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Changes in Circulating Levels of Soluble Cell Adhesion Molecules Following Highly Active Antiretroviral Treatment of HIV-1-Infected Patients
Clinical Immunology Vol. 95, No. 3, June, pp. 212–217, 2000 doi:10.1006/clim.2000.4865, available online at http://www.idealibrary.com on
Changes in Circulating Levels of Soluble Cell Adhesion Molecules Following Highly Active Antiretroviral Treatment of HIV-1-Infected Patients Claudio M. Mastroianni, Miriam Lichtner, Fabio Mengoni, Claudia D’Agostino, Gabriella d’Ettorre, Gabriele Forcina, Paola Santopadre, Anna P. Massetti, and Vincenzo Vullo Department of Infectious and Tropical Diseases, La Sapienza University, Rome, Italy
Increased levels of soluble cell adhesion molecules (sCAM) have been reported in HIV-1 infection and may possibly contribute to altering the adhesion mechanisms of phagocytic cells. We evaluated the effect of highly active antiretroviral therapy (HAART) on plasma levels of sL-selectin, sE-selectin, intercellular cell adhesion molecule-1 (sICAM-1), sICAM-3, and vascular cell adhesion molecule-1 (sVCAM-1). Study participants included 22 HIV-1-infected patients with a CD4 ⴙ T-cell count/l below 500 who were started on a HAART regimen and followed up for 9 months. After the initiation of therapy, plasma sL-selectin concentrations progressively decreased to normal ranges in the majority of our patients (P < 0.001), while no changes in sE-selectin were found. In all patients sICAM-1 remained relatively constant at significantly elevated concentrations during the 9 months of therapy. A significant reduction in plasma concentrations of both sICAM-3 and sVCAM-1 was found; however, the levels of these sCAM were not normalized by HAART and remained significantly elevated throughout the study (P < 0.001). The reduced release of sL-selectin could improve the ability of phagocitic cells to migrate in response to chemotactic stimuli after starting HAART. On the other hand, the persistent elevation of sICAM-1, sICAM-3, and sVCAM-1 could reflect continuous HIV-1-mediated immune activation, despite adequate control of plasma HIV-1 replication by therapy. © 2000 Academic Press
Key Words: adhesion molecules; innate immunity; anti-HIV-1 therapy.
INTRODUCTION
Neutrophils and monocytes are involved in the innate immune response during the progression of human immunodeficiency virus type 1 (HIV-1) infection. A normal function of polymorphonuclear leukocytes This work was supported by a grant from Ministero della Sanita`, ISS, Progetto AIDS 1997 (30A.0.74) and 1998 (30B.88), Rome, Italy. 1521-6616/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
(PMNL) is crucial for the clearance and killing of fungal and bacterial opportunistic pathogens that frequently affect AIDS patients (1). Monocytes also play an important role in HIV-1 infection as a reservoir for viral replication, mediators of local tissue damages, and as effector cells against intracellular microrganisms (2). To exert a variety of immunoregulatory and phagocyte functions, neutrophils and monocytes emigrate from the bloodstream and accumulate in body tissues. Leukocyte adherence to endothelial cells and transendothelial migration to inflammatory sites are mediated by the expression of various cell-surface proteins that are part of three families of cell adhesion molecules (CAM): selectins, integrins, and the immunoglobulin (Ig)-related superfamily (3–5). During the course of HIV-1 infection dysregulation of CAM synthesis as well as abnormal levels of soluble isoforms of CAM have been reported (6 –11). The alteration in leukocyte adhesion mechanisms may lead to dysfunction in phagocyte diapedesis, chemotaxis, and migration to the site of infection. In addition, up-regulation of CAM expression may facilitate extravasation of HIV-1-infected leukocytes into underlying tissues, contributing to disease spread and specific tissue damage (12). In recent years, dramatic immunological improvements have been shown in HIV-1-infected individuals who received highly active antiretroviral therapy (HAART), including HIV-1 protease and reverse transcriptase inhibitors (13). There is great interest in the quantitative and qualitative changes in host immune response following these potent therapies. In order to investigate the effect of HAART on leukocyte adhesion system, we have assessed changes in circulating levels of soluble forms of L-selectin (sL-selectin), sE-selectin, intercellular cell adhesion molecule-1 (sICAM-1), sICAM-3, and vascular cell adhesion molecule-1 (sVCAM-1) in a cohort of patients with moderately advanced HIV-1 infection undergoing treatment with a triple combination regimen (one protease inhibitor and two nucleoside analogues).
