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AIDS:
Research Letters

Altered lymphocyte heat shock protein 70 expression in patients with HIV disease

Agnew, Linda La; Kelly, Markb; Howard, Jonathana; Jeganathan, Sarangapanyb; Batterham, Marijkab,c; Ffrench, Rosemary Ad; Gold, Julianb; Watson, Kennetha

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aSchool of Biological, Biomedical and Molecular Sciences, University of New England, Armidale, NSW 2350, Australia; bAlbion Street Centre, Surry Hills, Sydney, NSW 2010, Australia; cSmart Foods Centre, University of Wollongong, Wollongong, NSW 2522, Australia; and dDepartment of Immunology and Infectious Diseases, Sydney Children's Hospital, Randwick, Sydney and School of Women's and Children's Health, University of New South Wales, Sydney, NSW 2031, Australia.

Sponsorship: This research was supported by grants from the AIDS Trust of Australia and University Research Grants from the University of New England.

Received: 20 February 2003; revised: 14 March 2003; accepted: 15 April 2003.

Heat shock protein (HSP) expression in lymphocytes isolated from 20 patients with HIV disease and 15 age-matched controls was determined. Fold increases in lymphocyte hsp70 expression after heat shock were 4.52 ± 2.97 in HIV-positive individuals compared with 2.60 ± 1.29 for HIV-negative controls (P = 0.001). Given clear roles for HSP in the cross-presentation of antigens, α-defensin internalization and pro-inflammatory cytokine production, a further investigation of HSP in HIV patients is merited.

Heat shock proteins (HSP) are highly evolutionary conserved proteins found in all organisms from bacteria to humans. HSP synthesis is also induced by cellular stressors other than heat, including heavy metal exposure, oxidative stress, viral and bacterial infection and thus the more general term ‘stress protein’ has been applied to this class of protein [1]. HSP perform essential functions in the cell associated with protein folding and assembly and the prevention of protein aggregation and degradation. Under normal conditions they act as molecular chaperones in ensuring that newly formed polypeptides are correctly transported to appropriate cellular organelles. They also act as cytokines and induce proinflammatory cytokine production in human monocytes [2].

The role of HSP in HIV disease pathogenesis is only beginning to be appreciated. A very recent report that the expression of the HSP receptor, CD91, is increased in monocytes from patients with long-term non-progressive HIV disease [3] is of particular importance given that key components of the soluble factor, termed CAF, which suppresses HIV replication, and which is secreted from stimulated CD8 T lymphocytes in high amounts from such individuals have been identified as α-defensins [4]. The latter have in turn been demonstrated to be associated with CD91, which mediates the internalization of α-defensins [5]. Furthermore, HSP are selectively incorporated into the HIV virion during the assembly process [6].

To date, HSP expression in patients with HIV disease has not been reported. Early studies have demonstrated the upregulation of hsp27 and hsp70 messenger RNA transcription in CD4 T-cell lines infected with HIV [7]. In this report, we demonstrated that the expression of lymphocyte hsp70 is altered in patients with HIV disease compared with uninfected age-matched controls.

Twenty patients (male, mean age 47 years) with HIV disease and 15 age-matched controls (male, mean age 45 years) were recruited. Fourteen of the HIV-positive cohort were currently receiving combination antiretroviral therapy and 13 had an undetectable viral load. The mean CD4 T cell count was 500 cells/mm3 (SD ± 304).

Lymphocytes from 20 ml of venous blood were isolated using Ficoll-Paque gradient centrifugation and were incubated at either 37°C for 1 h (control) or heat shocked at 42.5°C for 1 h. Lymphocytes were then allowed to recover at 37°C for 3 h and proteins were extracted and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Hsp70 expression was measured as previously described [8] using both Western immunoblots (β-actin used as an internal control) and enzyme-linked immunosorbent assay (StressGen Biotechnologies, Victoria, Canada). The fold increase in lymphocyte hsp70 expression was calculated by densitometric analysis and was expressed as the ratio of hsp70 expression at 42.5°C to that at 37°C. Correlations were sought between hsp70 expression and CD4 cell count, plasma viral load and antiretroviral treatment status. All procedures were approved by the Human Research Ethics Committee, University of New England (HEO 1/216) and the Research Ethics Committee-Eastern Section, Sydney (01/198).

