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How HIV may escape the activating effects of TNF
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60th F O R U M I N I M M U N O L O G Y
FINAL STATEMENT BY KAPLAN AND MOREIRA
Cytokines form an intricate regulatory network, w h e r e b y single c o m p o n e n t s o f the system exert multiple functions in the promotion or in the downm o d u l a t i o n o f the i m m u n e and i n f l a m m a t o r y response. Thalidomide has been shown to have no direct effect on the production of cytokines other than TNFtx, including ILl, IL6, I L l 0 or GM-CSF, all produced in response to the same in vitro stimuli. However, TNFt~ regulates the production of many other cytokines including ILl and IL6. Thus, it is expected that long-term downregulation of TNFct production would ultimately result in a reduction in serum levels o f o t h e r i n f l a m m a t o r y c y t o k i n e s .
Indeed, following one week therapy with thalidomide, s e r u m l e v e l s o f I L l w e r e also r e d u c e d , although to a lesser extent than TNFct, and monocytes which are stimulated ex-vivo release less ILl. This suggests that the impact of the drug on TNFo~ levels ultimately also reduces other inflammatory cytokines downstream in the cascade. In contrast, IFN T production is elevated in patient serum when TNFcx is reduced by thalidomide treatment. Thus, in our in vivo studies, a switch from TH 1 to TH2 cytokines f o l l o w i n g t h a l i d o m i d e treatment was not o b s e r v e d ; rather, i n f l a m m a t o r y c y t o k i n e s were reduced and protective cytokines were enhanced.
How HIV may escape the activating effects of TNF J.L. Virelizier Immunologie virale, Institut Pasteur, 75724 Paris C e d e x 15
Does HIV infection induce TNF secretion or TNF-mediated lesions ?
As early as 1989, it was realized that tumour necrosis factor-or (TNF) stimulation of human lymphoblastoid T-cell lines could induce nuclear transl o c a t i o n o f the t r a n s c r i p t i o n f a c t o r NF-kB and increase transcriptional activity of the HIV regulatory region (LTR) (Duh et al., 1989; Osborne et al., 1989; Israel et al., 1989). As one of the teams participating in this original demonstration, we, like others, were tempted to speculate that TNF secretion, induced by HIV infection itself and/or opportunistic infections, might increase HIV replication in the body, thus acting as a cofactor in the pathogenesis of AIDS. Since then, the literature has been flooded with publications aimed at documenting that T N F is i n d e e d e x c e s s i v e l y e x p r e s s e d in H I V infected individuals as well as in normal leukocytes infected in vitro with HIV. Others have aimed at finding correlations between TNF production in the body and immunopathological lesions in infected patients, in particular AIDS encephalopathy and cachexia, since TNF does induce cachexia in experi-
mental animals. This abundant literature, however, is not always entirely convincing and is somewhat contradictory. To quote a few recent examples, it is puzzling that cachexia was reported to correlate with increased serum TNF levels in patients from Ethiopia (Ayehunie et al., 1993) but not in children in the US (Ellaurie and Rubinstein, 1992). In vitro attempts to show a role for TNF in the toxic effects on n e u r o n c u l t u r e s o f s u p e r n a t a n t s f r o m H I V infected m o n o c y t e s found that the neuronotoxic activity was due to contamination of the HIV virus stock with mycoplasma (Bemton et al., 1992). Some authors reported that B lymphocytes analysed ex vivo from HIV-infected patients spontaneously produce TNF (Kehrl et al., 1992), whereas others do not find differences between patients and controls in TNF secretion from unstimulated or LPS-stimulated whole blood cultures (Jones et al., 1992). TNF production was not induced in normal, blood-derived m a c r o p h a g e s i n f e c t e d with H I V isolates f r o m patients at different stages of disease (Valentin et al., 1992). The situation is thus confused, both in vitro and in vivo, in this complex disease associating persistent viral infection, an early and progressive
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immunodeficiency state and opportunistic infections which may remain subclinical for a long time. Data f r o m e x p e r i m e n t a l m o d e l s do not s u p p o r t the hypothesis that TNF secretion is induced on a permanent basis by lentiviml infections. During the first six months after experimental feline immunodeficiency virus (FIV) infection, no differences in serum T N F values were observed between infected and non-infected cats (Lehmann et al., 1992). It could be argued, however, that even if HIV infection does not induce soluble forms of TNF, it might increase the membrane expression of this cytokine. It was indeed shown that a small proportion of cells from HIVinfected T-cell clones express membrane TNF and stimulate immunoglobulin synthesis by B lymphocytes through a contact-dependent mechanism (Macchia et al., 1993). The intriguing possibility that TNF participates in the dysregulation of T-cell functions (anergy and/or