Dynamics of cytokine expression in HIV productively infected primary CD4+ T cells

Abstract

Using intracellular p24 staining to discriminate between bystander and HIV productively infected cells, we evaluated the properties of HIV productively infected cells in terms of cytokine expression, activation status, apoptosis, and cell proliferation. We demonstrate that HIV productively infected primary CD4 ؉ T cells express 12-to 47-fold higher type 1 cytokines than bystander or mock-infected cells. The frequency of HIV productive replication occurred predominantly in Thelper 1 (Th1), followed by Th0, then by Th2 cells. These productively infected cells expressed elevated levels of CD95, CD25, CXC chemokine receptor 4 (CXCR4), and CC chemokine receptor 5 (CCR5). While productively infected cells were only 1.8-fold higher in apoptosis frequency, they up-regulated the antiapoptotic protein B-cell leukemia 2 (Bcl-2) by 10-fold. Up-regulation of interleukin-2 (IL-2) and Bcl-2 were dependent on phosphatidylinositol-3-kinase signal transduction, given that it was down-regulated by Wortmanin treatment. Additionally, 60% of productively infected cells entered the cell cycle, as evaluated by Ki67 staining, but none divided, as evaluated by carboxyfluoresccin diacetate succinimidyl ester (CFSE) staining. Evaluation of cell cycle progression by costaining for DNA and RNA indicated that the cells were arrested in G 2 /M. Collectively, these data indicate that HIV replication occurs predominantly in Th1 cells and is associated with immune activation and up-regulation of Bcl-2, conferring a considerable degree of protection against apoptosis in the productively infected subpopulation. IntroductionThe interaction between cytokines and HIV expression is multifaceted. Approaches to evaluate the relationship between cytokines and HIV have relied on (1) evaluating the impact of a particular cytokine on HIV replication in vitro, [1][2][3][4][5] (2) comparing the cytokine expression profile of HIV-infected and uninfected donors, 6,7 and (3) measuring cytokine production from in vitro infected cells. [8][9][10] Analysis of the impact of cytokines on HIV-1 replication in vitro have generally demonstrated that a number of type 1 cytokines (interleukin-1 [IL-1], tumor necrosis factor-␣ [TNF-␣], IL-2, IL-12) up-regulate HIV replication while type 2 cytokines down-modulate HIV (IL-10, transforming growth factor-␤ [TGF-␤]). This relationship is not simplistic given that a combination of type 1 and type 2 cytokines may lead to a synergistic or antagonistic effects on HIV replication [11][12][13][14] ; this result often depends on the combination of cytokines and on the cell type examined, whether lymphocytic or monocytic.Earlier studies describing the cytokine profile in purified peripheral blood mononuclear cells (PBMCs) from HIV ϩ patients have led to discordant findings. Some suggested that HIV-1 infection leads to a shift from type 1 (IL-2, interferon-␥ [IFN-␥]) to type 2 (IL-4, IL-10) cytokines with HIV disease progression. 6 By contrast, others have not been able to document such a type 1-to-type 2 cytokine shift with disease pro...

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“…This seems unlikely, since we examined the cells for early, intermediate, and late activation markers, and we found that the p24 high T cells did not express CD25, HLA-DR, or VLA-1 and only small amounts of CD69. Our results using T cells activated by ECs stand in striking contrast to previous studies, which used similar FACS-based techniques to show that viral replication under other conditions is restricted to highly activated T cells (4,44).…”

“…Since activated CD4 ϩ T cells are a primary source of HIV production in vitro (4,44) and in vivo (21,46,65), it is reasonable to hypothesize that ECs support viral replication in TCR-activated T cells. The productively infected cells observed on days 6 and 9 of culture, i.e., the p24 high T cells, could represent the progeny of the cells that had been fully activated and proliferated in response to high-affinity TCR binding to nonself (allogeneic) MHC molecules.…”

The activation and dynamics of cytokine expression by CD4+ T cells and AIDS progression in HIV-1-infected Chinese individuals

“…Similar to the findings of Bahbouhi et al [24], our results demonstrated that HIV-1 productively infected CD4 þ T cells expressed elevated levels of IL-2, IL-4, and IFN-g in comparison with bystander cells. It suggested that the infection of HIV-1 could up-regulate the expressions of both Th1and Th2 cytokines.…”

“…However, a physiologic response to the intact virus might not be accurately measured by molecular clones. The intact virus was used by Bahbouhi et al in 2004 to evaluate the dynamics of cytokine expression [24], inwhich the HIV productively infected CD4 þ T cells were identified by HIV-1 core antigen p24. Nevertheless, the in vitro HIV-1 infection model used by Bahbouhi et al might not represent the physiological conditions very well.…”

Immunophenotypic alterations in acute and early HIV infection

“…In the context of HIV, this may not be the case. We previously shown that Ki67 immunostaining and tracking of actual cell division through Carboxyfluoresccin Diacctate Succinimidyl Ester (CSFE) dye do not correlate in HIV + cells [34]. HIV infected CD4+ T cells stained strongly for Ki67 but there was no detectable cell division among cells productively infected by HIV [34].…”