Data were analysed using analysis of variance (general linear model) with control (non-heat shock) versus heat shock as the within-subject factor and HIV-positive versus HIV-negative as the between-subject factor.

Lymphocyte hsp70 expression after heat shock relative to baseline values was increased in both control and in patients with HIV disease. Significant augmentation in lymphocyte hsp70 expression after heat shock was demonstrated by Western blots. Representative Western immunoblots from HIV-positive and HIV-negative samples, together with corresponding β-actin controls, are presented in Fig. 1a,b. Fold increases in lymphocyte hsp70 expression (Fig. 1c,d) after heat shock were 4.52 ± 2.97 in HIV-positive individuals compared with 2.60 ± 1.29 for HIV-negative controls (P = 0.001). The increase in hsp70 expression in lymphocytes from HIV-positive individuals was primarily caused by the lower baseline level, rather than absolute amounts. Similar trends were demonstrated using enzyme-linked immunosorbent assay (results not shown). Using this assay, fold increases in lymphocyte hsp70 expression after heat shock were 4.88 ± 3.78 in HIV-positive individuals compared with 2.80 ± 1.21 in HIV-negative controls (P = 0.065).

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In this relatively small cohort, no correlation between fold increases in lymphocyte hsp70 expression and viral load, CD4 T cell count or antiretroviral treatment status could be determined. Larger studies will be required to examine such associations.

The determinants of altered hsp70 expression in patients with HIV have not been analysed. Alterations in viral replication or oxidative stress may influence hsp70 expression. Increases in oxidative stress, a characteristic of HIV-positive infection, may alter HSP expression. In this respect, the modulation of HSP expression by oxidative stress and antioxidants [9] has been reported. Although no correlation between lymphocyte hsp70 expression and viral load was demonstrated in this study, we did observe a direct correlation between lymphocyte hsp70 expression and measures of oxidative stress. In the present study, we have demonstrated increased plasma protein carbonyl formation (P < 0.05) and decreased plasma antioxidant status (P < 0.05), both measures of oxidative stress, in this HIV-infected cohort (data not shown), which correlated with fold increases in lymphocyte hsp70 expression. Further studies are required to define these interactions.

This is the first report of altered hsp70 expression in patients with HIV disease. Lymphocyte hsp70 expression in response to heat shock is significantly increased relative to baseline values in patients with HIV disease. The significance of altered lymphocyte HSP expression remains to be determined. However, given the clear roles of these proteins in the cross-presentation of antigens, α-defensin internalization and pro-inflammatory cytokine production, further investigation is merited.

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References

1. Watson K. Microbial stress proteins. Adv Microbrobrobial Physiol 1990, 31:183–223.

2. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, et al. HSP 70 stimulates cytokine production through CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat Med 2000, 6:435–442.

3. Stebbing J, Gazzard B, Kim L, Portsmouth S, Wildfire A, Teo I, et al. The heat shock protein receptor CD91 is up-regulated in monocytes of HIV-1-infected ‘true’ long term non-progressors. Blood 2003, 101:4000–4004.

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7. Wainberg Z, Oliviera M, Lerner S, Tao Y, Brenner BG. Modulation of stress protein (hsp 27 and hsp 70) expression in CD4+ lymphocytic cells following acute infection with human immunodeficiency virus type-1. Virology 1997, 233:364–373.

8. Rao DV, Watson K, Jones GL. Age-related attenuation in the expression of the major heat shock proteins in human peripheral lymphocytes. Mech Ageing Develop 1999, 107:105–118.

9. Peng J, Jones GL, Watson K. Stress proteins as biomarkers of oxidative stress: effects of antioxidant supplements. Free Rad Biol Med 2000, 28:1598–1606.#m AcknowledgementsThe authors would like to thank the nursing, medical and research staff at the Albion Street Centre for their continued support.

© 2003 Lippincott Williams & Wilkins, Inc.

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