Andrew J. Hapel

Enhanced Mucosal Cytokine Production in Inflammatory Bowel Disease

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohns disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.

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.

Macrophage-Induced Muscle Pathology Results in Morbidity and Mortality for Ross River Virus-Infected Mice

Ross River virus (RRV) is an Australian alphavirus that is often responsible for chronic epidemic polyarthritis and myalgia in humans. Past studies have shown severe disruption of striated muscle fibers to be prominent in RRV pathology in mice; in the present study, macrophages were directly implicated as the primary mediators of muscle damage. General immunosuppressive therapies had only minor effects on mortality and morbidity in RRV-infected mice, with no inhibition of muscle damage. Treatment of mice with macrophage-toxic agents (e.g., silica) prior to RRV infection completely abrogated disease symptoms without significantly affecting titers of virus in organs. Further studies found that clinical signs of infection and muscle damage correlated with a massive influx of macrophages into hind leg muscle, whereas no such infiltrate or damage was observed for silica-treated mice. These observations are significant for the human disease context, as monocytic cells have been detected in the synovial effusions of persons with epidemic polyarthritis.

Determination of the lipid peroxidation product trans-4-hydroxy-2-nonenal in biological samples by high-performance liquid chromatography and combined capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry

trans-4-Hydroxy-2-nonenal (HNE) is an aldehyde end-product of lipid peroxidation in biological systems which is capable of producing a range of powerful biological effects. We wish to describe a sensitive and selective strategy for the determination of HNE in biological samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivatives of HNE and its deuterated internal standard which, after sample clean-up by solid-phase extraction and purification by high-performance liquid chromatography (HPLC), were derivatised further to trimethylsilyl ethers. Subsequent capillary column gas chromatography-negative-ion chemical ionisation mass spectrometry (GC-NICIMS) using selected-ion monitoring allowed quantitation in the low ng/ml range. The use of an internal standard and the O-PFB oxime derivatives circumvented the problems encountered previously by other workers because of the volatility and instability of HNE. The syn-isomer of HNE O-PFB oxime followed the anti-isomer on the HPLC and GC columns used, giving a distinctive pair of peaks of characteristic relative proportion. Moreover, the NICI mass spectra of the geometrical isomers were significantly different, providing further evidence to validate the identity of any endogenous HNE recovered. The method was used to identify and quantify HNE in platelets, monocytes, plasma and oxidised low-density lipoprotein.

Immune mechanisms associated with the rejection of fetal murine proislet allografts and pig proislet xenografts: comparison of intragraft cytokine mrna profiles1

BACKGROUND Previous in vivo depletion studies of CD4 and CD8 T cells indicated that different rejection mechanisms operate for proislet allografts and xenografts. The cellular and molecular mechanisms of acute proislet allograft and xenograft rejection have therefore been characterized and directly compared. METHODS The intragraft cytokine mRNA profile in rejecting BALB/c (H-2d) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2k) recipient mice using semi-quantitative reverse transcriptase-assisted polymerase chain reaction (RT-PCR). The cytokine profiles for proislet allografts and pig proislet xenografts at 3-10 days posttransplant were directly compared and correlated with graft histopathology. RESULTS Allograft rejection was protracted (2-3 weeks), characterized by infiltrating CD8 T cells and CD4 T cells (no eosinophils) and was associated with a Th1-type CD4 T cell response (IL-2, IFN-gamma, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-gamma, IL-3, and IL-10 mRNA). Xenograft rejection was rapid (6-8 days), involved predominantly CD4 T cells and eosinophils, and in contrast to allografts, exhibited intragraft mRNA expression for the Th2 cytokines IL-4 and IL-5. CONCLUSIONS Proislet allograft and xenograft rejection differ in the tempo of destruction, phenotype of the cellular response and intragraft profile of cytokine mRNA. The recruitment of eosinophils only to the site of xenorejection correlates with IL4 and IL-5 mRNA expression. These findings suggest that different anti-rejection strategies may need to be developed to optimally target the allograft and the xenograft response.

