-2 and IFN-gamma production were found. IL-10 secretion increased with the acquisition of the CD45RBdim phenotype and, within the CD45RBdim cells, with the loss of CD27. Staining for cytoplasmic cytokines indicated that individual populations of CD27-CD4+ helper T cells produced either IL-4 or IFN-gamma, whereas more than half of the IL-4 producers also synthesized IL-2. Finally, the different abilities of CD4+ memory T cell subsets to support B cell differentiation did not relate to variations in the expression of CD40 ligand. These results indicate that within the CD4+ memory T cell population an increase of helper activity associates with the shift from a CD45RBbright to a CD45RBdim phenotype. Within the CD45RBdim subset, the loss of CD27 is associated with a reduction of suppressive activity.

Antigen-specific activation of B cells in vitro after recruitment of T cell help with superantigen.

Human B cells can proliferate in vitro after stimulation with anti-Ig and via the CD40 molecule. Superantigens like SEA which bind to MHC class II antigens on, e.g. B cells can polyclonally activate T cells via interaction with their TcR. The activated T cell subsequently activates the B cells to proliferation and Ig-production. To investigate whether superantigen could be used to direct polyclonal T cell help to human B cells stimulated by antigen in a restricted manner resulting in production of antigen-specific antibodies in vitro. Purified B cells were preincubated with the antigen in manners allowing crosslinking of surface-Ig. The antigen exposed B cells were then cultured together with autologous CD4+ helper T cells and in the presence of various concentrations of SEA. Antibody production was measured by ELISA after 7-12 days of culture. Antigen-specific activation of B cells could be obtained after stimulating the B cells with antigen or anti-surface-Ig antibodies in the presence of T helper cells and SEA. The degree of B cell activation (proliferation as well as antibody production) depended on the dose of antigen as well as on the dose of SEA used. Increased crosslinking of surface-Ig on antigen-specific B cells enhanced Ig production. Specific antibody production to a secondary recall antigen (tetanus toxoid) and to primary antigens (DNP and GM2) were obtained. The specific B cell response was dependent on contact between T and B cells. the results obtained demonstrate that the superantigen SEA can recruit T cell help to human B cells specifically stimulated by antigens, resulting in production of antigen reactive antibodies in vitro.

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)-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, interferon (IFN)-alpha and IFN-gamma, 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.

The Interplay of Interleukin-10 (IL-10) and Interleukin-2 (IL-2) in Humoral Immune Responses: IL-10 Synergizes with IL-2 to Enhance Responses of Human B Lymphocytes in a Mechanism Which Is Different from Upregulation of CD25 Expression

The role of the interplay of interleukin-2 (IL-2) and interleukin-10 (IL-10) in responses of human peripheral blood B cells stimulated by ligation of antigen receptors was examined in detail. Highly purified peripheral blood B cells from normal human individuals were cultured with Staphylococcus aureus Cowan I (SA) in the presence or absence of IL-10 and IL-2. Although IL-10 alone could modestly induce Ig production by SA-activated B cells, IL-10 markedly enhanced the Ig production in synergism with IL-2. This synergistic effect between IL-2 and IL-10 was completely abrogated by addition of either anti-CD25 mAb or anti-IL-10 mAb, indicating signals through high-affinity IL-2 receptors are involved in the synergism. In fact, IL-10 upregulated the expression of α-chain of IL-2 receptors (CD25) on B cells stimulated by SA alone or SA + IL-2 for 72 hr. However, such upregulation of CD25 expression did not result in significant enhancement of subsequent responses of the activated B cells to IL-2. Of note, during the initial activation phase of B cells, IL-2 was much more effective than IL-10 in enhancing the subsequent responses to either IL-2 or IL-10. By contrast, IL-10 was much more effective than IL-2 in supporting the maturation of B cells following the initial activation. Finally, the synergy between IL-2 and IL-10 was not observed in the initial activation phase, but in the subsequent maturation phase of activated B cells. These results indicate that IL-10 and IL-2 synergistically enhance Ig production of SA-activated B cells in a mechanism which is different from the upregulation of IL-2 receptors. Moreover, the data emphasize the importance of the interplay of IL-2 and IL-10 in determining the outcome of humoral immune responses.

