Therapeutic Potential of Targeting Interleukin 5 in Asthma

It is recognised that airway inflammation is key to asthma pathogenesis. Biopharmaceutical approaches have identified new therapies that target key cells and mediators that drive the inflammatory responses in the asthmatic lung. Such an approach resulted in the development of biologics targeted at inhibition of interleukin (IL)-4, IL-5 and IL-13. However, early clinical trials with these biologics in patients with asthma were, for the most part, disappointing even though they were highly effective in animal models of asthma. It is becoming apparent that significant clinical effects with anti-cytokine-based therapies are more likely in carefully selected patient populations that take asthma phenotypes into account. The development of discriminatory biomarkers and genetic profiling may aid identification of such patients with asthma. This review summarises the current evidence, demonstrating the effectiveness or otherwise of the targeting of IL-5 in patients with asthma.     

Expression of B220 Antigen on an Interleukin 4- and Interleukin 5-Producing T-Cell Line

The murine T-cell line 2.19, originally used for cloning of interleukin 4 (IL-4) and IL-5, was analysed for surface marker characteristics using monoclonal antibodies and a FACS-4 analyser and found to be positive for the B220 antigen. Thus, this lymphokine-producing cell with characteristic T-cell markers expresses an antigen earlier thought to be specific for B cells. With two-colour fluorescence it was found that a small fraction (2-4%) of Thy 1+ spleen cells from normal CBA mice were also positive for B220.     

Interleukin 5 Regulation of Peritoneal B-Cell Proliferation and Antibody Secretion

The influence of recombinant interleukin 5 (rIL-5) on murine peritoneal B-cell proliferation and antibody secretion was examined. Larger, low buoyant density peritoneal B cells proliferated better with rIL-5 than the smaller resting B cells. this was also true for splenic B cells; however, comparison of the respective populations showed the large peritoneal B-cell responses to be superior. Limiting dilution analyses showed that from 25% to about 40% of large peritoneal B cells proliferated in response to rIL-5 when lipopolysaccharide (LPS) was present. No detectable difference in the fraction of proliferating splenic B cells was seen in the presence of rIL-5. These results are consistent with expression of IL-5 receptors on about 70% of low-density peritoneal B cells as determined by fluorescent staining with anti-Il-5 receptor monoclonal antibody (MoAb). IL-5 also enhanced spontaneous and mitogen-driven IgM secretion by both peritoneal and splenic B lymphocytes; the increases exhibited by peritoneal B cells, however, were at least twice those exhibited by splenic B cells. Spontaneous and mitogen-driven secretion of auto-antibodies to bromelain-treated mouse erythrocytes (BrMRBC) by peritoneal B cells were also increased by this interleukin. Furthermore, rIL-5 enhanced peritoneal B-cell plaque-forming cell (PFC) responses to TNP-LPS but not to TNP-Ficoll. Both an anti-IL-5R MoAb and an anti-IL-5 MoAb blocked the rIL-5-induced enhancement of proliferation and auto-antibody PFC responses. Hence, IL-5 appears to be important for the regulation of proliferation and antibody secretion by many murine peritoneal B cells.     

Interleukin 5 is a differentiation factor for cytotoxic T lymphocytes

The role of IL-5 on the generation of cytotoxic T lymphocytes (CTL) was analysed using a culture system in which production of T helper cell factors was abrogated by exposure of the stimulator cells to ultraviolet irradiation. Supernatants from a T helper cell line (2.19 sup), recombinant (r) IL-2, rIL-4 and rIL-5 were then tested for the capacity to replace T cell help, on the generation of CTL. The results showed that the specific CTL response, in unseparated spleen cells, could be reconstituted by either 2.19 sup, IL-2 or IL-4. However, if the responder cells were purified in nylon wool, only 2.19 sup or rIL-4 plus rIL-5, but not each lymphokine per se, reconstituted the CTL response. Because IL-5 does not support T cell proliferation, it is suggested that IL-5 induces differentiation of immature precursors into CTL. Based on these findings and in an attempt to conciliate the conflicting views that have emerged from different reports, as to whether IL-2 by itself could support generation of CTL in purified T cells, a hypothesis is formulated, suggesting that T cell at different stages of differentiation require distinct lymphokines to acquire CTL effector function.     