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SOLUBLE CELL ADHESION MOLECULES DURING HAART MATERIALS AND METHODS
Patients and Controls Study participants included 22 HIV-1-infected patients (13 males and 9 females) with a CD4 ⫹ T-cell count/l below 500 who were started on an HAART regimen containing a protease inhibitor and followed up for 9 months. The median age was 38.5 years (range, 21– 65). Patients with concomitant ongoing acute infection and prior use of protease inhibitors were excluded. HIV-1 seropositivity was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot analysis (Chiron Corp., Emeryville, CA). Risk factors for HIV-1 infection were injecting drug use in 3 cases, homosexuality in 7 cases, heterosexuality in 12 patients, and hemophilia in 1 case. Fifteen patients were naive for any antiretroviral drugs and 7 had previously been treated with reverse transcriptase inhibitors for more than 1 year. The HAART regimen consisted of two reverse transcriptase inhibitors and one protease inhibitor (indinavir in 18 patients, ritonavir in 3 patients, and nelfinavir in 1 case). Ten age- and sex-matched healthy blood donors were included as controls. A fully informed consent was obtained from all study participants. Sample Handling Venous blood samples were collected from each patient into EDTA-containing tubes (Becton–Dickinson Systems, San Jose, CA) at enrollment and at months 3, 6, and 9 of follow-up. For the enumeration of CD4 ⫹ and CD8 ⫹ lymphocyte numbers, venous blood was collected into 3-ml tubes and processed within 4 h of handling. For the assessment of HIV-1 RNA and sCAM levels, venous blood was collected into 15-ml tubes and processed within 2 h of collection. The cell-free plasma was stored at ⫺70°C until use. HIV-1 RNA Levels and CD4 ⫹ and CD8 ⫹ Lymphocyte Counts Plasma HIV-1-RNA levels were measured by using a quantitative reverse polymerase chain reaction (Amplicor HIV Monitor; Roche Diagnostic Systems, Branchburg, NJ). The limit of detection was 200 (2.30 log 10) copies/ml. Enumeration of CD4 ⫹ and CD8 ⫹ lymphocyte numbers was assessed using two-color flow cytometric analysis with the Becton–Dickinson flow cytometer FACScan. The whole blood lysis procedure and SimulTEST reagents (Becton–Dickinson) were used.
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Assays for Detection of Soluble Cell Adhesion Molecules (sCAM) Commercially available ELISA kits were used for the quantitative detection of plasma levels of sL-selectin, sE-selectin, sICAM-1, sICAM-3, and sVCAM-1 (Bender MedSystems, Boehringer Ingelheim Group, Vienna, Austria), according to the manufacturer’s instructions. The lower limit of sensitivity for assays was as follows: 0.3 ng/ml for sL-selectin; 0.5 ng/ml for sE-selectin; 3.3 ng/ml for sICAM-1; 0.58 ng/ml for sICAM-3; and 0.9 ng/ml for sVCAM-1. The concentrations of sCAM in the samples were calculated by interpolation from a standard curve constructed with specific sCAM standards. Statistical Analysis Results are presented as the median and interquartile range (IQR). Data at multiple time points in the treatment group were compared using the Friedman two-way analysis of variance by ranks with pairwise comparisons on treatment versus baseline values (Dunnett’s test). Differences between healthy controls and treated patients at baseline and at the end of study were evaluated by the nonparametric Mann–Whitney U test. The significance of correlation study parameters was estimated using the Spearman rank correlation test. RESULTS
Plasma HIV-1 RNA Measurements and Changes in CD4 ⫹ and CD8 ⫹ Lymphocytes The baseline median plasma viral load was 5.47 log 10 copies/ml (range, 2.62– 6.91). After initiation of HAART, HIV-1 load fell below the assay’s level of detection (2.30 log 10/ml) in 15/22 (68.1%) patients at 3 months (median decrease, 2.46 log 10/ml; range, 0.32– 4.0), in 17/22 (77.2%) at 6 months (median decrease, 2.5 log 10/ml; range, 0.23–5.45), and in 20/22 (90.9%) subjects at 9 months (median decrease, 2.83 log 10/ml; range, 0.24 –5.47). The median absolute CD4 ⫹ T-cell count increased from 117/l at baseline to 232/l at month 3, 306/l at month 6, and 369/l at month 9 (range, 4 to 644/l increase at month 9; P ⬍ 0.001). The absolute CD8 ⫹ T-cell count also rose from a median of 529/l at baseline to 765/l at month 3, 853/l at month 6, and 916/l at month 9 (range, 1125/l) decrease to 791/l increase at month 9; P ⬍ 0.001). Finally, there was also a significant increase in CD4:CD8 ratio from a median of 0.24 at baseline to 0.32 at month 6 and 0.36 at month 9 (P ⬍ 0.05).
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TABLE 1 Plasma Levels of sCAM in 10 HIV-Seronegative Controls and in 22 HIV-Infected Patients of a Treatment Group at Baseline (Tx 0) and after 9 Months of Therapy (Tx 9) sCAM (ng/ml)
Controls
Tx 0
Tx 9
sL-selectin sE-selectin sICAM-1 sICAM-3 sVCAM-1
518 (487–750)* 24.5 (21–52) 251 (173–293)* 52 (37–57)* 690 (601–790)*
942 (782–1157) 39.6 (22–52) 449 (392–557) 138 (107–176) 1116 (974–1391)
720 (620–812) 44.4 (37.9–72) 472 (383–548) 97 (63–110) 1016 (774–1069)
Note. Data are expressed as medians (interquartile range). * P ⬍ 0.001 versus Tx 0 and Tx 9 (Mann–Whitney U test).