apoptosis) underlying the immunodeficiency of AIDS patients is a matter of speculation. While no decisive evidence supports a role, beneficial or deleterious, for TNF in the pathogenesis of AIDS, patients have been submitted to phase I/II trials of TNF therapy. It is comforting to learn that 16-week TNF administration did not hasten the progression of HIV infection, nor did it modify the markers o f HIV infection (Agosti et al., 1992). Resting CD4 lymphocytes as a TNF-R-negative niche for HIV latency CD4 lymphocytes are one of the main targets of HIV infection. Most of these lymphocytes, whether circulating or located in lymphoid organs, are in a non-proliferating, non-activated state in the body. To better understand the role of the normal T-cell environment in the transcriptional latency of HIV in these resting cells, we have transfected purified p r e p a r a t i o n s o f c i r c u l a t i n g l y m p h o c y t e s with reporter gene (luciferase) expression vectors under the control of HIV LTR (Alcami et al., in preparation). We found very little or no activity of the HIV LTR in these cells. In contrast, transfection of the same constructs into transformed lymphoblastoid Tcell lines resulted in a high background of lucifemse activity. Deletion o f the kB-responsive elements responsible for the response to the transcription factor NF-kB did not decrease the spontaneous activity o f the HIV LTR detected in transformed T cells. This indicated that, in lymphoblastoid T-cell lines, the basal transcriptional activity o f the HIV L T R was not due to the NF-kB-responsive HIV enhancer, but rather to spontaneous activity of the HIV promoter domain. In resting CD4 cells, however, neither the enhancer nor the promoter domain of the HIV genome was spontaneously active, which is r e m i n i s c e n t o f the latency o f HIV transcription o b s e r v e d in c i r c u l a t i n g l e u k o c y t e s f r o m H I V -
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infected patients. This was not due to an inability of the HIV enhancer region to respond to membrane stimuli, since phorbol ester (PMA) stimulation of resting CD4 lymphocytes resulted in both nuclear translocation of NF-~B and induction of kB-responsive element-dependent HIV-LTR activity (Alcami et al., in preparation). Addition of TNF to the culture medium of resting CD4 lymphocytes did not i n d u c e d e t e c t a b l e a c t i v a t i o n o f the H I V L T R (unpublished). This is easily understandable, since resting CD4 lymphocytes do not express detectable TNF receptors (TNF-R) (Scheurich et al., 1987). These results indicate that HIV proviruses integrated into the cell DNA of resting CD4 lymphocytes remain quiescent because the cell nucleus does not provide the transcription factors needed for the activity of either the enhancer or promoter domains of the HIV LTR. Furthermore, TNF molecules, even if they were transiently or permanently circulating in the blood, would not be able to reactivate the HIV genome from its natural latenty in this cellular environment, since resting CD4 lymphocytes lack TNF receptors. Transcriptional latency of HIV in normal lymphocytes totally escapes the activating effects of TNF.
TNF activates NK-kB, but not HIV LTR transcription in human CD4 IL2-independent T-cell clones T h e raison d ' e t r e of T lymphocytes is to respond to s p e c i f i c a n t i g e n r e c o g n i t i o n t h r o u g h the TCR/CD3 transmembrane receptor. Antigen recognition, however, cannot be tested in transient transfection assays of normal CD4 cells, because the proportion of circulating lymphocytes with a receptor for any given antigen is too low to permit such analysis. We thus transfected a human, IL2-dependent CD4 T-cell clone specific for tetanus toxoid with HIV-LTR-driven vectors. Antigen presentation by autologous macrophages to this T-cell clone resulted in a clear transactivation of the HIV LTR (Hazan et al., 1990). This T-cell clone, in contrast to resting CD4 l y m p h o c y t e s , expresses detectable TNF-R. Indeed, addition of recombinant TNF to the culture medium resulted in clear translocation of p50/p65 NF-kB heterodimers into the cell nucleus. This NF~B activation did not result in HIV LTR transactivation. In contrast, PMA induced both NF-kB activation and HIV L T R activity. This indicated that nuclear translocation of NF-kB p e r se is necessary, but not sufficient, to activate HIV genome expression. Moreover, it was found that stimulation of the clone with antibody to CD3 induced NF-kB in a TNF- and lymphotoxin (LT)-independent way, as shown by the inability of neutralizing antibodies to these two lymphokines to suppress NF-kB induction (Hazan et al., 1990).
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IL2
NF-kB
CD4
50 / n 65 TCR
CD45
HIV TRANSCRIPTION /
T cell activation Im RESTING CD4 LYMPHOCYTES N.__oomembrane expression of TNF Receptors
Antibody to TNF / LT
TNF___~/ LT_T ANTIGEN-ACTIVATED CD4 T CELL CLONE Specific antigen, but not TNF stimulation activates HIV transcription, although both induce nuclear translocation of NF-kB
Fig. 1. Inability of TNF to activate HIV transcription in normal, non-transformed CD4 T lymphocytes.