Interleukin 5 is a differentiation factor for IgA B cells

Interleukin 5 (IL5) stimulates murine B cells to IgA production in vitro. The present study examined the B cell target of IL5 activity, and asked whether IL5 acts as an isotype switch factor, a growth factor, or a differentiation factor on B cells that produce the IgA isotype. Lipopolysaccharide-activated surface IgM-positive, surface IgA-positive or surface IgA-negative B cell populations from spleen or Peyer's patches were stimulated with IL5, after which the expression of cell surface IgA, IgA secretion, and numbers of IgA-secreting cells were determined. IL5 increased the IgA response by acting on B cells that are already committed to IgA expression. Thus, with regard to the IgA response, IL5 acts as a differentiation factor, rather than a growth or IgA isotype switch factor.     

Regulation of Interleukin 2 Receptor Expression by Interleukin 2

The regulatory influence of Interleukin 2 (IL-2) on the expression of IL-2 receptors (IL-2R) was studied using long-term cultured T-cell lines and recombinant IL-2 (r-IL-2). Three T-cell lines with different growth requirements were used as model systems: insulin-specific BK-BI-1.2 cells express IL-2R transiently after antigenic restimulation, ovalbumin-reactive BK-OVA-1R cells express IL-2R permanently, and BK-BI-2.6.C6 cells bear IL-2R constitutively but do not exhibit antigen reactivity. All three T-cell lines exhibited the property of increased IL-2R expression in the presence of r-IL-2, as tested by cytofluorometry employing monoclonal antibody AMT-13 directed at the murine IL-2R. IL-2R density was influenced selectively by r-IL-2, because the level of Thy-1.2 molecules was similar in the presence and absence of r-IL-2. With BK-BI-2.6.C6 cells, r-IL-2 was shown to upregulate high-affinity receptors. Since BK-BI-2.6.C6 and BK-OVA-1R cells were grown in the absence of feeder cells, these data show that r-IL-2 can regulate the expression of its own receptor without the participation of monokines. Results obtained with the T-cell line BK-BI-1.2, representing insulin-specific T cells with transient IL-2R expression, show that the presence of r-IL-2 did not prevent a decline in IL-2R density occurring on day 5 after antigenic stimulus. This indicates that additional mechanisms besides antigen- and IL-2-induced IL-2R upregulation are operative in controlling IL-2R density on the cell surface.     

Interleukin 10 Inhibits TNF-Alpha Production in Human Monocytes Independently of Interleukin 12 and Interleukin 1 Beta

Previously we demonstrated that endogenously produced Interleukin (IL-)IO suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via downregulation of paracfine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, EL-10 inhibited monocyte TNF-alpha production by about half Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the EL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interleukin 10 Inhibits TNF-Alpha Production in Human Monocytes Independently of Interleukin 12 and Interleukin 1 Beta

Previously we demonstrated that endogenously produced Interleukin (IL-)IO suppressed the production of tumor necrosis factor-alpha (TNF-alpha) in CD3 activated T-cells via downregulation of paracfine IL-12 secretion from APC. Here we investigated the effect of endogenous IL-10 on TNF-alpha production in purified lipopolysaccharide (LPS) stimulated monocytes and its mechanism. Similarly to its effects on T-cells, EL-10 inhibited monocyte TNF-alpha production by about half Unlike in T-cells, however, this effect was not mediated via IL-12. While blockade of endogenous IL-10 binding to the IL-10 receptor enhanced the autocrine production of TNF-alpha, IL-12 and IL-1 beta, the neutralization of IL-12 or IL-1 beta did not affect the EL-10 effects on TNF-alpha production. This suggests that despite its inhibitory effects on IL-12 and IL-1 beta, which is quite similarly observed in T-cells, in purified monocytes IL-10 does not effect its TNF-alpha suppression by this mechanism. These findings indicate that IL-10 regulates production of pro-inflammatory cytokines by distinct mechanisms in different cells and tissues. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interaction Between Interleukin 10 and Interleukin 6 in Human B-Cell Differentiation