Plasma Levels of sCAM Plasma concentrations of sCAM in the healthy controls and in the treatment group, at baseline and after 9 months of HAART, are reported in Table 1. The plasma levels of sL-selectin, sICAM-1, sICAM-3, and sVCAM-1 in HIV-1-infected patients at baseline were significantly higher than that in control (P ⬍ 0.01), while no significant differences were found in sE-selectin concentrations. Changes in levels of soluble forms of selectin in the 22 HIV-1-infected patients receiving HAART are shown in Fig. 1. The HAART regimen was associated with a significant decrease of sL-selectin over time. The median (IQR) sL-selectin levels decreased to 796 ng/ml (620 –920) at month 3, 701 ng/ml (615–787) at month 6, and to 720 ng/ml (620 – 812) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 1a). At the end of follow-up, only 4/22 (18%) patients had sL-selectin above the 90th percentile of healthy controls, which was 66 ng/ml. No significant changes in sE-selectin levels were observed during treatment (median, IQR: 45 ng/ml, 27– 60 at month 3; 43.5 ng/ml, 25– 82 at month 6, and 44.4 ng/ml, 37.9 –72 at month 9 (P ⬎ 0.05) (Fig. 1b). Levels of sICAM-1, sICAM-3, and sVCAM-1 prior to and after HAART are reported in Fig. 2. HAART did not have any significant effect on plasma sICAM-1 concentrations which persisted in being elevated in all patients over time (median, IQR: 452 ng/ml, 395–500, at month 3; 459 ng/ml, 301– 624, at month 6, and 472 ng/ml, 383–534, at month 9 (P ⬎ 0.05) (Fig. 2a). After the initiation of HAART regimens, the median (IQR) sICAM-3 levels decreased to 105 ng/ml (89 –128) at month 3, 102 ng/ml (83–116) at month 6, and 97 ng/ml (63–110) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 2a). The median (IQR) sVCAM-1 concentrations also diminished to 1087 ng/ml (920 – 1212) at month 3, 974 ng/ml (840 –1111) at month 6, and to 1016 ng/ml (774 –1069) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 2c). However, while HAART induced a reduction in plasma concentrations of both sCAM, the levels of sICAM-3 and
sVCAM-1 remained significantly elevated compared with healthy controls throughout the study (P ⬍ 0.001 for both) (Table 1). In particular, after 9 months of therapy, 16 (72.7%) and 12 (54.5%) of the 22 patients had concentrations of sICAM-3 and sVCAM-1 above the 90th percentile of healthy controls (66.5 ng/ml for sICAM-3 and 942 ng/ml for sVCAM-1).
FIG. 1. Plasma levels of sL-selectin (A) and sE-selectin (B) in HIV-1-infected patients (n ⫽ 22) before initiation of and during HAART and in 10 HIV-seronegative controls. Box plots show the 10th, 25th, 50th (median), 75th, and 90th percentiles and outlying values. Significant differences in sL-selectin concentrations between multiple time points by Friedman test (P ⬍ 0.01); P ⬍ 0.05 for months 3, 6, and 9 versus baseline (Dunnett’s test for pairwise comparison).
SOLUBLE CELL ADHESION MOLECULES DURING HAART
FIG. 2. Plasma levels of sICAM-1 (A), sICAM-3 (B), and sVCAM-1 (C) in HIV-1-infected patients (n ⫽ 22) before initiation of and during HAART and in 10 HIV-seronegative controls. Box plots show the 10th, 25th, 50th (median), 75th, and 90th percentiles and outlying values. Significant differences in sICAM-3 and sVCAM-1 concentrations between multiple time points by Friedman test (P ⬍ 0.01); P ⬍ 0.05 for months 3, 6, and 9 versus baseline (Dunnett’s test for pairwise comparison).
No correlation was found between CD4 ⫹ and CD8 ⫹ counts and all measured sCAM (data not shown). On the other hand, HIV-1 RNA levels correlated significantly with sL-selectin (r ⫽ 0.34; P ⬍ 0.01), sICAM-3 (r ⫽ 0.32; P ⬍ 0.01), and sVCAM-1 (r ⫽ 0.25; P ⬍ 0.05), but not with sICAM-1 (r ⫽ 0.10; P ⬎ 0.05). DISCUSSION
Increased levels of soluble isoforms of CAM have been reported in HIV-1 infection and may possibly
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contribute to altering the adhesion mechanisms of phagocytic cells, including defects in chemotaxis and diapedesis (9, 10, 14 –16). In the present study we have evaluated the effect of potent antiretroviral treatment, including one protease inhibitor and two reverse transcriptase inhibitors, on circulating levels of sCAM in HIV-1-infected patients with a CD4 ⫹ T-cell count/l below 500. Levels of sL-selectin were significantly elevated in HIV-1-infected patients before starting HAART compared with healthy controls, while no differences were found in sE-selectin concentrations. After the initiation of the HAART regimen, plasma sL-selectin concentrations progressively decreased to normal ranges in the majority of our patients, while no therapy-induced changes of sE-selectin were found. It has been shown that Lselectin is shed from neutrophils soon after activation induced by inflammatory stimuli and may represent a useful marker of neutrophil function (17). In AIDS patients, the abnormal production of sL-selectin may interfere with neutrophil function, impairing the ability of the cells to migrate in response to chemotactic stimuli. In this respect, several studies have shown that neutrophils from adults and children with HIV-1 infection exhibited decreased chemotactic activity, thereby increasing susceptibility to bacterial and fungal infections (18 –20). Recently, we have demonstrated that the restoration of normal chemotactic responses of neutrophils can be achieved in most patients with moderately advanced HIV-1 infection following the use of potent antiretroviral treatments containing protease inhibitors (21). The results of the present study indicate that the suppression of HIV-1 replication by HAART was associated with a reduced accumulation of sL-selectin in the circulation, a phenomenon that could improve the ability of neutrophils to respond to chemotactic stimuli. In agreement with our findings, Moore et al. have recently shown that abnormalities of neutrophil adhesion molecule expression may be reversed in HIV-1-infected patients who showed significant CD4 ⫹ T-cell responses following the use of protease inhibitor therapy (22). In the present study, we have also analyzed HAARTinduced changes in the levels of soluble forms of ICAM-1, ICAM-3, and VCAM-1. We found that both sICAM-1 and sVCAM-1 were significantly elevated in blood plasma of patients before therapy when compared with uninfected controls. In all patients sICAM-1 remained relatively constant at significantly elevated concentrations during the 9 months after the initiation of HAART. When changes of sVCAM-1 were assessed, the levels of this molecule significantly decreased, but they were still high in about 50% of the patients at the end of follow-up. A similar pattern was found for sICAM-3, which decreased to normal levels