Differential Effect of Growth Hormone and Insulin-Like Growth Factor-I, Insulin-Like Growth Factor-II, and Insulin on Ig Production and Growth in Human Plasma Cells

AbstractThe effect of human growth hormone (GH) and insulin-like growth factor-l (IGF-I), IGF-II, and insulin on human plasma cell responses was studied. GH enhanced Ig production and thymidine uptake in the human plasma cell lines, IM-9 and AF-10. IGF-I, but not IGF-II or insulin, also enhanced Ig production and proliferation in them. However, enhancement by GH was not mediated by IGF-I, because enhancement was blocked by anti-GH antibody (Ab), but not by Ab to IGF-I or IGF-I receptor. Conversely, the enhancement by IGF-I was blocked by either Ab to IGF-I or IGF-I receptor but not by anti-GH Ab. GH and IGF-I also enhanced production of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and thymidine uptake in PCA-1+ plasma cells generated in vitro. Again, enhancement by GH was specifically blocked by anti-GH Ab, whereas enhancement by IGF-I was specifically blocked by either Ab to IGF-I or IGF-I receptor. These results indicate that GH and IGF-I may play important roles in plasma cell responses.

Differential effect of growth hormone and insulin-like growth factor-I, insulin-like growth factor-II, and insulin on Ig production and growth in human plasma cells

The effect of human growth hormone (GH) and insulin-like growth factor- I (IGF-I), IGF-II, and insulin on human plasma cell responses was studied. GH enhanced Ig production and thymidine uptake in the human plasma cell lines, IM-9 and AF-10. IGF-I, but not IGF-II or insulin, also enhanced Ig production and proliferation in them. However, enhancement by GH was not mediated by IGF-I, because enhancement was blocked by anti-GH antibody (Ab), but not by Ab to IGF-I or IGF-I receptor. Conversely, the enhancement by IGF-I was blocked by either Ab to IGF-I or IGF-I receptor, but not by anti-GH Ab. GH and IGF-I also enhanced production of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and thymidine uptake in PCA-1+ plasma cells generated in vitro. Again, enhancement by GH was specifically blocked by anti-GH Ab, whereas enhancement by IGF-I was specifically blocked by either Ab to IGF-I or IGF-I receptor. These results indicate that GH and IGF-I may play important roles in plasma cell responses.

Isotype-restricted hyperimmunity in a murine model of the toxic oil syndrome.

The toxic oil syndrome is characterized by IgE elevation and eosinophilia, as well as scleroderma-like skin manifestations and other symptoms of autoimmune disease. Fatty acid anilides, found in large amounts in adulterated cooking oil, were suspected to be the etiologic agent in this disease. The capacity of oleic acid anilide to induce features of autoimmunity in vivo was investigated. B10.S mice were continuously treated i.p. with oleic acid anilide for 6 wk by using osmotic pumps. A significant increase in IgE and IgM serum levels was observed after 1 to 3 wk; subsequently five of six mice developed IgG1 levels 3.5- to 10-fold higher than the controls. Anilide-treated mice developed splenomegaly with a 2.1- and a 3.5-fold increase in IgM- and IgG-bearing splenocytes, respectively, and a 5.6- and 29-fold elevation in functional IgM- and IgG-secreting cells, respectively. Increased serum levels of predominantly IgM antibodies to histone, denatured DNA, and DNP as well as rheumatoid factor were detected. In vivo expression in the spleen of 10 cytokine genes was also examined, and mRNA encoding IL-1 beta and IL-6 were significantly elevated in splenocytes of anilide-treated mice. The enhanced Ig production suggests that anilide induced a cytokine-mediated polyclonal activation of B cells. Elicitation of IgM antibodies to denatured forms of autoantigens indicates that anilide treatment partially broke autoimmune tolerance in these mice. Anilide-treated mice may be a useful animal model for further exploring the mechanism and pathogenesis of systemic autoimmunity in the toxic oil syndrome.

Polyclonal B cell activation by the Eta-1 cytokine and the development of systemic autoimmune disease.