Altogether, as depicted in figure 1, the above results indicate that integrated HIV proviruses use T lymphocytes for their transcriptional latency. The resting CD4 lymphocyte environment offers a niche where latency is profound or absolute. IL2-activated T cells proliferate, but do not show NF-kB activation. The latter ceils can reactivate HIV transcription only when multiple transduction signals provided by antigen recognition, but not TNF stimulation alone, induce both NF-kB and other molecular events (possibly SP1 phosphorylation (Vlach et al., in preparation) which induce HIV L T R activity. This is in keeping with results showing that isolation of virus from patient leukocytes is optimally achieved by stimulation with phytoaemagglutinin (PHA), a lymphokine mimicking full T-cell activation induced by antigen recognition.
Chronic infection of m o n o c y t i c cell lines activates NF-kB and H I V L T R activity in a T N F - i n d e p e n dent m a n n e r
It could be argued that normal T cells are not the appropriate e n v i r o n m e n t for T N F - i n d u c e d HIV
genome activation, whereas cells of the myelomonocyte lineage, due to their permanent expression of TNF-R, represent the cell environment in which TNF exerts its activating effects on HIV transcription. Indeed, macrophages are CD4 antigen-bearing cells, and harbour HIV DNA in patients. A subclone of the human monocytic cell line U937 (the U 1 line) chronically but latently infected with HIV is inducible for HIV replication by TNF (Folks et al., 1988). We reported that chronic HIV replication itself induces HIV LTR activation through permanent NFkB translocation in the U937 monocytic cell line ( B a c h e l e r i e e t al., 1991). T h i s p h e n o m e n o n , observed in monocytic but not lymphoblastoid Tcell lines, suggests that HIV replication is self-perpetuated in monocytes. The mechanism of the phenomenon is not yet elucidated, but is underlaid by NF-kB-dependent induction of the p105 gene, which produces the precursor protein of this p50 subunit of NF-kB (Paya et al., 1992). We could eliminate a role for autocrine secretion of TNF (and also IL I) in this cell system, since we did not detect secretion of these cytokines in HIV-infected U937 cells, and TNF neutralizing antibody did not interfere with the ability of chronic HIV replication to induce NF-kB
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(Bachelerie et al., 1991). W h a t e v e r the intimate mechanism(s) in cause, it appears that the phenomenon is endogenous to each infected monocyte and is independent of TNF secretion. Our in vitro results obviously do not rule out a role for TNF in activating HIV replication o f normal m o n o c y t e / m a c r o phages in patients, but they do suggest that such a role does not need to be postulated if normal monocytes, like U937 cells, activate NF-kB and self-perpetuate HIV genome transcription in a TNF-independent manner, upon induction by HIV itself.
Conclusion No definitive conclusion should be drawn from the data discussed above, since most of the evidence published is from in vitro work, and since in vivo data remain controversial. However, we have discussed evidence from our own work suggesting that the "appealing" concept of TNF as a cofactor in the pathogenesis of AIDS, through its ability to induce NF-kB and HIV replication, is not supported by molecular analysis of HIV genome activation in its two main, and possibly sole, target cells in the body, namely CD4 T lymphocytes and macrophages. The HIV transcriptional strategy appears to involve the use of normal lymphocytes for latency, an ideal manner to escape humoral and cell-mediated immunity, and also permanent, self-perpetuated, TNF-independent replication in macrophages, another manner for HIV to escape immune effector mechanisms if this replication occurs (as is well documented) in microglial cells of the brain, a well-known niche secluded from intervention by antibodies and T lymphocytes.
References Agosti, J.M., Coombs, R.W., Collier, A.C., Paradise, M.A., Jaffe, H.S. & Corey, L. (1992), A randomized, double-blind blind phase 1/1I trial of tumor necrosis factor and interferon gamma for treatment of AIDSrelated complex. AIDS Res. Hum. Retroviruses, 8, 581-587. Ayehunie, S., Sonnerborg, A., Yemane-Berhan, T., Zewdie, D.W., Britton, S. & Strannegard, O. (1993), Raised levels of tumor necrosis factor-alpha and neopterin, but not interferon-alpha, in serum of HIV1-infected patients from Ethiopa. Clin. Exp. lmmunol., 91, 37-42. Bachelerie, F., Alcami, J., Arenzana-Seisdedos, F., Hazan, U. & Virelizier, J.L. (1991), HIV enhancer activity perpetuated by NF-kb induction on infection of monocytes. Nature (Lond.), 350, 709-712. Bernton, E.W., Bryant, H.U., Decoster, M.A., Orenstein, J.M., Ribas, J.L., Meltzer, M.S. & Gendelman, H.E. (1992), No direct neuronotoxicity by HIV-1 virions or culture fluids from HIV-I infected T cells or monocytes. AIDS Res. Hum. Retroviruses, 8, 495-5031.