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only in one-third of cases. Visser et al., in a longitudinal analysis of blood samples collected from 11 patients before and during 15 months of HAART, have documented an early decrease of sICAM-1 and sVCAM-1 only during the first 2 months after initiation of therapy, after which the levels remained elevated (23). In another study, Bucy et al. have shown that ICAM-1 and VCAM-1 were expressed in high concentrations in lymph node tissue before HAART and substantially declined after 10 weeks of therapy (24). In both these studies, the early decline of inflammatory adhesion molecules ICAM-1 and VCAM-1 on HAART may support the hypothesis that the initial rise in circulating CD4 ⫹ T-lymphocytes is likely due to redistribution of sequestered lymphocytes from lymphoid tissue to blood. Despite the differences between the single molecules, it is generally assumed that the presence of high levels of circulating forms of the Ig superfamily of adhesion molecules is related to the cell immune activation status occurring in HIV-1 infection. In our study, plasma concentrations of these sCAM were not normalized by HAART and remained significantly elevated throughout the study, thus indicating persistent immune activation despite control of plasma HIV-1 replication by therapy. The persistence of elevated levels of sICAM-1, sICAM-3, and sVCAM-1 on HAART could reflect continuous HIV-1-mediated immune activation in response to low peripheral viral replication that is below the detection limit of our assay. Several lines of evidence support the concept that immune activation in HIV-1 disease may arise as consequence of immune response to HIV-1 replication and it may implicated in the pathogenesis of T-cell immune dysfunction (25). Down-regulation of markers of immune activation has been reported in HIV-1-infected patients receiving HAART, especially in those with short-term suppression of HIV-1 replication (27, 28). On the other hand, Aukrust et al. recently found that plasma levels of soluble markers of immune activation, such as tumor necrosis factor components, were elevated after 52 weeks of HAART (29). This elevation was more relevant in patients who had virologic treatment failure at the end of follow-up and it was present even when HIV-1 RNA was undetectable in plasma. In the present study we provided evidence of virologic treatment failure (detectable plasma HIV-1 RNA) only in two of the patients who had concentrations of sICAM-1, sICAM-3, and sVCAM-1 above the 90th percentile of healthy controls after 9 months of HAART. Long-term studies are needed to assess whether the persistent elevation of sICAM-1, sICAM-3, and sVCAM-1, despite an adequate virologic response by HAART, may be predictive for the development of treatment failure.
ACKNOWLEDGMENTS This work was presented in part at The Sixth Conference on Retrovirus and Opportunistic Infections, January 31–February 4, 1999, Chicago, IL. REFERENCES 1. Shyur, S-D., and Hill, H. R., Polymorphonuclear leukocyte function in HIV. In “Immunology of HIV Infection” (S. Gupta, Ed.), pp. 377–386, Plenum, New York, 1996. 2. Wahl, S. M., and Orenstein, J. M., Macrophage functions in HIV-1 infection. In “Immunology of HIV Infection” (S. Gupta, Ed.), pp. 303–336, Plenum, New York, 1996. 3. Springer, T. A., Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76, 301–314, 1994. 4. Adams, D. A., and Shaw, S., Leucocyte-endothelial interactions and regulation of leucocyte migration. Lancet 343, 831– 836, 1994. 5. Imhof, B. A., and Dunon, D., Leukocyte migration and adhesion. Adv. Immunol. 58, 345– 416, 1995. 6. Go`rski, A., The role of cell adhesion molecules in immunopathology. Immunol. Today 15, 251–255, 1994. 7. Stent, G., Cameron, P. U., and Crowe, S. M., Expression of CD11/CD18 and ICAM-1 on monocytes and lymphocytes of HIV1-infected individuals. J. Leukocyte Biol. 56, 304 –309, 1994. 8. Tzavara, V., Vlachoyaiannopoulos, G., Kordossis, T., et al., Evidence for non-adaptive immune response in HIV infection. Eur. J. Clin. Invest. 27, 846 – 849, 1997. 9. Galea, P., Vermot-Desroches, C., Le Contel, C., Wijdenes, J., and Chermann, J-C., Circulating cell adhesion molecules in HIV-1infected patients as indicator markers for AIDS progression. Res. Immunol. 148, 109 –117, 1997. 10. Nordoy, I., Aukrust, P., Muller, F., and Froland, S. S., Abnormal levels of circulating adhesion molecules in HIV-1 infection with characteristic alterations in opportunistic infections. Clin. Immunol. Immunopathol. 81, 16 –21, 1997. 11. Moore, D. A. J., Henderson, D., and Gazzard, B. G., Neutrophil adhesion molecules in HIV diseases. Clin. Exp. Immunol. 114, 73–77, 1998. 12. Weeks, B. S., The role of HIV-1 activated leukocyte adhesion mechanisms and matrix metalloproteinase secretion in AIDS pathogenesis (review). Int. J. Mol. Med. 1, 361–366, 1998. 13. Powderly, W. G., Landay, A., and Lederman, M. M., Recovery of the immune system with antiretroviral therapy. The end of opportunism? J. Am. Med. Assoc. 280, 72–77, 1998. 14. Gearing, A. J. H., and Newman, W., Circulating adhesion molecules in disease. Immunol. Today 14, 506 –512, 1993. 15. Most, J., Zangerle, R., Herold, M., et al., Elevated concentrations of circulating intercellular adhesion molecule 1 (ICAM-1) in HIV-1 infection. J. Acquired Immune Defic. Syndr. 6, 221–226, 1993. 16. Diez-Ruiz, A., Kaiser, G., Jager, H., et al., Increased levels of serum intercellular adhesion molecules 1 in HIV infection are related to immune activation. Int. Arch. Allergy Immunol. 102, 52– 60, 1993. 17. Rosenberg, H. E., and Gallin, J. I., Inflammation. In “Fundamental Immunology” (W. E. Paul, Ed.), pp. 1051–1066, Lippincott– Raven, Philadelphia, 1999. 18. Ellis, M., Gupta, S., and Galant, S., et al., Impaired neutrophil function in patients with AIDS or AIDS-related complex: A comprehensive evaluation. J. Infect. Dis. 158, 1268 –1276, 1988.
