Publisher Summary This chapter focuses on structure and expression of interleukin-6 (IL-6), which is a cytokine with pleiotropic activities that plays a central role in host defense. IL-6 can exert growth-inducing, growth-inhibitory, and differentiation-inducing activities, depending on the target cells. These activities include (1) terminal differentiation (secretion of immunoglobulins) in B cells and (2) growth promotion on various B cells. IL-6 has been implicated in the pathology of many diseases including multiple myeloma, mesangial proliferative glomerulonephritis, rheumatoid arthritis, and acquired immunodeficiency syndrome (AIDS). Selective inhibition of the synthesis or of the action of IL-6 may have therapeutic benefit against the IL-6-associated diseases. On the other hand, IL-6 has potent antitumor activity against certain types of tumors. Application of IL-6 is promising in cancer treatment as well as in treatment of radiation- or chemotherapy-induced myelosuppression. The cell biology of the intracellular events that link transduction to gene regulation is an important area, and work on these topics helps to understand such phenomena as multifunction of IL-6 and bidirectional effects of cell growth depending on the cell type.

Interleukin-6 in biology and medicine

Variant immunoglobulins, particularly humanized antibody polypeptides are provided, along with methods for their preparation and use. Consensus immunoglobulin sequences and structural models are also provided.

Method for making humanized antibodies

Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-β2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human γ-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.

Production of hybridoma growth factor by human monocytes.

Vitamin C is an essential micronutrient for humans, with pleiotropic functions related to its ability to donate electrons. It is a potent antioxidant and a cofactor for a family of biosynthetic and gene regulatory enzymes. Vitamin C contributes to immune defense by supporting various cellular functions of both the innate and adaptive immune system. Vitamin C supports epithelial barrier function against pathogens and promotes the oxidant scavenging activity of the skin, thereby potentially protecting against environmental oxidative stress. Vitamin C accumulates in phagocytic cells, such as neutrophils, and can enhance chemotaxis, phagocytosis, generation of reactive oxygen species, and ultimately microbial killing. It is also needed for apoptosis and clearance of the spent neutrophils from sites of infection by macrophages, thereby decreasing necrosis/NETosis and potential tissue damage. The role of vitamin C in lymphocytes is less clear, but it has been shown to enhance differentiation and proliferation of B- and T-cells, likely due to its gene regulating effects. Vitamin C deficiency results in impaired immunity and higher susceptibility to infections. In turn, infections significantly impact on vitamin C levels due to enhanced inflammation and metabolic requirements. Furthermore, supplementation with vitamin C appears to be able to both prevent and treat respiratory and systemic infections. Prophylactic prevention of infection requires dietary vitamin C intakes that provide at least adequate, if not saturating plasma levels (i.e., 100–200 mg/day), which optimize cell and tissue levels. In contrast, treatment of established infections requires significantly higher (gram) doses of the vitamin to compensate for the increased inflammatory response and metabolic demand.

Vitamin C and Immune Function

The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8+ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8+ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8+ T cells compared with cytomegalovirus (CMV)-specific CD8+ T cells in HIV chronic infection. We show that the majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8+ T cells express significantly lower levels of perforin than CMV-specific CD8+ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8+ T cells are impaired in cytolytic activity.

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HIV-Specific Cd8+T Cells Produce Antiviral Cytokines but Are Impaired in Cytolytic Function

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen.

Human endothelial culture supernatant (HECS) had strong growth-promotion activity for hybridoma cells: the yield of hybridomas after fusion was increased at least 2-fold over that in the presence of feeder cells, such as mouse macrophages and spleen cells. Moreover, HECS could substitute for feeder cells when hybridomas were cultured at the single-cell level, and strongly enhanced the proliferation of hybrid cells. furthermore, the presence of human endothelial cells prolonged the stability of human-mouse hybridomas, producing human immunoglobulin, hybrids known to survive poorly during culture in vitro.

Human endothelial culture supernatant (HECS): a growth factor for hybridomas.

Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells.

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells.

Human peripheral blood lymphocytes were separated into subpopulations enriched or depleted with respect to B lymphocytes (Ig-bearing cells), T lymphocytes, (cells forming rosettes with sheep erythrocytes: E-RFC) and Fc receptor-bearing lymphocytes (EA-RFC).



From the distributions and recoveries of the various cell types it could be concluded that there was very little overlap between Ig-bearing lymphocytes and EA-RFC. The latter cells partly belonged to “null” (non-T, non-B) cells; it was however demonstrated that 30 % of the EA-RFC were T cells (E-RFC).



The lytic capacity in antibody-dependent lymphocytotoxicity (ADL) was shown to correspond with the proportions of EA-RFC in the various fractions. Non-T cells showed enhanced ADL activity when compared to the unseparated cells. Purified T cell populations also displayed ADL activity. Since the latter could not be due to contaminating non-T cells, this activity was ascribed to Fc receptor-bearing T lymphocytes.

Wim P. Zeijlemaker

Papers

Tissue distribution and biochemical and functional properties of Tp55 (CD27), a novel T cell differentiation antigen.

Human endothelial culture supernatant (HECS): a growth factor for hybridomas.

Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells.

Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigens. Quantitation of binding and staining of individual cells.

Separation and properties of EA‐rosette‐forming lymphocytes in humans