Contrary to their opposing action on human T-lymphocytes and monocytes, both Interleukin (IL-)10 and IL-6 are potent differentiation factors of human B-cells. Both are known to induce immunoglobulin (Ig) production. The precise mechanism of this converging effect of IL-6 and IL-10 remains elusive, however. Here we investigated the role of IL-6 in the IL-10 dependent B-cell differentiation into Ig secreting cells. We found that co-stimulation of SAC-stimulated human peripheral B-lymphocytes with IL-10 and IL-6 exhibited no additive effect on Ig production over stimulation with IL-10 alone, and that IL-6 receptor blockade only mildly inhibited IL-10 induced Ig synthesis. In fact, we could show that stimulation of B-cells with IL-10 somewhat suppressed SAC induced autocrine IL-6 production. Despite this suppression IL-6 levels remained sufficiently high to stimulate its receptor, and IL-6 binding to the B-cell surface was not affected. The failure of IL-6 to exert an additional effect on SAC+IL-10 induced Ig production suggests that IL-10 may recruit components of the IL-6 intracellular pathway for Ig induction. In conclusion we could demonstrate that IL-10 acts on B-cell differentiation independently of autocrine IL-6 and that it had a considerably mild effect on B lymphocytic autocrine IL-6 secretion. This still allows an IL-6 effect in the presence of IL-10 which appears adaptive with a view to the converging effects of these two cytokines on human B lymphocytes. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interaction Between Interleukin 10 and Interleukin 6 in Human B-Cell Differentiation

Contrary to their opposing action on human T-lymphocytes and monocytes, both Interleukin (IL-)10 and IL-6 are potent differentiation factors of human B-cells. Both are known to induce immunoglobulin (Ig) production. The precise mechanism of this converging effect of IL-6 and IL-10 remains elusive, however. Here we investigated the role of IL-6 in the IL-10 dependent B-cell differentiation into Ig secreting cells. We found that co-stimulation of SAC-stimulated human peripheral B-lymphocytes with IL-10 and IL-6 exhibited no additive effect on Ig production over stimulation with IL-10 alone, and that IL-6 receptor blockade only mildly inhibited IL-10 induced Ig synthesis. In fact, we could show that stimulation of B-cells with IL-10 somewhat suppressed SAC induced autocrine IL-6 production. Despite this suppression IL-6 levels remained sufficiently high to stimulate its receptor, and IL-6 binding to the B-cell surface was not affected. The failure of IL-6 to exert an additional effect on SAC+IL-10 induced Ig production suggests that IL-10 may recruit components of the IL-6 intracellular pathway for Ig induction. In conclusion we could demonstrate that IL-10 acts on B-cell differentiation independently of autocrine IL-6 and that it had a considerably mild effect on B lymphocytic autocrine IL-6 secretion. This still allows an IL-6 effect in the presence of IL-10 which appears adaptive with a view to the converging effects of these two cytokines on human B lymphocytes. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interleukin 2, 4 and 5 are sequentially produced in mitogen-stimulated murine spleen cell cultures

Lymphokine production was analyzed in murine spleen lymphocytes stimulated with different T cell mitogens. Using in situ hybridization, frequencies of cells and the kinetics of production of interleukin (IL) 2, 4 and 5 were analyzed. The different mitogens varied in their ability to induce the three interleukins. IL2 was most successfully induced with a high dose of the calcium ionophore A23187 combined with phorbol 12-myristate 13-acetate (PMA). Significant frequencies of cells containing IL4 or IL5 mRNA were found among cells stimulated with an anti-CD3 antibody together with PMA, or pokeweed mitogen. The combination of anti-CD3 and PMA induced relatively high frequencies of all three cytokines. The production was sequential with the highest levels of IL2 mRNA present during the first 24 h, IL4 mRNA reaching a peak on day 2 and finally IL5 peaking on day 3. When cells that had been stimulated with mitogens in vitro were restimulated, the lymphokines were produced more rapidly. The order of production was maintained with IL2 mRNA reaching a maximum already at 3 h of culture, IL4 mRNA at 8 h and IL5 mRNA at 24 h.     