SOLUBLE CELL ADHESION MOLECULES DURING HAART 19. Roilides, E., Mertins, S., Eddy, J., Walsh, T. J., Pizzo, P. A., and Rubin, M., Impairment of neutrophil chemotactic and bactericidal function in children infected with human immunodeficiency virus type 1 and partial reversal after in vitro exposure to granulocyte-macrophage colony-stimulating factor. J. Pediatr. 117, 531–540, 1990. 20. Flo, R. W., Naess, A., Nilsen, A., Harthug, S., and Solberg, C. O., A longitudinal study of phagocyte function in HIV-infected patients. AIDS 8, 771–777, 1994. 21. Mastroianni, C. M., Lichtner, M., Mengoni, F., et al., Improvement in neutrophil and monocyte function during highly active antiretroviral treatment of HIV-1-infected patients. AIDS 13, 883– 890, 1999. 22. Moore, D. A. J., Henderson, D., Gotch, F., and Gazzard, B. G., Reversal of abnormalities of neutrophil adhesion molecule expression in HIV infection following protease inhibitor therapy. AIDS 12, 2083–2084, 1998. 23. Visser, C. J. T., Cohen Stuart, J. W. T., De Jong, T. C. M., et al., A rapid decrease of sICAM-1 and sVCAM-1 may explain the redistribution of CD4⫹ T-cells in HIV patients early on HAART. Sixth Conference on Retrovirus and Opportunistic infections, January 31–February 4, 1999, Chicago, IL (Abstract 320). Received December 28, 1999; accepted with revision March 8, 2000
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24. Bucy, P. R., Hockett, R. D., Derdeyn, C. A., et al., Initial increase in blood CD4⫹ lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues. J. Clin. Invest. 103, 1391–1398, 1999. 25. Fauci, A. S., Pantaleo, G., Stanley, S., and Weissman, D., Immunopathogenetic mechanisms of HIV infection. Ann. Intern. Med. 124, 654 – 663, 1996. 26. Kelleher, A. D., Carr, A., Zaunders, J., and Cooper, D. A., Alterations in the immune response of human immunodeficiency virus (HIV)-infected subjects treated with an HIVspecific protease inhibitor, ritonavir. J. Infect. Dis. 173, 321– 329, 1996. 27. Lederman, M. M., Connick, E., Landay, A., et al., Immunologic responses associated with 12 weeks of combination antiretroviral therapy consisting of zidovudine, lamivudine, and ritonavir: Results of AIDS clinical trials group protocol 315. J. Infect. Dis. 178, 70 –79, 1998. 28. Aukrust, P., Muller, F., Lien, E., et al., Tumor necrosis factor (TNF) system levels in human immunodeficiency virus-infected patients during highly active antiretroviral therapy: Persistent TNF activation is associated with virologic and immunologic treatment failure. J. Infect. Dis. 179, 74 – 82, 1999.
Changes in Circulating Levels of Soluble Cell Adhesion Molecules Following Highly Active Antiretroviral Treatment of HIV-1-Infected Patients Claudio M. Mastroianni, Miriam Lichtner, Fabio Mengoni, Claudia D’Agostino, Gabriella d’Ettorre, Gabriele Forcina, Paola Santopadre, Anna P. Massetti, and Vincenzo Vullo Department of Infectious and Tropical Diseases, La Sapienza University, Rome, Italy
Increased levels of soluble cell adhesion molecules (sCAM) have been reported in HIV-1 infection and may possibly contribute to altering the adhesion mechanisms of phagocytic cells. We evaluated the effect of highly active antiretroviral therapy (HAART) on plasma levels of sL-selectin, sE-selectin, intercellular cell adhesion molecule-1 (sICAM-1), sICAM-3, and vascular cell adhesion molecule-1 (sVCAM-1). Study participants included 22 HIV-1-infected patients with a CD4 ⴙ T-cell count/l below 500 who were started on a HAART regimen and followed up for 9 months. After the initiation of therapy, plasma sL-selectin concentrations progressively decreased to normal ranges in the majority of our patients (P < 0.001), while no changes in sE-selectin were found. In all patients sICAM-1 remained relatively constant at significantly elevated concentrations during the 9 months of therapy. A significant reduction in plasma concentrations of both sICAM-3 and sVCAM-1 was found; however, the levels of these sCAM were not normalized by HAART and remained significantly elevated throughout the study (P < 0.001). The reduced release of sL-selectin could improve the ability of phagocitic cells to migrate in response to chemotactic stimuli after starting HAART. On the other hand, the persistent elevation of sICAM-1, sICAM-3, and sVCAM-1 could reflect continuous HIV-1-mediated immune activation, despite adequate control of plasma HIV-1 replication by therapy. © 2000 Academic Press
Key Words: adhesion molecules; innate immunity; anti-HIV-1 therapy.