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Duh, E.J., Maury, W.J., Folks, T.M., Fauci, A.S. & Rabson, A.B. (1989), Tumor necrosis factor-~ activates human immunodeficiency virus through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA, 86, 5974-5978. Ellaurie, M. & Rubinstein, A. (1992), Tumor necrosis factor-alpha in pediatric HIV-I infection. AIDS 6, 11 1265-1268. Folks, T.M., Justement, J., Kinter, A., Schnittman, S.M., Oreinstein, J., Poli, G. & Fauci, A.S. (1988), Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol -12myristate acetate. J. Immunol., 140, 1117-1122. Hazan, U., Thomas, D., Alcami, J., Bachelerie, F., IsraSl, N., Yssel, H., Virelizer, J.L. & Arenzana-Seisdedos, F. (1990), Stimulation of a human T cell clone with anti-CD3 or tumor necrosis factor induces NF-kB translocation but not human immunodeficiency virus1 enhancer-dependent transcription. Proc. Natl. Acad. Sci. USA, 87, 7861-7865. IsraSl, N., Hazan, U., Alcami, J., Munnier, A., ArenzanaSeisdedos, F., Bachelerie, F., Israel, A. & Virelizier, J.L. (1989), Tumor necrosis factor stimulates transcription of HIV-I in human T lymphocytes, independently and synergistically with mitogens. J. Immunol., 143, 3956-3960. Jones, P.D., Shelley, L. & Wakefield, D. (1992), Tumor necrosis factor-alpha in advanced HIV infection in the absence of AIDS-related secondary infections. J. Acquir. Immune Defic. Syndr., 5, 1266-1271. Kehrl, J.H., Rieckmann, P., Koslow, E. & Fauci, A.S. (1992), Lymphokine production by B cells from normal and HIV-infected individuals. Ann. N. Y. Acad. Sci., 651,220-227. Lehmann, R., Joller, H., Jaagmans, B.L. & Lutz, H. (1992), Tumor necrosis factor alpha levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection. Vet. lmmunol. Immunopathol., 35, 61-91. Macchia, D., Amerigogna, F., Parronchi, P., Ravina, A., Maggi, E. & Romagnani, S. (1993), Membrane tumor necrosis factor-alpha is involved in the polyclonal B-cell activation induced by HIV-infected human T cells. Nature (Lond.), 363, 464-466. Osborne, L., Kunkel, S. & Nabel, G.J. (1989), Tumor necrosis factor and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear kB. Proc. Natl. Acad. Sci. USA, 86, 23362340. Paya, C.V., Ten, R.M., Bessia, C., Alcami, J., Hay, R.T. & Virelizier, J.L. (1992), NF-kB-dependent induction of the NF-kB p50 subunit underlies self-perpetuation of human immunodeficiency virus transcription in monocyte cells. Proc. Natl. Acad. Sci. USA, 89, 7826-7830. Scheurich, P.B., Thoma, B., Ucer, U. & Pfizenmaier (1987), Immunoregulatory activity of recombinant tumor necrosis factor (TNF)-0t: induction of TNF receptors on human T cells and TNF-mediated enhancement of T cell responses. J. Immunol., 138, 1786-1800. Valentin, A., Albert, J., Svenson, S.B. & Asjo, B. (1992), Blood-derived macrophages produce IL-1, but not TNF-alpha, after infection with HIV-1 isolates from patients at different stages of disease. Cytokine, 4, 185-191.
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SPECIFIC COMMENTARYON VIRELIZIER
By Poli, Kinter, Vicenzi and Fauci
By Kaplan and Moreira
Virelizier offers a critical reading of the literature concerning the role of TNF as an inducer of HIV replication in vitro and as a potential determinant of in vivo pathogenesis, as a consequence of its activating effect on the virus LTR. We fully agree with Virelizer's view that the above mentioned concepts have been likely too oversimplified by an abundant literature, and that the existing gaps are perhaps more important than the few acquired notions. However, a few remarks should be mentioned in this regard. First, NF-kB, the universally recognized mediator of TNF effect on virus replication, has rapidly evolved from being a heterodimeric transcription factor maintained in an inactivated from by the bound I-kB molecule to an extremely complex and still rapidly growing family of multiple related transcription factors. This fact per se suggests that some of the discrepancies described in the recent past by Virelizier's group and others may be the result of molecular interactions unknown at the time of the original studies. Second, we fully agree that the "TNF/NF-kB/HIV" connection has been taken too dogmatically as the modality through which cytokines in general and/or other cell activators induce HIV expression. For example, we have described how ILl and IL6, alone or in synergistic combination, can activate HIV production in U1 cells without activating TNFo~ secretion and/or NF-kB activation (see Poll et al., this Forum). Third, although the experiments conducted in resting T lymphocytes are certainly indicative of one possible modality of HIV to "evade" TNF-mediated virus production, it should be noted that the majority of infected T cells are present in the lymphoid organs, and not in the peripheral blood compartment (PBC), of HIV-infected individuals, and that a sizeable fraction of these cells appear to be activated, as judged by the constitutive expression of class II MHC molecules (see Graziosi et al., Forum). Thus, TNF as well as other factors leading to T-cell activation may act in the tissues and may not necessarily be detected in the PBC. Finally, as discussed by several authors in this issue, although autocrine/paracrine TNFo~-driven HIV expression can be demonstrated in several models of acute and chronic infection of both primary and immortalized T lymphocytic and monocytic cells, we fully agree that TNF-independent pathways of HIV expression certainly exist and should be an important focus of future research.