Interleukin 4 induces CD8 alpha expression on T cell receptor V gamma 5 thymocytes

It is generally accepted that most T cell receptor (TcR) gamma/delta cells are CD4-CD8-. After in vitro culture; however, a low percentage of these cells express the CD8 alpha subunit. We show here that addition of recombinant interleukin (IL) 4 to IL 2-cultured murine TcR V gamma 5 thymocytes induces the expression of CD8 alpha; CD8 beta is not expressed. Co-addition of the anti-IL 4 mAb 11B11 abrogates the induction of CD8 alpha expression, ruling out the possibility of a contaminant. Furthermore, we demonstrate that a substantial part of freshly prepared TcR V gamma 5 thymocytes express CD8 alpha.     

Interleukin 5 regulation of peritoneal Ly-1 B lymphocyte proliferation, differentiation and autoantibody secretion

The in vitro effects of recombinant interleukin (IL) 5 on proliferation and maturation of mouse Ly-1 B cells were studied. Most freshly isolated peritoneal Ly-1 B cells expressed high levels of IL5 receptor (R). Limiting dilution analyses showed that mitogens could reveal IL5 responsiveness in more than half of low density peritoneal Ly-1 B cells. IL 5 was able not only to increase the proportion of these Ly-1 B cells induced to proliferate, but it also shifted the clone size distribution of proliferating cells towards larger clone sizes. Splenic Ly-1 B cells also proliferated in response to mitogens plus IL5. Spontaneous and polyclonal activator-induced plaque-forming cell responses of Ly-1 B cells were increased by IL5. Furthermore, IL5 increased the frequency of peritoneal Ly-1 B cells induced to secrete certain autoantibodies. IL5 was certainly the agent responsible since its effects on both proliferation and differentiation were inhibited by either anti-IL5R monoclonal antibodies or by anti-IL5 monoclonal antibodies. Hence, Ly-1 B cells, IL5 and the IL5R appear to constitute a system of cellular proliferation, differentiation and some autoantibody production. Strategies specifically targeting the interleukin and receptor elements of this system might afford external control of these cellular responses.     

Interleukin 1alpha and interleukin 6 protect human neuronal SH-SY5Y cells from oxidative damage

Interleukin (IL)-1alpha and IL-6 are powerful inflammatory cytokines produced in brain primarily by microglia and astrocytes. Here we demonstrate, using an in vitro assay system, that they can have a direct neuroprotective action against oxidative attack. Exposure of retinoic acid-differentiated human SH-SY5Y neuroblastoma cells to 270 microM hydrogen peroxide caused activation of caspase 3 and significant neuronal death. Treatment with IL-1alpha or IL-6 caused a dose-dependent increase in survival as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. An antibody against single-strand DNA demonstrated many apoptotic neuroblastoma cells following exposure to hydrogen peroxide, with a decrease following cytokine treatment. These data indicate that IL-1alpha and IL-6 can, under appropriate circumstances, protect neurons from oxidative damage in addition to their well-known action of stimulating inflammation.     

Human Peripheral Blood-Derived Dendritic Cells do not Produce Interleukin 1α, Interleukin 1β, or Interleukin 6

Little is known about the non-antigen-specific signals delivered to T cells by dendritic cells (DC). Because several monocyte-derived factors like interleukins 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6) enhance the T-cell proliferative responses, we studied the production of the above-mentioned cytokines by DC separated from human peripheral blood. The intracellular expression of the proteins (IL-1 alpha and IL-6) was studied at a single-cell level using an immunolabelling technique. The supernatants and cell lysates were studied with ELISA (IL-1 alpha and IL-1 beta). Northern blotting analysis was used to quantitate the mRNA levels. Several approaches were taken to stimulate the production of IL-1 alpha, IL-1 beta, and IL-6 by DC. These included the incubation of the DC in the presence of either LPS, rIL-1, or monoclonal anti-HLA-DR antibody, or the stimulation of cells with resting allogeneic T cells. None of the stimuli was able to induce the production of IL-1 alpha, IL-1 beta, or IL-6 by DC, whereas LPS-stimulated monocytes were strong producers of these mentioned cytokines and expressed the respective mRNA. Thus we concluded that IL-1 alpha, IL-1 beta, and IL-6 are primarily monocyte-derived factors and that these factors are not needed or produced during the activation of resting T cells by DC.