INTRODUCTION
Neutrophils and monocytes are involved in the innate immune response during the progression of human immunodeficiency virus type 1 (HIV-1) infection. A normal function of polymorphonuclear leukocytes This work was supported by a grant from Ministero della Sanita`, ISS, Progetto AIDS 1997 (30A.0.74) and 1998 (30B.88), Rome, Italy. 1521-6616/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
(PMNL) is crucial for the clearance and killing of fungal and bacterial opportunistic pathogens that frequently affect AIDS patients (1). Monocytes also play an important role in HIV-1 infection as a reservoir for viral replication, mediators of local tissue damages, and as effector cells against intracellular microrganisms (2). To exert a variety of immunoregulatory and phagocyte functions, neutrophils and monocytes emigrate from the bloodstream and accumulate in body tissues. Leukocyte adherence to endothelial cells and transendothelial migration to inflammatory sites are mediated by the expression of various cell-surface proteins that are part of three families of cell adhesion molecules (CAM): selectins, integrins, and the immunoglobulin (Ig)-related superfamily (3–5). During the course of HIV-1 infection dysregulation of CAM synthesis as well as abnormal levels of soluble isoforms of CAM have been reported (6 –11). The alteration in leukocyte adhesion mechanisms may lead to dysfunction in phagocyte diapedesis, chemotaxis, and migration to the site of infection. In addition, up-regulation of CAM expression may facilitate extravasation of HIV-1-infected leukocytes into underlying tissues, contributing to disease spread and specific tissue damage (12). In recent years, dramatic immunological improvements have been shown in HIV-1-infected individuals who received highly active antiretroviral therapy (HAART), including HIV-1 protease and reverse transcriptase inhibitors (13). There is great interest in the quantitative and qualitative changes in host immune response following these potent therapies. In order to investigate the effect of HAART on leukocyte adhesion system, we have assessed changes in circulating levels of soluble forms of L-selectin (sL-selectin), sE-selectin, intercellular cell adhesion molecule-1 (sICAM-1), sICAM-3, and vascular cell adhesion molecule-1 (sVCAM-1) in a cohort of patients with moderately advanced HIV-1 infection undergoing treatment with a triple combination regimen (one protease inhibitor and two nucleoside analogues).
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SOLUBLE CELL ADHESION MOLECULES DURING HAART MATERIALS AND METHODS
Patients and Controls Study participants included 22 HIV-1-infected patients (13 males and 9 females) with a CD4 ⫹ T-cell count/l below 500 who were started on an HAART regimen containing a protease inhibitor and followed up for 9 months. The median age was 38.5 years (range, 21– 65). Patients with concomitant ongoing acute infection and prior use of protease inhibitors were excluded. HIV-1 seropositivity was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot analysis (Chiron Corp., Emeryville, CA). Risk factors for HIV-1 infection were injecting drug use in 3 cases, homosexuality in 7 cases, heterosexuality in 12 patients, and hemophilia in 1 case. Fifteen patients were naive for any antiretroviral drugs and 7 had previously been treated with reverse transcriptase inhibitors for more than 1 year. The HAART regimen consisted of two reverse transcriptase inhibitors and one protease inhibitor (indinavir in 18 patients, ritonavir in 3 patients, and nelfinavir in 1 case). Ten age- and sex-matched healthy blood donors were included as controls. A fully informed consent was obtained from all study participants. Sample Handling Venous blood samples were collected from each patient into EDTA-containing tubes (Becton–Dickinson Systems, San Jose, CA) at enrollment and at months 3, 6, and 9 of follow-up. For the enumeration of CD4 ⫹ and CD8 ⫹ lymphocyte numbers, venous blood was collected into 3-ml tubes and processed within 4 h of handling. For the assessment of HIV-1 RNA and sCAM levels, venous blood was collected into 15-ml tubes and processed within 2 h of collection. The cell-free plasma was stored at ⫺70°C until use. HIV-1 RNA Levels and CD4 ⫹ and CD8 ⫹ Lymphocyte Counts Plasma HIV-1-RNA levels were measured by using a quantitative reverse polymerase chain reaction (Amplicor HIV Monitor; Roche Diagnostic Systems, Branchburg, NJ). The limit of detection was 200 (2.30 log 10) copies/ml. Enumeration of CD4 ⫹ and CD8 ⫹ lymphocyte numbers was assessed using two-color flow cytometric analysis with the Becton–Dickinson flow cytometer FACScan. The whole blood lysis procedure and SimulTEST reagents (Becton–Dickinson) were used.
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Assays for Detection of Soluble Cell Adhesion Molecules (sCAM) Commercially available ELISA kits were used for the quantitative detection of plasma levels of sL-selectin, sE-selectin, sICAM-1, sICAM-3, and sVCAM-1 (Bender MedSystems, Boehringer Ingelheim Group, Vienna, Austria), according to the manufacturer’s instructions. The lower limit of sensitivity for assays was as follows: 0.3 ng/ml for sL-selectin; 0.5 ng/ml for sE-selectin; 3.3 ng/ml for sICAM-1; 0.58 ng/ml for sICAM-3; and 0.9 ng/ml for sVCAM-1. The concentrations of sCAM in the samples were calculated by interpolation from a standard curve constructed with specific sCAM standards. Statistical Analysis Results are presented as the median and interquartile range (IQR). Data at multiple time points in the treatment group were compared using the Friedman two-way analysis of variance by ranks with pairwise comparisons on treatment versus baseline values (Dunnett’s test). Differences between healthy controls and treated patients at baseline and at the end of study were evaluated by the nonparametric Mann–Whitney U test. The significance of correlation study parameters was estimated using the Spearman rank correlation test. RESULTS
Plasma HIV-1 RNA Measurements and Changes in CD4 ⫹ and CD8 ⫹ Lymphocytes The baseline median plasma viral load was 5.47 log 10 copies/ml (range, 2.62– 6.91). After initiation of HAART, HIV-1 load fell below the assay’s level of detection (2.30 log 10/ml) in 15/22 (68.1%) patients at 3 months (median decrease, 2.46 log 10/ml; range, 0.32– 4.0), in 17/22 (77.2%) at 6 months (median decrease, 2.5 log 10/ml; range, 0.23–5.45), and in 20/22 (90.9%) subjects at 9 months (median decrease, 2.83 log 10/ml; range, 0.24 –5.47). The median absolute CD4 ⫹ T-cell count increased from 117/l at baseline to 232/l at month 3, 306/l at month 6, and 369/l at month 9 (range, 4 to 644/l increase at month 9; P ⬍ 0.001). The absolute CD8 ⫹ T-cell count also rose from a median of 529/l at baseline to 765/l at month 3, 853/l at month 6, and 916/l at month 9 (range, 1125/l) decrease to 791/l increase at month 9; P ⬍ 0.001). Finally, there was also a significant increase in CD4:CD8 ratio from a median of 0.24 at baseline to 0.32 at month 6 and 0.36 at month 9 (P ⬍ 0.05).
