Human immunodeficiency virus type-1 Nef protein induces blood–brain barrier disruption in the rat: role of matrix metalloproteinase-9
Introduction
Over 50% of adult patients infected with the human immunodeficiency virus (HIV) develop a variety of central nervous system (CNS) disorders (Price, 1996), including opportunistic infections, neoplasms and chronic HIV encephalopathy (Levy and Bredesen, 1988; Price, 1995). A blood–brain barrier (BBB) leakage (detected by an elevated cerebrospinal fluid [CSF] to serum albumin ratio) was observed more frequently in neurologically symptomatic than in asymptomatic HIV-infected patients (Singer et al., 1994). Furthermore, BBB disturbances were detected in approximately 50% of AIDS patients in a postmortem study on 33 brains using immunohistochemical methods that identified the presence of extravasated fibrinogen and immunoglobulin G (Petito and Cash, 1992). The definitive pathophysiologic mechanisms causing an altered BBB permeability during HIV infection are not yet known in detail.
Matrix metalloproteinases (MMPs), a large family of Zn2+-binding endopeptidases, can degrade components of the BBB, e.g., endothelial basement membrane, and thus increase the cerebral capillary permeability (Rosenberg et al., 1992) (for a review see: Woessner, 1994; Cawston, 1996). We reported recently that MMP-9 activity can be detected in the CSF of approximately 50% of neurologically symptomatic HIV-infected patients, and that CSF MMP-9 activity was associated with BBB breaching, as measured by an increased CSF to serum albumin ratio (Sporer et al., 1998).
HIV-1 proteins, such as the regulatory protein Nef, have been implicated in the neuropathogenesis of HIV infection. Persistently HIV-1-infected astrocytes in vivo and in vitro have been reported to express Nef protein when little or no viral structural protein could be detected (Brack et al., 1992; Tornatore et al., 1994). Furthermore, Nef mRNA and protein were detectable in astrocytes in brain sections selected for extensive histopathology, suggesting that Nef plays a role in AIDS neuropathogenesis (Saito et al., 1994; Tornatore et al., 1994). In a recent study, we described chemotactic activities of Nef protein in vivo and in vitro. Intracisternal (i.c.) application of Nef protein caused the recruitment of leukocytes into the subarachnoid space in rats (Koedel et al., 1999).
In this study, we investigated whether i.c. injection of recombinant HIV-1 Nef protein induces MMP-9 activity in the CSF of rats and compared its effect to that of other HIV proteins (gp120 and gp160). Since Nef induced BBB leakage in the rat model, we investigated the pathophysiological role of MMPs using the MMP inhibitor batimastat (BB-94). Finally, different cell types were assayed in order to identify possible cellular sources of MMP release in the CNS and to test whether Nef directly induces MMP-9 release in different cell culture systems.
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Experimental rat model
A total of 38 male Wistar rats were used in this study (approved by the Government of Upper Bavaria). The experiments were performed using a well-characterized rat model of meningitis, which was previously described in detail (Pfister et al., 1992). Briefly, rats (250–300 g) were anesthetized (100 mg/kg thiopental; Byk Gulden, Constance, Germany), tracheotomized, and artificially ventilated with a small animal ventilator (Ap-10, K. Effenberger, Pfaffing, Germany). Mean arterial blood pressure
HIV-1 Nef protein induces CSF MMP-9 activity after i.c. injection in the rat
Six hours after i.c. injection of 100 ng (but not 10 ng) Nef protein, MMP-9 activity was detected in CSF samples by zymography. Inactivation of the protein by heat abolished Nef-induced CSF MMP-9 activity. The i.c. injection of other HIV-1 proteins (gp120, gp160) and PBS (not shown) did not induce CSF MMP-9 activity. All examined CSF samples showed a constitutive expression of the 72 kDa-gelatinase MMP-2 (Fig. 1).
MMP-9 contributes to HIV-1 Nef protein-induced BBB disruption in the rat
After 6 h, the i.c. injection of 100 ng HIV-1 protein Nef (but not
Discussion
Major results of our study were (1) that HIV-1 Nef protein (but not gp120 and gp160) caused an increase in BBB permeability, which was paralleled by detectable CSF MMP-9 activity and CSF pleocytosis, and (2) that Nef-induced changes were inhibited by the pretreatment with the MMP inhibitor BB-94.
According to our results, previous experimental and clinical studies have indicated that MMPs play a critical role in BBB leakage of different etiology. For example, intracerebral injection of TNF-α in
Acknowledgements
We thank Ms. B. Angele for her excellent technical assistance. Furthermore, we thank Prof. L. Gürtler, Max von Pettenkofer-Institute, Munich, for outstanding clinical cooperation and Ms. J. Benson for editing the manuscript. Supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 464, project B3 to H.-W. Pfister; project B2 to V. Erfle).
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