Surendran Mahalingam

Intrinsic Defect in T Cell Production of Interleukin (IL)-13 in the Absence of Both IL-5 and Eotaxin Precludes the Development of Eosinophilia and Airways Hyperreactivity in Experimental Asthma

Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.

Cytokines and immunity to viral infections

In this review, we discuss two broad approaches we have taken to study the role of cytokines and chemokines in antiviral immunity. Firstly, recombinant vaccinia viruses were engineered to exit encoding cytokines and chemokines of interest. Potent antiviral activity was mediated by many of these encoded factors, including IL‐2, IL12. IFN‐γ. TNF‐α. CD40L. Mig and Crg‐2, In some cases, liosi defense mechanisms were induced (IL‐2, IL‐t2. Mig and Crg‐2), whilst for others, a direct antiviral effect was demonstrated (IFN‐γ. TNF‐α and CD40L), In sharp contrast, vector‐directed expression of IL 4, a type 2 factor, greatly increased virus virulence, due 10 a downregulation of host type 1 immune responses. Our second experimental approach involved the use of strains of mice deficient for the production of particular cytokines or their receptors, often in combination with our engineered viruses. Mice deficient in either IFN‐γ, IFN‐γR, IFN‐α/βR, TNFFRs, CD40 or IL‐6 were, in general, highly susceptible to poxvirus infection. Surprisingly, not only the TNFR1, but also the TNFR2, was able to mediate the antiviral effects of TNF‐α in viv, whilst the antiviral activity observed following CD40‐CD40L interaction is a newly defined function which may involve apoptosis of infected cells. Through the use of perforin‐deficient mice, we were able to demonstrate a requirement for this molecule in the clearance of some viruses. such as ectromelia virus, whilst for others, such as vaccinia virus, perforin was less important but IFN‐γ was essential.

Elemental signals regulating eosinophil accumulation in the lung

Summary: In this review we identify the elemental signals that regulate eosinophil accumulation in the allergic lung. We show that there are two interwoven mechanisms for the accumulation of eosinophils in pulmonary tissues and that these mechanisms are linked to the development of airways hyperreactivity (AHR). Interleukin‐(IL)‐5 plays a critical role in the expansion of eosinophil pools in both the bone marrow and blood in response to allergen provocation of the airways. Secondly, IL‐4 and IL‐13 operate within the allergic lung to control the transmigration of eosinophils across the vascular bed into pulmonary tissues. This process exclusively promotes tissue accumulation of eosinophils. IL‐13 and IL‐4 probably act by activating eosinophil‐specific adhesion pathways and by regulating the production of IL‐5 and eotaxin in the lung compartment. IL‐5 and eotaxin co‐operate locally in pulmonary tissues to selectively and synergistically promote eosinophilia. Thus, IL‐5 acts systemically to induce eosinophilia and within tissues to promote local chemotactic signals. Regulation of IL‐5 and eotaxin levels within the lung by IL‐4 and IL‐13 allows Th2 cells to elegantly co‐ordinate tissue and peripheral eosinophilia. Whilst the inhibition of either the IL‐4/IL‐13 or IL‐5/ eotaxin pathways resulted in the abolition of tissue eosinophils and AHR, only depletion of IL‐5 and eotaxin concurrently results in marked attenuation of pulmonary inflammation. These data highlight the importance of targeting both IL‐5 and CCR3 signalling systems for the resolution of inflammation and AHR associated with asthma.

Chemokines and chemokine receptors in infectious diseases

Today, 10 years after the discovery of IL‐8, chemokines (chemotactic cytokines) are seen as the stimuli that largely control leucocyte migration. Chemokines are low molecular weight chemoattractant cytokines secreted by a variety of cells, including leucocytes, epithelial cells, endothelial cells, fibroblasts and numerous other cell types. They are produced in response to exogenous stimuli, such as viruses and bacterial LPS, and endogenous stimuli, such as IL‐1, TNF and IFN. These factors mediate chemotaxis and leucocyte activation. They also regulate leucocyte extravasation from the blood and/or lymph vessel luminal surface to the tissue space, the site of inflammation. There is no doubt that chemokines and chemokine receptors are critical for defence against infectious pathogens. It is also clear that these pathogens have evolved to accommodate the workings of the host immune system. Survival of these infectious agents appears dependent upon strategies that can evade, suppress, counteract or otherwise confound the constellation of host responses to invading pathogens. In this regard, the chemokines and their receptors are a major target. Reviewed in the present paper are several examples in which microbial pathogens have usurped the mammalian chemokine system to subvert the host immune response.