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TABLE 1 Plasma Levels of sCAM in 10 HIV-Seronegative Controls and in 22 HIV-Infected Patients of a Treatment Group at Baseline (Tx 0) and after 9 Months of Therapy (Tx 9) sCAM (ng/ml)
Controls
Tx 0
Tx 9
sL-selectin sE-selectin sICAM-1 sICAM-3 sVCAM-1
518 (487–750)* 24.5 (21–52) 251 (173–293)* 52 (37–57)* 690 (601–790)*
942 (782–1157) 39.6 (22–52) 449 (392–557) 138 (107–176) 1116 (974–1391)
720 (620–812) 44.4 (37.9–72) 472 (383–548) 97 (63–110) 1016 (774–1069)
Note. Data are expressed as medians (interquartile range). * P ⬍ 0.001 versus Tx 0 and Tx 9 (Mann–Whitney U test).
Plasma Levels of sCAM Plasma concentrations of sCAM in the healthy controls and in the treatment group, at baseline and after 9 months of HAART, are reported in Table 1. The plasma levels of sL-selectin, sICAM-1, sICAM-3, and sVCAM-1 in HIV-1-infected patients at baseline were significantly higher than that in control (P ⬍ 0.01), while no significant differences were found in sE-selectin concentrations. Changes in levels of soluble forms of selectin in the 22 HIV-1-infected patients receiving HAART are shown in Fig. 1. The HAART regimen was associated with a significant decrease of sL-selectin over time. The median (IQR) sL-selectin levels decreased to 796 ng/ml (620 –920) at month 3, 701 ng/ml (615–787) at month 6, and to 720 ng/ml (620 – 812) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 1a). At the end of follow-up, only 4/22 (18%) patients had sL-selectin above the 90th percentile of healthy controls, which was 66 ng/ml. No significant changes in sE-selectin levels were observed during treatment (median, IQR: 45 ng/ml, 27– 60 at month 3; 43.5 ng/ml, 25– 82 at month 6, and 44.4 ng/ml, 37.9 –72 at month 9 (P ⬎ 0.05) (Fig. 1b). Levels of sICAM-1, sICAM-3, and sVCAM-1 prior to and after HAART are reported in Fig. 2. HAART did not have any significant effect on plasma sICAM-1 concentrations which persisted in being elevated in all patients over time (median, IQR: 452 ng/ml, 395–500, at month 3; 459 ng/ml, 301– 624, at month 6, and 472 ng/ml, 383–534, at month 9 (P ⬎ 0.05) (Fig. 2a). After the initiation of HAART regimens, the median (IQR) sICAM-3 levels decreased to 105 ng/ml (89 –128) at month 3, 102 ng/ml (83–116) at month 6, and 97 ng/ml (63–110) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 2a). The median (IQR) sVCAM-1 concentrations also diminished to 1087 ng/ml (920 – 1212) at month 3, 974 ng/ml (840 –1111) at month 6, and to 1016 ng/ml (774 –1069) at month 9 (P ⬍ 0.05 for months 3, 6, and 9 versus baseline) (Fig. 2c). However, while HAART induced a reduction in plasma concentrations of both sCAM, the levels of sICAM-3 and
sVCAM-1 remained significantly elevated compared with healthy controls throughout the study (P ⬍ 0.001 for both) (Table 1). In particular, after 9 months of therapy, 16 (72.7%) and 12 (54.5%) of the 22 patients had concentrations of sICAM-3 and sVCAM-1 above the 90th percentile of healthy controls (66.5 ng/ml for sICAM-3 and 942 ng/ml for sVCAM-1).
FIG. 1. Plasma levels of sL-selectin (A) and sE-selectin (B) in HIV-1-infected patients (n ⫽ 22) before initiation of and during HAART and in 10 HIV-seronegative controls. Box plots show the 10th, 25th, 50th (median), 75th, and 90th percentiles and outlying values. Significant differences in sL-selectin concentrations between multiple time points by Friedman test (P ⬍ 0.01); P ⬍ 0.05 for months 3, 6, and 9 versus baseline (Dunnett’s test for pairwise comparison).
SOLUBLE CELL ADHESION MOLECULES DURING HAART
FIG. 2. Plasma levels of sICAM-1 (A), sICAM-3 (B), and sVCAM-1 (C) in HIV-1-infected patients (n ⫽ 22) before initiation of and during HAART and in 10 HIV-seronegative controls. Box plots show the 10th, 25th, 50th (median), 75th, and 90th percentiles and outlying values. Significant differences in sICAM-3 and sVCAM-1 concentrations between multiple time points by Friedman test (P ⬍ 0.01); P ⬍ 0.05 for months 3, 6, and 9 versus baseline (Dunnett’s test for pairwise comparison).
