8th International Workshop on Adverse Drug Reactions and Lipodystrophy in HIV San Francisco, California - September 24 - 26, 2006

LUMINEX CYTOKINE EXPRESSION ARRAY IN SUBCUTANEOUS FAT ADIPOCYTES, PREADIPOCYTES AND MACROPHAGES IN HIV-LIPOATROPHIC PATIENTS

Antiviral Therapy 2006; 11:L25 (abstract no. 37)

DA Killebrew, CM Shikuma and M Gerschenson
Hawaii AIDS Clinical Research Program and the Department of Medicine, John A. Burns School of Medicine, University of Hawaii-Manoa, Honolulu, HI, USA

BACKGROUND: An increase in proinflammatory cytokines has been found in fat tissue of HIV+ lipoatrophic patients. However, the etiology of the origin of this cytokine production has yet to be elucidated. Our hypothesis is that cells in fat tissue, like adipocytes, preadipocytes, and macrophages can be isolated and cultured separately to assess their pro-inflammatory cytokine production.

METHODS: Subcutaneous (SC) adipose tissue biopsies were obtained from HIV+ lipoatrophic and HIV-subjects for flow cytometry analysis (n=5) and Luminex cytokine array assessment (n=6). Tissue weight excised ranged from 350–950 mg. Adipocytes and preadipocyte pellets (including macrophages) were isolated from the SC tissue using collagenase and centrifugation. Preadipocyte fractions were analysed by flow cytometer for CD68+ (macrophage) cells. Adipocytes and the preadipocyte (+ macrophage) cells were cultured and supernatants were obtained at 24 hours. Cytokine/chemokine levels were then assessed using the Luminex multi-cytokine panel protein array for: Eotaxin, GM-CSF, IFNγ, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1, RANTES, and TNFα.

RESULTS: There is a 50-70% higher level of CD68+ cells in HIV+ (n=3) adipose tissue than HIV-(n=2), as shown by flow cytometry. The cytokine panels for HIV+lipoatrophic versus HIV negative indicated that out of 22 cytokine/chemokines: 17 recorded above background for preadipocytes: Eotaxin, GM-CSF, INFγ, IL-10, IL12(p40), IL-12(p70), IL-1α, IL-1β, IL-2, IL-6, IL-8, Rantes, IL-15, IP-10, MCP-1, MIP-1, TNF-α), and 12 for adipocytes: Eotaxin, GM-CSF, INFγ, IL-10, IL12(p40), IL-12(p70), IL-1α, IL-1β, IL-2, IL-6, IL-8, Rantes). The cytokine/chemokine pg/ml indicated a trend for HIV+lipoatrophic versus HIV negative showing differential expression in both adipocytes and the preadipocyte (+ macrophage) cells. In HIV+lipoatrophic there was a 100% decrease in IL-1α, IL-6, and IL-8 and a 100% increase in IL-1β, in both adipocytes and the preadipocyte (+ macrophage) fraction, compared to HIV- individuals.

CONCLUSIONS: This technology will facilitate further analyses of cytokine expression in different cell components of fat tissue.

Acknowledgements: This research was supported by NIH, USA (HL085025, AI068525, AI060409, and RR16467).

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2006-09-24
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