Dr Virelizier poses the question of how HIV might escape the effects of TNF, and then proceeds to challenge the importance of TNF in the regulation of HIV infection. One of the u n d e r l y i n g p r o b l e m s not addressed by this review is the difficulty many investigators have had in measuring TNF~ production in vivo and in vitro. This situation has resulted in extensive confusion concerning the importance of TNF(~ in HIV1 infection and other diseases. Indeed, activation of virus in vivo may not be exclusively dependent on TNF(z. However, evidence does exist for a role of this cytokine in HIV1 activation and disease progression. The author states that he would eliminate a role of autocrine secretion of the cytokine in the monocytic cell system infected with HIV, since he did not detect secretion of TNFo~ in U937 cells, and TNFo~ neutralizing antibodies did not interfere with chronic HIV replication. However TNF~ mRNA can be shown to be chronically produced in cell lines and primary cells infected with HIV (Makonkawkeyoon et aL (1993), Proc. Natl. Acad. ScL (USA) 90, 5974; and A. Moreira, manuscript in preparation). Also, it is possible that an endogenous TNFo~ regulatory pathway is utilized and TNFo~ does not need to be secreted in order to activate HIV1 replication. The author is aware of the possibility that there are differences in the regulatory pathway of HIV1 replication in T cells and monocytes, as well as differences in the regulation of TNFo~ gene expression in primary cells and cell lines (Griffin et al. (1989), Nature, 339, 70). Thus, the review seems a bit too negative as evidenced by the selection of publications which is clearly aimed at negating a role for TNFe~. For every paper referred to in this review, there are many other publications that provide evidence to the contrary.
By Torbett and Mosier: How important is TNF in the pathogenesis of AIDS? It is still difficult to know, and this review correctly points out the confusion in the literature. I am not sure that anything (good or bad) can be concluded from the 25-patient study of Agosti et al., since low doses of TNF (10 lig/m 2) were administered at the same time as IFNI,, and the patients were followed for only 16 weeks. A
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negative effect of TNF could have been masked by a positive benefit of IFN?. The cited paper of Macchia et al. may be the most important contribution to this literature, particularly if polyclonal activation of B cells contributes to HIV pathogenesis, as might be the case if VH3 immunoglobulins target virus to macrophages, for example (Berberian et al., 1993).
References Agosti, J.M., Coombs, R.W., Collier, A.C., Paradise, M.A., Benedetti, J.K., Jaffe, H.S. & Corey, L. (1992), A randomized, double-blind, phase 1/11trial of tumor necrosis factor and interferon gamma for treatment of AIDS-related complex. AIDS Res. Hum. Retroviruses, 8, 581-587. Berberian, L., Goodglick, L., Kipps, T. & Braun, J. (1993), Immunoglobulin VH3 gene products: natural ligands for HIV gp120. Science, 261, 15881591. Macchia, D., Almerigogna, F., Parronchi, P., Ravina, A., Maggi, E. & Romagnani, S. (1993), Membrane tumour necrosis factor-alpha is involved in the polyclonal B-cell activation induced by HIVinfected human T cells. Nature, 363, 464-466.