No correlation was found between CD4 ⫹ and CD8 ⫹ counts and all measured sCAM (data not shown). On the other hand, HIV-1 RNA levels correlated significantly with sL-selectin (r ⫽ 0.34; P ⬍ 0.01), sICAM-3 (r ⫽ 0.32; P ⬍ 0.01), and sVCAM-1 (r ⫽ 0.25; P ⬍ 0.05), but not with sICAM-1 (r ⫽ 0.10; P ⬎ 0.05). DISCUSSION
Increased levels of soluble isoforms of CAM have been reported in HIV-1 infection and may possibly
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contribute to altering the adhesion mechanisms of phagocytic cells, including defects in chemotaxis and diapedesis (9, 10, 14 –16). In the present study we have evaluated the effect of potent antiretroviral treatment, including one protease inhibitor and two reverse transcriptase inhibitors, on circulating levels of sCAM in HIV-1-infected patients with a CD4 ⫹ T-cell count/l below 500. Levels of sL-selectin were significantly elevated in HIV-1-infected patients before starting HAART compared with healthy controls, while no differences were found in sE-selectin concentrations. After the initiation of the HAART regimen, plasma sL-selectin concentrations progressively decreased to normal ranges in the majority of our patients, while no therapy-induced changes of sE-selectin were found. It has been shown that Lselectin is shed from neutrophils soon after activation induced by inflammatory stimuli and may represent a useful marker of neutrophil function (17). In AIDS patients, the abnormal production of sL-selectin may interfere with neutrophil function, impairing the ability of the cells to migrate in response to chemotactic stimuli. In this respect, several studies have shown that neutrophils from adults and children with HIV-1 infection exhibited decreased chemotactic activity, thereby increasing susceptibility to bacterial and fungal infections (18 –20). Recently, we have demonstrated that the restoration of normal chemotactic responses of neutrophils can be achieved in most patients with moderately advanced HIV-1 infection following the use of potent antiretroviral treatments containing protease inhibitors (21). The results of the present study indicate that the suppression of HIV-1 replication by HAART was associated with a reduced accumulation of sL-selectin in the circulation, a phenomenon that could improve the ability of neutrophils to respond to chemotactic stimuli. In agreement with our findings, Moore et al. have recently shown that abnormalities of neutrophil adhesion molecule expression may be reversed in HIV-1-infected patients who showed significant CD4 ⫹ T-cell responses following the use of protease inhibitor therapy (22). In the present study, we have also analyzed HAARTinduced changes in the levels of soluble forms of ICAM-1, ICAM-3, and VCAM-1. We found that both sICAM-1 and sVCAM-1 were significantly elevated in blood plasma of patients before therapy when compared with uninfected controls. In all patients sICAM-1 remained relatively constant at significantly elevated concentrations during the 9 months after the initiation of HAART. When changes of sVCAM-1 were assessed, the levels of this molecule significantly decreased, but they were still high in about 50% of the patients at the end of follow-up. A similar pattern was found for sICAM-3, which decreased to normal levels
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only in one-third of cases. Visser et al., in a longitudinal analysis of blood samples collected from 11 patients before and during 15 months of HAART, have documented an early decrease of sICAM-1 and sVCAM-1 only during the first 2 months after initiation of therapy, after which the levels remained elevated (23). In another study, Bucy et al. have shown that ICAM-1 and VCAM-1 were expressed in high concentrations in lymph node tissue before HAART and substantially declined after 10 weeks of therapy (24). In both these studies, the early decline of inflammatory adhesion molecules ICAM-1 and VCAM-1 on HAART may support the hypothesis that the initial rise in circulating CD4 ⫹ T-lymphocytes is likely due to redistribution of sequestered lymphocytes from lymphoid tissue to blood. Despite the differences between the single molecules, it is generally assumed that the presence of high levels of circulating forms of the Ig superfamily of adhesion molecules is related to the cell immune activation status occurring in HIV-1 infection. In our study, plasma concentrations of these sCAM were not normalized by HAART and remained significantly elevated throughout the study, thus indicating persistent immune activation despite control of plasma HIV-1 replication by therapy. The persistence of elevated levels of sICAM-1, sICAM-3, and sVCAM-1 on HAART could reflect continuous HIV-1-mediated immune activation in response to low peripheral viral replication that is below the detection limit of our assay. Several lines of evidence support the concept that immune activation in HIV-1 disease may arise as consequence of immune response to HIV-1 replication and it may implicated in the pathogenesis of T-cell immune dysfunction (25). Down-regulation of markers of immune activation has been reported in HIV-1-infected patients receiving HAART, especially in those with short-term suppression of HIV-1 replication (27, 28). On the other hand, Aukrust et al. recently found that plasma levels of soluble markers of immune activation, such as tumor necrosis factor components, were elevated after 52 weeks of HAART (29). This elevation was more relevant in patients who had virologic treatment failure at the end of follow-up and it was present even when HIV-1 RNA was undetectable in plasma. In the present study we provided evidence of virologic treatment failure (detectable plasma HIV-1 RNA) only in two of the patients who had concentrations of sICAM-1, sICAM-3, and sVCAM-1 above the 90th percentile of healthy controls after 9 months of HAART. Long-term studies are needed to assess whether the persistent elevation of sICAM-1, sICAM-3, and sVCAM-1, despite an adequate virologic response by HAART, may be predictive for the development of treatment failure.
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