The failure of highly active antiretroviral therapy (HAART) is ascribed to the emergence of resistant HIV mutants. However, despite plasma RNA level rebound some patients show a persistent and significant increase in CD4 cell count and appear to be doing well clinically, suggesting that some benefits of HAART are not only due to its antiviral action . To investigate whether HAART has direct immunomodulatory effects, cytokine production was assessed in two HIV-uninfected subjects who received HAART prophylaxis.
After conjunctival exposure to HIV-1-contaminated blood, a nurse was treated for 4 weeks with zidovudine (ZDV) (300 mg twice a day) plus lamivudine (150 mg twice a day) plus indinavir (800 mg three times a day). A similar prophylactic regimen (ZDV 270 mg/m2 × 2 + lamivudine 4 mg/kg × 2 + indinavir 500 mg/m2 × 3) was given to a parenterally exposed seronegative child. Cytokine synthesis was evaluated on the last day of treatment and 2 months later in parallel with normal controls. Briefly, freshly isolated peripheral blood mononuclear cells were cultured for 4 h at 37°C with optimum stimulating concentrations of phorbol ester (10 ng/ml, Sigma, St Louis, MO, USA) plus calcium ionophore (500 ng/ml, Sigma). Cells were then incubated with Brefeldin A (10 m/ml, Sigma) for 2 h and fixed with 4% formaldehyde; a suspension of 2 × 105 cells in saponin buffer was incubated with 5 μl of phycoerythrin-mouse anti-human monoclonal antibody to IL-2, IL-4, IFN-γ, or TNF-α (PharMingen, San Diego, CA, USA) together with 5 μl of fluorescein-isothiocyanate CD4 monoclonal cell antibody (Becton–Dickinson, San Jose, CA, USA). After 30 min at room temperature, cell suspension was scored by FACScan.
As reported in Table 1, at the end of prophylaxis in both subjects, who did not acquire HIV, the percentage of peripheral blood mononuclear cells expressing TNF-α, IL-2 and IFN-γ was reduced when compared with simultaneously tested normal controls. The impaired cytokine synthesis mostly involved the CD4 cell subset and was no longer detectable 2 months after the suspension of prophylaxis. The production of IL-4 was less affected, although the low number of specifically reactive cells even in normal subjects does not allow any conclusion to be drawn for this cytokine. Total lymphocyte and CD4 cell counts did not change after HAART.
The clinical significance of the direct immunosuppressive effect of HAART remains to be established. In view of autoreactive immunopathology occurring in AIDS, however, it raises intriguing speculations on anti-HIV strategy. Cell activation is a mandatory step in HIV replication. Pro-inflammatory cytokines, such as TNF-α, and type 1 cytokines, such as IL-2 and IFN-γ, promote viral expression in latently infected, resting CD4 cells by increasing cell activation . Indeed only a minority of CD4 cells are infected with HIV, and their depletion is not explained by direct viral cytolysis, but rather by cytokine-driven effects, such as immune-mediated killing or the induction of apoptosis by TNF-α. In HIV-infected individuals there is an impressive activation of the immune system, and lymphoid tissues are characterized by extensive cytokine expression and a high degree of apoptosis when compared with control tissue . The initiation of HAART induces a rapid reduction of activation markers with downregulation of inflammatory and type 1 cytokines and programmed cell death [4,5], which precede the decrease of HIV expression in lympoid tissues . The immunomodulatory activity of HAART may thus contribute to correct the HIV-driven cytokine dysregulation and account for some clinical and immunological benefits of therapy even when failing virologically.
An approach to anti-HIV therapy, in which host cells instead of the virus are targeted through immunosuppressive drugs, has several supports. In-vitro cyclosporine or tacrolimus (FK 506) exhibit a potent antiretroviral action . Immunological and clinical improvements have been reported in HIV-1-infected patients treated with glucocorticoids , and hydroxyurea at lower doses than those used in cancer protocols is effective in combination with antiretroviral drugs . Of course, careful exploration of this strategy and optimal regimens are required to maximize the beneficial effects by minimizing cellular activation without unduly compromising the host's defence mechanisms. In the sole patient tested, the proliferative responses to mitogens and tetanus toxoid were intact despite the decreased cytokine production. This suggests that at concentrations at which vital housekeeping functions are not yet blocked, HAART can inhibit the persistent, uncontrolled immune activation with a downregulation of cytokine expression. In this context, the waning efficiency of chemotherapy could be due not only to the emergence of resistant viruses, but also to cellular factors. Long-term treatment with ZDV may induce decreased activity of cellular thymidine kinase . The induction of cellular efflux transporters, such as P-glycoprotein, can also interfere with the activity of nucleosides  and protease inhibitors . By developing drug resistance the cell will also recover its normal reactive functions, including intact cytokine production, resulting in a relapse of cell activation, immune-mediated damage, and apoptosis. The connected practical implication is that multi-drug resistance should be investigated not only for the virus, but also for the cell.
The increased production of TNF-α and type 1 cytokines is the hallmark of some transplantation reactions and autoimmune diseases. HAART is sufficiently well tolerated in most patients for prolonged periods, thus, in the future, it might be employed in autoimmune diseases, transplantation medicine, or other viral infections characterized by immune-mediated pathology.
The author would like to thank T. Bertolino and M. Ricotti for their technical support, and Drs M.G. Roncarolo and G. Basso for help with the analysis of the results.
1. Ledergerber B, Egger M, Opravil M, et al
. Clinical progression and virologic failure on highly active antiretroviral therapy in HIV-1 patients:a prospective cohort study.Swiss Cohort Study.Lancet
2. Chun TW, Engel D, Mizell SB, Ehler La, Fauci AS. Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med 1998, 188: 83–91.
3. Badley AD, Dockrell DH, Algeciras A. et al
. In vivo analysis of FAS/FasL interactions in HIV-infected patients. J Clin Invest 1998, 102: 79–87.
4. Tenner-Racs K, Stellbricnk H-J, van Lunzen J, et al
. The enlarged lymph nodes of HIV-1-infected, asymptomatic patients with high CD4 T-cell counts are sites for virus replication and cell proliferation.The impact of highly active antiretroviral therapy
. J Exp Med
5. Andersson J, Fehniger TE, Patterson BK. et al
. Early reduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS 1998, 30: F123–F129.
6. Borvak J, Chou C-S, Van Dyke G, Rosenwirth B, Vitetta ES, Ramilo O. The use of cyclosporine, FK506, and SDZ NIM811 to prevent CD25−
quiescent peripheral blood mononuclear cells from producing human immunodeficiency virus. J Infect Dis 1996, 174: 850–853.
7. Andrieux JM, Lu W, Levy R. Sustained increases in CD4 cell counts in asymptomatic human immunodeficiency virus type 1-seropositive patients treated with prendisone for 1 year. J Infect Dis 1995, 171: 523–530.
8. Rutschman OT, Opravil M, Iten A, et al.A placebo-controlled trial of didanosine plus stavudine, with and without hydroxyurea, for HIV infection.The Swiss HIV Cohort Study.AIDS
9. Groschel B, Cinatl J, Cinatl J. Viral and cellular factors for resistance against antiretroviral agents. Intervirology 1997, 40: 400–407.
10. Friedland A, Paibir S, Srinivas M. et al
. Involvement of an active efflux pump in the cellular resistance of antiretroviral nucleoside analogs. Antiviral Res 1998, 37: 40.40.
11. Lee C, Gottesman M, Cardellir C. et al
. HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 1998, 37: 3594–3601.