By A. Lau: This paper reviews some of the controversies surrounding the role of TNFo~ in HIV infection. The arguments are well stipulated by the author. However, we wish to point out some underlying reasons that may explain these controversies. Studying cytokine levels in body fluids or tissues can be confusing. Different investigators used different assay techniques including ELISA, radioimmunoassays, bioassays with different indicator cell lines. Northern analysis for mRNA, or RT-PCR with or without semi-quantification. In addition, the patients studied were far from being homogenous with variations in sampling time at different stages of HIV infection, opportunistic infections, or associated cancers. The kind of primary cells or tissues, or tumour cell lines also used added another layer of confusion. Moreover, the biological system can be more complicated, for instance, simultaneous induction of soluble TNF receptors (sTNF-R) in vivo would neutralize the biological activity of TNF(~ and yet the TNF~ = sTNF-R complex may still be detectable by ELISA. With regard to cytokine mRNA, detection of messages does not necessarily mean that there w i l l be s u b s e q u e n t translation and release of the cytokine. TNF(~, a prototype of proinflammatory cytokines, is cons t i t u t i v e l y transcribed at low levels; but its mRNA are rapidly degraded due to the presence
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of UA sequence in its 3' untranslated end of the message. With respect to the role of NF-kB in activating HIV-LTR, it remains possible that other transcription factors are involved. For instance, it has been postulated that oncogenes, jun/fos, may be involved in the mediation of HIV activation in addition to NF-kB.
By A. Vyakarnam: This paper deals with the question of the potential importance of the cytokine TNF in the pathogenesis of AIDS and concludes that the cytokine is unlikely to play an important role due to the author's data s h o w i n g the failure of recombinant TNF to activate the HIV1 LTR in Tcell clones or in macrophage cell lines. Furthermore, the author sites the example of cellular data showing a correlation between excess TNF production and disease to be equivocal due to contradictory observations, some showing this cytokine to be elevated in HIV infection whilst some others have shown the opposite. The rationale for believing that TNF may be important in the pathogenesis of AIDS is based on three sets of observations: (i) in vitro cellular data showing that HIV replication is enhanced or induced in PBMC (T cells and macrophages) as well as various cell lines cultured with recombinant TNF, an observation that is not reviewed in this manuscript (see Poli et al., this Forum); (ii) data showing excess TNF production in HIV by some groups; (iii) data showing that the HIV1 LTR can be directly activated by recombinant TNF (see Poli et aL, this Forum). In this context, studies of HIV replication in cell lines can only prove to be of limited value as tumour cell lines which are self-perpetuating cannot accurately represent the differentiation stage of a primary cell, e.g. resting T cells or macrophages. A more representative model would be the use of PBMC infected in vitro or derived from HIV patients. An extension would be to carry out ex vivo experiments on tissuederived cells from HIV patients. There are not many studies of this nature, but a few do show TNF to be a potent inducer of HIV replication in PBMC infected in v i t r o (see Poli et al., this Forum). In this context, the author's observations that TNF alone failed to activate the HIV1LTR in a T-cell clone despite inducing the translocation of NF-kB are interesting and suggest
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that the effects of TNF on HIV in T cells may be indirect. However, in the absence of parallel experiments on the effects of TNF directly on virus production in such clones, it is difficult to establish the implications of the HIV1-LTR-CAT studies. The contradictory observation of excess TNF production in HIV have to be viewed in the context of many observations in the past showing that the failure to detect this cytokine in chronic diseases is associated with high levels of soluble TNF receptors (also observed in HIV: Godfried et al. (1993) AIDS, 7, 93) to which the cytokine becomes bound, thereby escaping detection. Secondly, recent o b s e r v a t i o n s of p o l y m o r phisms in the TNF gene governing TNF protein levels (Wilson et aL (1994) Br. J. Rheumatol., 33, 89) might have important implications in HIV (see for recent discussion in malaria: McGuire et
al., 1994). Variation in the TNFo~ promoter is associated w i t h s u s c e p t i b i l i t y to cerebral malaria. Nature (in press). In summary, experiments to decide the relative importance of any cytokine in the pathogenesis of a given disease is a complex process and could be facilitated by appropriate animal models in which a given gene is deleted or in transgenic animals which overproduce a cytokine in tissues. In the case of HIV, data from in vitro experimental systems which are likely to represent the complex in in vivo situations consisting of defined cell populations mixed together and natural HIV isolates are still awaited. In the absence of such data, results of clinical trials with TNF inhibitors in HIV could serve as important markers. On the whole, therefore, the role of TNF in HIV pathogenesis is still a question mark.
Does preferential Th subset activation contribute to the murine acquired immunodeficiency disease (MAIDS)? B.E. T o r b e t t and D.E. M o s i e r
Department of lmmunology-IMM7, The Scripps Research Institute, La Jolla, CA 92037 (USA)
Introduction Is has recently been proposed that imbalances between the CD4 T h l and Th2 subsets and the unique cytokines they produce are important in the pathogenesis of AIDS and also in a related murine retrovirus-induced disease named MAIDS (Clerici and Shearer, 1993, Gazzinelli et al., 1992). We review here the relevance of the MAIDS model for studying HIV pathogenesis, and the data supporting the importance o f selective loss of T h l responses and increases in Th2 cytokines in allowing disease progression. We conclude that the cytokine changes seen by most groups in MAIDS are not compelling evidence for the hypothesized Thl to Th2 shift, and that MAIDS has sufficient differences from AIDS to necessitate caution in extrapolating results from this mouse disease to HIV infection of humans. Since the changes of cytokines in HIV infection are discussed elsewhere in this Forum, we will focus on the role of Thl and Th2 cells in MAIDS.