Dengue virus therapeutic intervention strategies based on viral, vector and host factors involved in disease pathogenesis

Dengue virus (DV) is the most widespread arbovirus, being endemic in over 100 countries, and is estimated to cause 50 million infections annually. Viral factors, such as the genetic composition of the virus strain can play a role in determining the virus virulence and subsequent clinical disease severity. Virus vector competence plays an integral role in virus transmission and is a critical factor in determining the severity and impact of DV outbreaks. Host genetic variations in immune-related genes, including the human leukocyte antigen, have also been shown to correlate with clinical disease and thus may play a role in regulating disease severity. The hosts immune system, however, appears to be the primary factor in DV pathogenesis with the delicate interplay of innate and acquired immunity playing a crucial role. Although current research of DV pathogenesis has been limited by the lack of an appropriate animal model, the development of DV therapeutics has been a primary focus of research groups around the world. In the past decade advances in both the development of vaccines and anti-virals have increased in dramatically. This review summarises the current understanding of viral, vector and host factors which contribute to dengue virus pathogenesis and how this knowledge is critically important in the development of pharmaceutical interventions.

The Protective Role of Thymus-Derived Lymphocytes in Arbovirus-Induced Meningoencephalitis

The inflammatory response in mouse brain and the elimination of virus from the brain following intracerebral inoculation of a group A arbovirus has been shown to be T‐cell dependent. Virus persists in the brain of T‐cell depleted mice, and inflammation is depressed. Inflammation is restored by transfer of immune cells but not by immune serum, and the transferred cells are effective in reducing virus titres in the absence of circulating antibody. Transferred antibody is not efficient in protecting infected mice. The cells that restore inflammation can come from mice immunized with a group A arbovirus of the same subtype but not from mice immunized with more distantly related viruses. Macrophages may be important effector cells working in collaboration with T cells.

Persistent noncytolytic togavirus infection of primary mouse muscle cells

Abstract Infection of confluent primary mouse muscle cell cultures with Ross river virus (RRV), an Australian alphavirus, resulted in a noncytolytic persistent virus infection. Analysis of the species of viral RNA synthesized in infected cells suggested that defective interfering particles of RRV do not play a role in the establishment or maintenance of viral persistence.

Appearance of Cytotoxic T Cells in Cerebrospinal Fluid of Mice with Ectromelia Virus‐Induced Meningitis

In mice with meningitis induced by ectromelia virus, the influx of inflammatory cells into cerebrospinal fluid (CSF) was shown to be triggered by an immunologically specific, T cell‐dependent mechanism. These cells were counted, and their cytotoxicity assayed in vitro. Cell numbers reached a peak 6 days after infection and showed specific cytotoxicity for ectromelia‐infected target cells, an effect that reached a peak at day 6 and was retained for at least a further 4 days. Treatment with anti‐thymocyte serum 2 to 3 days before harvest of CSF cells depressed the numbers found and their cytotoxicity, suggesting that they reached the exudate from the blood.

Blocking of delivery of the antigen-mediated signal to the nucleus of T cells by cyclosporine.

Cyclosporine (CsA) inhibits release of interleukin 2 (IL-2) and hemopoietic growth activities such as interleukin 3 (IL-3) from major histocompatibility complex (MHC)-antigen-activated T cells. Production of both lymphokines appears to be coordinately regulated; the antigen dose response, T cell dose response, and time course of lymphokine appearance are similar. The triggering of lymphokine production by these cells is solely dependent on T cell-target cell interaction, as the T cell dose response curve indicates that no cooperation occurs between T cells, and any metabolic contribution by the target cell was eliminated by ultraviolet irradiation. This interaction triggers the transcription of lympho-kine-encoding mRNA. The process of lymphokine release can be divided into 4 steps: Antigen binds to the T cell; a signal is transferred to the cell nucleus; transcription of lymphokine-encoding mRNA occurs; and intact lymphokine is synthesized and secreted. CsA inhibits antigen triggered lymphokine production. However, it does not inhibit lymphokine release from the constitu-tively producing tumor cell lines WEHI-3 (which releases IL-3) and MLA 144 (which produces IL-2). Thus CsA has no effects on the lymphokine secretion process or any direct action upon lymphokine-coding mRNA. CsA does not affect antigen recognition during cell-mediated cytotoxicity. Therefore, CsA acts after antigen binding and before transcription of lymphokine-encoding mRNA. That is CsA blocks the transmission of the antigen signal. This information is used to show that this CsA-sensitive signal is required continuously to maintain the T cell in a lymphokine-secreting state.