B Bennett

In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.

Recently, we described the cloning and expression of a human cDNA which is the homologue to P600, a gene transcribed by mouse Th2 clones. Based on its activities on human monocytes and B cells this gene was designated IL-13. In the present study we investigated the effects of IL-13 alone or in combination with IL-4, IFN-gamma, or IL-10 on human monocytes. IL-13 induced significant changes in the phenotype of monocytes. Like IL-4, it enhanced the expression of CD11b, CD11c, CD18, CD29, CD49e (VLA-5), class II MHC, CD13, and CD23, whereas it decreased the expression of CD64, CD32, CD16, and CD14 in a dose-dependent manner. IL-13 induced up-regulation of class II MHC Ag and its down-regulatory effects on CD64, CD32, and CD16 expression were prevented by IL-10. IFN-gamma could also partially prevent the IL-13-induced down-regulation of CD64, but not that of CD32 and CD16. However, IL-13 strongly inhibited spontaneous and IL-10- or IFN-gamma-induced ADCC activity of human monocytes toward anti-D coated Rh+ erythrocytes, indicating that the cytotoxic activity of monocytes was inhibited. Furthermore, IL-13 inhibited production of IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, macrophage inflammatory protein-1 alpha, granulocyte/macrophage-CSF, granulocyte-CSF, IFN-alpha, and TNF alpha by monocytes activated with LPS. In contrast, IL-13 enhanced the production of IL-1 ra by these cells. Similar results on cytokine production were observed or have been obtained with IL-4. Thus IL-13 shares most of its activities on human monocytes with IL-4, but no additive or synergistic effects of IL-4 and IL-13 on human monocytes were observed, suggesting that these cytokines may share common receptor components. Taken together, these results indicate that IL-13 has anti-inflammatory and important immunoregulatory activities.

To gain insights into a possible immune defect predisposing to disseminated mycobacteria infection, we studied three of six surviving children with disseminated Mycobacterium avium complex infection, who had no recognized form of immunodeficiency. We used mycobacteria isolated from the patients and diphtheria, tetanus and pertussis vaccine (DTP) to study antigen-specific T lymphocyte responses. We observed that interferon-gamma (IFN-gamma) production by T cells in response to antigens (both mycobacteria and DTP) in these patients with disseminated infection was greatly impaired. This defect did not seem to be the result of T cell unresponsiveness, as phytohaemagglutinin (PHA) stimulation was able to induce high levels of IFN-gamma comparable to those seen in control patients with localized infection. Further experiments showed that peripheral blood mononuclear cells (PBMC) from patients with disseminated infection were able to present influenza haemagglutinin (HA) peptides to specific T cell clones. However, this ability was lost when the whole HA protein was used as source of antigen. Taken together, these observations support the notion that the primary immune defect in these patients with disseminated mycobacterial infection rests in the antigen-processing functions of their antigen-presenting cells (APC). These findings may provide clues to the wider problem of susceptibility to mycobacteria and other intracellular pathogens and have implications in designing therapy for these patients.

Abstract Activation and proliferation of antigen specific T cells depends on the interaction of multiple signaling molecules with the TCR/CD3 complex. We report that expression of connexin 43 (Cx43), a gap junction protein, is critical for the expansion of antigen-specific or self-reactive effector CD4+ T cells. While gap junction mediated intercellular communication has been the main focus of connexin research, the cytoplasmic C-terminus of Cx43 has multiple functions independent of membrane channel forming properties and may act as a docking molecule for assembling signaling complexes including the immunological synapse. We also report that antigen-specific Cx43-deficient CD4+ T cells transferred into recipient mice immunized with cognate antigenic peptide and adjuvant expanded poorly compared to wild type cells. This protected mice from experimental autoimmune encephalomyelitis (EAE). Decreased proliferation and expansion also protects lymphopenic mice repopulated with naive, Cx43 deficient CD4+ T cells, from inflammatory bowel disease. We have found that Cx43-deficient CD4+ T cells have a signaling defect affecting Erk, p38 and Akt signaling pathways. Transcriptome RNA-seq analysis comparing gene expression profiles of activated Cx43 sufficient and deficient CD4+ T cells shows that differentially expressed genes are enriched in genes involved in G protein and Ca2+ signaling, ATP metabolism, genes regulating cytoskeleton reorganization and T cell homing. In summary, our data demonstrate that Cx43 has a major impact on the generation of effector CD4+ T cells, their ability to respond to antigenic stimulation, or to precipitate autoimmune disease.