Expression of CTLA-4 by human monocytes

Cytotoxic T lymphocyte‐associated molecule‐4 (CTLA‐4) is a receptor present on T cells that plays a critical role in the downregulation of antigen‐activated immune responses. CTLA‐4 interacts with the ligands CD80 and CD86 on antigen‐presenting cells (APC), and also directs the assembly of inhibitory signalling complexes that lead to quiescence or anergy. In this study, we show that human monocytes constitutively express CTLA‐4. About 3% of monocytes expressed CTLA‐4 on the cell surface, whereas the intracellular expression was higher and present in about 20% of the monocytes. The sequences of the cDNAs from human monocytes were identical to the sequences of CTLA‐4 from T cells. Expression of CTLA‐4 was also confirmed in the activated myelomonocytic cell lines U937 and THP‐1. Monocytes, but not T cells, activated by interferon (IFN)‐γ also secreted soluble CTLA‐4 in vitro. The CTLA‐4 expression was upregulated upon treatment with phorbol 12‐myristate 13‐acetate (PMA) and IFN‐γ. This increased expression could be partially abolished by staurosporine, an inhibitor of protein kinase C (PKC). Ligation of CTLA‐4 in the monocyte‐like cell‐line U937 with antibodies against CTLA‐4 partially inhibited the proliferation of cells and the upregulation of cell‐surface markers CD86, CD54, HLA‐DR and HLA‐DQ induced by IFN‐γ and Staphylococcus aureus, Cowan I strain (SAC). Ligation of CTLA‐4 suppressed the PMA‐stimulated activation of transcription activator protein 1 (AP‐1) and nuclear factor (NF)‐κB in the U937 cell line, indicating the involvement of an inhibitory signal transduction. These data provide the first evidence that CTLA‐4 is constitutively expressed by monocytes and thus might be important for the regulation of immune mechanisms associated with monocytes.

Active Vaccination Against IL-5 Bypasses Immunological Tolerance and Ameliorates Experimental Asthma

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by ∼50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to β-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

Suppression of lipopolysaccharide-induced antiviral transcription factor (STAT-1 and NF-κB) complexes by antibody-dependent enhancement of macrophage infection by Ross River virus

Subneutralizing concentrations of antibody may enhance virus infection by bringing the virus–antibody complex into contact with the cell surface Fc receptors; this interaction facilitates entry of virus into the cell and is referred to as antibody-dependent enhancement (ADE) of infection. Northern analysis of macrophage RNA demonstrated that ADE infection by the indigenous Australian alphavirus Ross River (RRV-ADE) ablated or diminished message for tumor necrosis factor α (TNF-α), nitric-oxide synthase 2 (NOS2), and IFN regulatory factor 1 (IRF-1), as well as for IFN-inducible protein 10 (IP-10) and IFN-β; the transcription of a control gene was unaffected. Additionally, electrophoretic mobility-shift assay (EMSA) studies showed that transcription factor IFN-α-activated factor (AAF), IFN-stimulated gene factor 3 (ISGF3), and nuclear factor-κB (NF-κB) complex formation in macrophage nuclear extracts were specifically suppressed post-RRV-ADE infection, emphasizing the capacity for ADE infections to compromise antiviral responses at the transcriptional level. The suppression of antiviral transcription factor complexes was shown to depend on replicating virus and was not simply a result of general antibody–Fc–receptor interaction. Although only a minority of cells (≈15%) were shown to be positive for RRV by immunostaining techniques post ADE, molecular (RT-PCR) analysis showed that unstained cells carried RRV-RNA, indicating a higher level of viral infectivity than previously suspected. Electron microscopy studies confirmed this observation. Furthermore, levels of cellular IL-10 protein were dramatically elevated in RRV-ADE cultures. This evidence demonstrates that RRV can potently disrupt the activation of specific antiviral pathways via ADE infection pathways, and may suggest a significant mechanism in the infection and pathogenesis of other ADE viruses.