Characteristics o f both MAIDS and AIDS are summarized in table I. One of the arguments cited in support of a T h l to Th2 shift in HIV infection is the importance of a similar shift in MAIDS. Even if one were convinced that an imbalance of T h l and Th2 cells is important in the pathogenesis of MAIDS (a conclusion we will challenge), on would also have to argue that MAIDS is a relevant animal model for AIDS. As table I illustrates, there are some superficial similarities (e.g., hypergammaglobulinaemia, Tcell based immunodeficiency) but many more differences, both in the disease process and the viruses involved. MAIDS is a disease where B-cell proliferation is critical and is directly linked to virus infection ( H u a n g et al., 1989, 1991, 1992). B cells become unresponsive to stimulation with agents like lipopolysaccharide (Mosier et al., 1985), and B-cell lymphomas harbouring integrated provirus appear late in disease (Jolictxur, 1991 ; Morse et al., 1992). The critical open question in MAIDS is why CD4 and CD8 T cells exposed to virus-infected B cells
60th F O R U M I N I M M U N O L O G Y
FINAL STATEMENT BY KAPLAN AND MOREIRA
Cytokines form an intricate regulatory network, w h e r e b y single c o m p o n e n t s o f the system exert multiple functions in the promotion or in the downm o d u l a t i o n o f the i m m u n e and i n f l a m m a t o r y response. Thalidomide has been shown to have no direct effect on the production of cytokines other than TNFtx, including ILl, IL6, I L l 0 or GM-CSF, all produced in response to the same in vitro stimuli. However, TNFt~ regulates the production of many other cytokines including ILl and IL6. Thus, it is expected that long-term downregulation of TNFct production would ultimately result in a reduction in serum levels o f o t h e r i n f l a m m a t o r y c y t o k i n e s .
Indeed, following one week therapy with thalidomide, s e r u m l e v e l s o f I L l w e r e also r e d u c e d , although to a lesser extent than TNFct, and monocytes which are stimulated ex-vivo release less ILl. This suggests that the impact of the drug on TNFo~ levels ultimately also reduces other inflammatory cytokines downstream in the cascade. In contrast, IFN T production is elevated in patient serum when TNFcx is reduced by thalidomide treatment. Thus, in our in vivo studies, a switch from TH 1 to TH2 cytokines f o l l o w i n g t h a l i d o m i d e treatment was not o b s e r v e d ; rather, i n f l a m m a t o r y c y t o k i n e s were reduced and protective cytokines were enhanced.
How HIV may escape the activating effects of TNF J.L. Virelizier Immunologie virale, Institut Pasteur, 75724 Paris C e d e x 15
Does HIV infection induce TNF secretion or TNF-mediated lesions ?
As early as 1989, it was realized that tumour necrosis factor-or (TNF) stimulation of human lymphoblastoid T-cell lines could induce nuclear transl o c a t i o n o f the t r a n s c r i p t i o n f a c t o r NF-kB and increase transcriptional activity of the HIV regulatory region (LTR) (Duh et al., 1989; Osborne et al., 1989; Israel et al., 1989). As one of the teams participating in this original demonstration, we, like others, were tempted to speculate that TNF secretion, induced by HIV infection itself and/or opportunistic infections, might increase HIV replication in the body, thus acting as a cofactor in the pathogenesis of AIDS. Since then, the literature has been flooded with publications aimed at documenting that T N F is i n d e e d e x c e s s i v e l y e x p r e s s e d in H I V infected individuals as well as in normal leukocytes infected in vitro with HIV. Others have aimed at finding correlations between TNF production in the body and immunopathological lesions in infected patients, in particular AIDS encephalopathy and cachexia, since TNF does induce cachexia in experi-
mental animals. This abundant literature, however, is not always entirely convincing and is somewhat contradictory. To quote a few recent examples, it is puzzling that cachexia was reported to correlate with increased serum TNF levels in patients from Ethiopia (Ayehunie et al., 1993) but not in children in the US (Ellaurie and Rubinstein, 1992). In vitro attempts to show a role for TNF in the toxic effects on n e u r o n c u l t u r e s o f s u p e r n a t a n t s f r o m H I V infected m o n o c y t e s found that the neuronotoxic activity was due to contamination of the HIV virus stock with mycoplasma (Bemton et al., 1992). Some authors reported that B lymphocytes analysed ex vivo from HIV-infected patients spontaneously produce TNF (Kehrl et al., 1992), whereas others do not find differences between patients and controls in TNF secretion from unstimulated or LPS-stimulated whole blood cultures (Jones et al., 1992). TNF production was not induced in normal, blood-derived m a c r o p h a g e s i n f e c t e d with H I V isolates f r o m patients at different stages of disease (Valentin et al., 1992). The situation is thus confused, both in vitro and in vivo, in this complex disease associating persistent viral infection, an early and progressive
