Reply to Klasse et al.

    Department of Medicine, University of Minnesota, Minneapolis

    To the EditorWe appreciate the comments of Klasse et al. [1] regarding our study [2]. However, we believe that their conclusions are based on an inaccurate view of our work.

    Although it is difficult to determine the concentration of gp120 in vivo, we used a concentration that is in the range used by several investigators [3, 4]. We do not feel that serum levels of gp120 are a good indicator of its concentration in tissues. The level of gp120 is likely to be much more concentrated around cells that are productively infected, leading to high local concentrations that may be intensified by binding to extracellular matrix components. An excellent review [5] discusses several factors that may lead to increased local concentrations of viral proteins in the brain. Our concentrations of gp120 have been shown to induce intracellular signaling [6]. Further dose-response studies are needed before any conclusions can be drawn about the minimal amount of gp120 needed to affect neural progenitor cells (NPCs). Of note, in brain-slice cultures, the concentration of gp120 around NPCs is likely to be <800 pmol/L, because of inhibition of diffusion of the large gp120 protein. Another argument is that gp120 only initiates a cascade of events, one of which could be the release of chemokines that inhibit proliferation of NPCs [7].

    To address the question of how much gp120 is present in vivo, we tested frozen sections of tissue from a healthy human brain and from a brain from a patient with HIV dementia. After they were fixed with acetone, we incubated sections of healthy brain tissue with 8-mmol/L, 800-pmol/L, and 80-pmol/L concentrations of gp120 from the IIIb strain of HIV-1. After allowing the gp120 to bind for 1 h, we washed the sections several times with PBS to remove unbound gp120. Next, a cross-reactive monoclonal antibody (National Institutes of Health AIDS Research and Reference Reagent Program catalog no. 1101) was used to detect gp120. The sections of brain from the HIV-infected patient were similarly incubated with this antibody. After the sections were washed with PBS, a 35S-labeled goat antimouse IgG antibody was added. Labeled cells were then detected by autoradiography, as described elsewhere [8]. This method has been shown to quantitate amounts of antigen in a linear fashion [9]. We found that the 800-pmol/L concentration of gp120 bound to the tissue and showed levels of staining that were similar to those observed in the HIV-infected brain (figure 1). We conclude that our concentration of gp120 reproduces the concentration seen in vivo around infected cells.

    Although Klasse et al. suggest that binding of gp120 to neurons in the absence of CD4 is unlikely, in fact, signaling in neurons has been demonstrated by 2 other groups and has been found to be independent of CD4 [4, 10].

    As for the ability of the cross-reactive antibody that we used to bind to CM235, other studies have found antibodies to the V3 loop to be broadly cross-reactive [10]. In any event, we tested, by blocking CCR3 with a specific antagonist, SB328437 (Calbiochem), the involvement of CCR3 in the ability of gp120 of the CM235 strain of HIV-1 to inhibit proliferation of NPCs. Using the plating assay that we have described elsewhere [2], we found that, although gp120 of the CM235 strain inhibited proliferation of NPCs by 43%, the CCR3-specific antagonist SB328437, at a concentration of 500 nmol/L, reversed this inhibitory effect, resulting in a nonsignificant, 8% reduction of proliferation of NPCs. Therefore, further studies have supported our original conclusionthat gp120 affects NPCs through chemokine receptors.

    We believe that our original study is valid and that further experiments confirm our original conclusions.

    References

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