Interleukin-5 and eosinophils as therapeutic targets for asthma

Extensive clinical investigations have implicated eosinophils in the pathogenesis of asthma. In a recent clinical trial, humanized monoclonal antibody to interleukin (IL)-5 significantly limited eosinophil migration to the lung. However, treatment did not affect the development of the late-phase response or airways hyperresponsiveness in experimental asthma. Although IL-5 is a key regulator of eosinophilia and attenuation of its actions without signs of clinical improvement raises questions about the contribution of these cells to disease, further studies are warranted to define the effects of anti-IL-5 in the processes that lead to chronic asthma. Furthermore, eosinophil accumulation into allergic tissues should not be viewed as a process that is exclusively regulated by IL-5 but one in which IL-5 greatly contributes. Indeed, data on anti-IL-5 treatments (human and animal models) are confounded by the failure of this approach to completely resolve tissue eosinophilia and the belief that IL-5 alone is the critical molecular switch for eosinophil development and migration. The contribution of these IL-5-independent pathways should be considered when assessing the role of eosinophils in disease processes.

Biochemical and functional characterization of human transmembrane tryptase (TMT)/tryptase γ: TMT is an exocytosed mast cell protease that induces airway hyperresponsiveness in vivo via an interleukin-13/interleukin-4 receptor α/signal transducer and activator of transcription (STAT) 6-dependent pathway

Transmembrane tryptase (TMT)/tryptase γ is a membrane-bound serine protease stored in the secretory granules of human and mouse lung mast cells (MCs). We now show that TMT reaches the external face of the plasma membrane when MCs are induced to degranulate. Analysis of purified recombinant TMT revealed that it is a two-chain neutral protease. Thus, TMT is the only MC protease identified so far which retains its 18-residue propeptide when proteolytically activated. The genes that encode TMT and tryptase βI reside on human chromosome 16p13.3. However, substrate specificity studies revealed that TMT and tryptase βI are functionally distinct even though they are ∼50% identical. Although TMT is rapidly inactivated by the human plasma serpin α1-antitrypsin in vitro, administration of recombinant TMT (but not recombinant tryptase βI) into the trachea of mice leads to airway hyperresponsiveness (AHR) and increased expression of interleukin (IL) 13. T cells also increase their expression of IL-13 mRNA when exposed to TMT in vitro. TMT is therefore a novel exocytosed surface mediator that can stimulate those cell types that are in close proximity. TMT induces AHR in normal mice but not in transgenic mice that lack signal transducer and activator of transcription (STAT) 6 or the α-chain of the cytokine receptor that recognizes both IL-4 and IL-13. Based on these data, we conclude that TMT is an exocytosed MC neutral protease that induces AHR in lungs primarily by activating an IL-13/IL-4Rα/STAT6-dependent pathway.

Specific Ablation of Antiviral Gene Expression in Macrophages by Antibody-Dependent Enhancement of Ross River Virus Infection

ABSTRACT Ross River virus (RRV) is an indigenous Australian arthropod-borne alphavirus responsible for epidemic polyarthritis (EPA), myalgia, and lethargy in humans. Macrophages and monocytes have been associated with human RRV disease, and previous studies have shown that RRV is capable of infecting macrophages via both a natural virus receptor and by Fc receptor-mediated antibody-dependent enhancement (ADE). Similar to other viruses, such as human immunodeficiency virus and dengue virus, ADE infection results in dramatic RRV growth increases for in vitro macrophage cultures. This study demonstrates that RRV could resist lipopolysaccharide (LPS)-induced antiviral activity in macrophage cultures when infection was via the ADE pathway. Investigation of this infection pathway found that RRV was able to suppress the transcription and translation of key antiviral genes (tumor necrosis factor and inducible nitric oxide synthase) in LPS-stimulated macrophages by disrupting the transcription into mRNA of the genes coding for the associated transcription factors IRF-1 and NF-κB. The transcription of non-antiviral control genes was not perturbed by RRV-ADE infection, and de novo protein synthesis also was not significantly affected in RRV-ADE infected cells. The ADE pathway of infection allowed RRV to specifically target antiviral genes in macrophages, resulting in unrestricted virus replication. As ADE has been observed for several virus families and associated with disease and adverse vaccination outcomes, these findings may have broad relevance to viral disease formation and antiviral vaccination strategies.