Expression of inducible nitric oxide synthase, interleukin-1 and caspase-1 in HIV-1 encephalitis

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Abstract

Inflammatory cytokines and enzymes such as IL-1 and inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of AIDS dementia, a condition associated with infection of the CNS cells by the HIV-1. In this report, we investigated the expression of iNOS, IL-1, and caspase-1 (interleukin-1 converting enzyme) in HIV-1 encephalitis (HIVE) by immunocytochemistry and analyzed their expression with respect to HIV-1 infection and glial activation. In HIVE, all three molecules were expressed at high levels in areas of HIV-1 infection (microglial nodules with HIV-1 p24 immunoreactivity) and in areas of diffuse white matter gliosis. Expression was cell-type specific, with IL-1 and caspase-1 being expressed in macrophages and microglia, and iNOS in activated astrocytes. Multinucleated giant cells, a hallmark of virally infected cells, showed intense staining for both IL-1 and caspase-1, suggesting induction of these molecules by HIV-1. Double immunocytochemistry demonstrated a regional co-localization of astrocyte iNOS and microglial IL-1 and caspase-1. These results support the notion that autocrine and paracrine interactions between HIV-1 infected macrophages and microglia, activated microglia, and astrocytes lead to expression of proinflammatory and neurotoxic molecules. iNOS and caspase-1 may provide additional therapeutic targets for HIVE.

Introduction

HIV-1 associated dementia (HAD: also termed AIDS–dementia complex) affects significant numbers of patients with AIDS Navia et al., 1986a, Navia et al., 1986b, Dickson et al., 1994, Lipton and Gendelman, 1995. Postmortem analysis of the CNS shows microglial nodules with multinucleated giant cells (MGC), diffuse microglial and astrocyte activation, and evidence for neuronal damage, a constellation of findings termed under “HIV-1 encephalitis (HIVE).” Studies in vivo and in vitro demonstrate that HIV-1 induces a number of inflammatory molecules in glia, which could play a pivotal role in the pathogenesis of HIVE. One of the candidate inflammatory molecules that may cause neuronal dysfunction in HIVE is inducible nitric oxide synthase (iNOS).

iNOS was first cloned in murine macrophages and its expression in murine cells is induced by cytokines and bacterial endotoxin Nathan, 1992, Nathan and Xie, 1994, MacMicking et al., 1997. A number of animal models of infectious and autoimmune diseases demonstrate that nitric oxide generated by iNOS has cytotoxic and immunoregulatory properties Wei et al., 1995, Murphy et al., 1993, Brosnan et al., 1994. Human iNOS was first cloned in hepatocytes by Geller et al. and its regulation of expression has been studied since Geller et al., 1993, Chartrain et al., 1994. Remarkably, some of these studies failed to show that human monocytes and macrophages can be induced to express iNOS Liu et al., 1996, Chao et al., 1996, Denis, 1994, Zhang et al., 1996. Analysis of human and murine iNOS promoter revealed a number of differences including the size of the promoter and the location of the critical NF B sites de Vera et al., 1996, Taylor et al., 1998, Marks-Konczalik et al., 1998. Although activation of NFκB is important in the transcription of iNOS in both species, induction of human iNOS requires IL-1 (shown in hepatocytes, chondrocytes and astrocytes), whereas IL-1 plays a non-essential role in the induction of murine iNOS Geller et al., 1995, Charles et al., 1993, Liu et al., 1996.

IL-1 is a macrophage cytokine whose expression is regulated at multiple steps (Dinarello, 1994). Following transcriptional activation and translation, generation of mature (active) IL-1 requires processing of proIL-1 by IL-1 converting enzyme (ICE: caspase-1). Active caspase-1 is generated also by proteolytic cleavage of procaspase-1 (Nicholson and Thornberry, 1997). In addition to IL-1 processing, caspase-1 is also implicated in macrophage apoptosis, as shown in vitro as well as in caspase-1-deficient mice Kuida et al., 1995, Li et al., 1995, Hogquist et al., 1991. In a number of human CNS diseases, IL-1 expression is induced in activated microglia at sites of tissue damage, and its expression may lead to ultimate neuronal damage. Given the importance of IL-1 in the induction of human iNOS, we determined the expression of iNOS in HIVE and analyzed its expression with respect to IL-1 and caspase-1.

Section snippets

Patient material

Paraffin-embedded formalin-fixed brain tissues from 19 patients were obtained from the Manhattan HIV-1 Brain Bank. These consisted of 9 patients with HIVE, and 10 HIV-1 seropositive and seronegative individuals without HIVE. Brains with HIV-1 associated opportunistic infections or tumors were excluded in this study, except for patient 3 who had documented neurosyphillis. History was obtained by retrospective chart analysis at the time of autopsy or collected prospectively (see tables). For each

iNOS immunoreactivity

The results of immunocytochemistry are summarized in Table 1, Table 2, and illustrated in Fig. 1. On H&E, HIVE cases had perivascular accumulations of multinucleated giant cells (MGC), activated macrophages, and microglia (microglial nodules: MN); diffuse myelin pallor associated with hypercellularity reflected diffuse gliosis (Fig. 1A). iNOS expression was present in all HIVE cases (Table 1). iNOS immunoreactivity was localized primarily to reactive astrocytes (Fig. 1B), based on typical

iNOS

Our results shown in this study are consistent with the notion that in HIVE, iNOS is expressed in glial cells as a result of viral infection and/or immune activation. Since we excluded cases with opportunistic infections and tumors that are often present in the CNS of AIDS patients, the extent of iNOS expression in our study most likely reflects the direct or indirect consequences of HIV-1 infection.

Previous studies using RT-PCR or Western blot analysis reported expression of iNOS mRNA and

Acknowledgements

This study was supported by MH55477. We are grateful to the staff and participants in the Manhattan HIV Brain Bank (R24MH59724), to Dr. Sue Griffin for providing the detailed protocol for IL-1 immunostain, Dr. David Goldman for critical reading of the manuscript and contributing Fig. 2, and Dr. Marty Sliwinski for the statistical analysis. We also thank Drs. Dennis Dickson, Celia Brosnan, Moon Shin, and Martha Downen for their support throughout the study.

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