Studies of systemic autoimmune disease have led to the view that initiation and progression of the disease process reflects chronic and sustained B cell activation by unidentified polyclonal activating agents. In earlier studies, we found that T cells from MRL/1 mice, which develop murine lupus, express very high levels of a newly defined T cell cytokine, Eta-1. Inasmuch as chronic and sustained B cell stimulation by T cells is a cardinal feature of MRL/1 disease, we determined the effects of this cytokine on Ig production by B cells. We show that both recombinant and biochemically purified natural Eta-1 stimulate IgM and IgG production by mixtures of B cells and macrophages from the autoimmune MRL/l strain. Additional studies suggest that optimal Ig production by Eta-1 may require macrophages and reflect enhanced Ig production by large B cells. These findings support the view that elevated levels of endogenous Eta-1 may cause chronic and sustained polyclonal B cell activation that leads to autoimmune disease in this murine model.

Lead and cadmium at very low doses affect in vitro immune response of human lymphocytes

The effect of lead chloride and cadmium chloride on in vitro immunoglobulin (Ig) production by human lymphocytes was investigated. After 7 days in culture, lead added in the range of human exposure (207-1035 micrograms/liter) significantly enhanced Ig production either when cells were activated by pokeweed mitogen (PWM) or not. The effect was dose-dependent and was related to the Pb we measured in the extracellular medium and in the cells. Independently of the mitogen addition, about 2% of the Pb added was accumulated in the cells, most being associated with the nuclear fraction. Those findings suggest that the Pb effects could depend on its uptake and distribution in the cells. Cadmium added in the 50-500 nM range exhibited a dose-independent mitogenic activity in unstimulated cells, whereas the Ig secretion was not significantly affected by Cd when cells were PWM-activated. A considerable intraindividual variability, however, was observed when blood donors were separately examined, with both an increase, a decrease, or no variation on Ig production. Furthermore, higher percentages of Cd were accumulated in the nuclear fraction, and lower in the cytosol and precipitate, in PWM-activated compared to resting lymphocytes. Genetic factors could be of importance for the observed variability of the immune response to cadmium, and we support the hypothesis that differences in the metallothionein (MT) inducibility could play a role.

Erythropoietin enhances immunoglobulin production and proliferation by human plasma cells in a serum-free medium

The effect of recombinant human erythropoietin (Epo) on plasma cells was studied in a serum-free medium, COSMEDIUM-001 (Cosmedium). Epo enhanced both Ig production and thymidine uptake by human plasma cell lines, AF-10 and IM-9. Interleukin-6 (IL-6) enhanced both Ig production and thymidine uptake by AF-10 and IM-9, while other cytokines, including IL-1β, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-α (IFN-α), or IFN-γ, failed to do so. However, the Epo effect was specific since Epo-induced enhancement of Ig production and thymidine uptake was blocked by the anti-Epo antibody but not by the anti-IL-6 antibody or the control antibody. Conversely, IL-6-induced enhancement was blocked by the anti-IL-6 antibody but not by the anti-Epo antibody. Epo also enhanced Ig production (IgG, IgM, and IgA) and thymidine uptake by PCA-1 + plasma cells generated in vitro. This enhancement was also blocked by the anti-Epo antibody but not by the anti-IL-6 antibody. Taken together, these results suggest that Epo enhances plasma cell responses by a different mechanism than does IL-6.

Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines.

Platelet-activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date, little is known of the effect of PAF on B lymphocytes. Using two Ig-secreting B lymphoblastoid cell lines, we have demonstrated that PAF can enhance Ig production by these cells in a dose-dependent fashion. PAF also causes significant alteration of the kinetics of Ig secretion in these lymphoblastoid cell lines. The effect of PAF is rapid, with detection of 6- to 12-fold increases in Ig production in the first 24 h of cell culture, followed by a plateau during the next 24 to 48 h. The specificity of the PAF effect on Ig secretion is emphasized by lyso-PAF having no Ig-enhancing properties and by the inhibition of Ig enhancement in the presence of the structural analogue PAF antagonist CV3988 and the soluble nonstructural analogue PAF receptor antagonist Web 2086. PAF does not cause an increase in the kinetics of cell proliferation or an increase in cell numbers at any time during a 72-h culture period. In an attempt to explain the increase in Ig secretion in the absence of changes in growth parameters, an ELISA spot assay for enumeration of Ig-secreting cells was developed. This assay demonstrated that the increase in Ig production is likely due to enhancement of single cell Ig secretion rather than an increase in cell number. These data indicate that PAF may have an important immunomodulatory role in the production of Ig by B lymphocytes.