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immunodeficiency state and opportunistic infections which may remain subclinical for a long time. Data f r o m e x p e r i m e n t a l m o d e l s do not s u p p o r t the hypothesis that TNF secretion is induced on a permanent basis by lentiviml infections. During the first six months after experimental feline immunodeficiency virus (FIV) infection, no differences in serum T N F values were observed between infected and non-infected cats (Lehmann et al., 1992). It could be argued, however, that even if HIV infection does not induce soluble forms of TNF, it might increase the membrane expression of this cytokine. It was indeed shown that a small proportion of cells from HIVinfected T-cell clones express membrane TNF and stimulate immunoglobulin synthesis by B lymphocytes through a contact-dependent mechanism (Macchia et al., 1993). The intriguing possibility that TNF participates in the dysregulation of T-cell functions (anergy and/or apoptosis) underlying the immunodeficiency of AIDS patients is a matter of speculation. While no decisive evidence supports a role, beneficial or deleterious, for TNF in the pathogenesis of AIDS, patients have been submitted to phase I/II trials of TNF therapy. It is comforting to learn that 16-week TNF administration did not hasten the progression of HIV infection, nor did it modify the markers o f HIV infection (Agosti et al., 1992). Resting CD4 lymphocytes as a TNF-R-negative niche for HIV latency CD4 lymphocytes are one of the main targets of HIV infection. Most of these lymphocytes, whether circulating or located in lymphoid organs, are in a non-proliferating, non-activated state in the body. To better understand the role of the normal T-cell environment in the transcriptional latency of HIV in these resting cells, we have transfected purified p r e p a r a t i o n s o f c i r c u l a t i n g l y m p h o c y t e s with reporter gene (luciferase) expression vectors under the control of HIV LTR (Alcami et al., in preparation). We found very little or no activity of the HIV LTR in these cells. In contrast, transfection of the same constructs into transformed lymphoblastoid Tcell lines resulted in a high background of lucifemse activity. Deletion o f the kB-responsive elements responsible for the response to the transcription factor NF-kB did not decrease the spontaneous activity o f the HIV LTR detected in transformed T cells. This indicated that, in lymphoblastoid T-cell lines, the basal transcriptional activity o f the HIV L T R was not due to the NF-kB-responsive HIV enhancer, but rather to spontaneous activity of the HIV promoter domain. In resting CD4 cells, however, neither the enhancer nor the promoter domain of the HIV genome was spontaneously active, which is r e m i n i s c e n t o f the latency o f HIV transcription o b s e r v e d in c i r c u l a t i n g l e u k o c y t e s f r o m H I V -
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infected patients. This was not due to an inability of the HIV enhancer region to respond to membrane stimuli, since phorbol ester (PMA) stimulation of resting CD4 lymphocytes resulted in both nuclear translocation of NF-~B and induction of kB-responsive element-dependent HIV-LTR activity (Alcami et al., in preparation). Addition of TNF to the culture medium of resting CD4 lymphocytes did not i n d u c e d e t e c t a b l e a c t i v a t i o n o f the H I V L T R (unpublished). This is easily understandable, since resting CD4 lymphocytes do not express detectable TNF receptors (TNF-R) (Scheurich et al., 1987). These results indicate that HIV proviruses integrated into the cell DNA of resting CD4 lymphocytes remain quiescent because the cell nucleus does not provide the transcription factors needed for the activity of either the enhancer or promoter domains of the HIV LTR. Furthermore, TNF molecules, even if they were transiently or permanently circulating in the blood, would not be able to reactivate the HIV genome from its natural latenty in this cellular environment, since resting CD4 lymphocytes lack TNF receptors. Transcriptional latency of HIV in normal lymphocytes totally escapes the activating effects of TNF.
TNF activates NK-kB, but not HIV LTR transcription in human CD4 IL2-independent T-cell clones T h e raison d ' e t r e of T lymphocytes is to respond to s p e c i f i c a n t i g e n r e c o g n i t i o n t h r o u g h the TCR/CD3 transmembrane receptor. Antigen recognition, however, cannot be tested in transient transfection assays of normal CD4 cells, because the proportion of circulating lymphocytes with a receptor for any given antigen is too low to permit such analysis. We thus transfected a human, IL2-dependent CD4 T-cell clone specific for tetanus toxoid with HIV-LTR-driven vectors. Antigen presentation by autologous macrophages to this T-cell clone resulted in a clear transactivation of the HIV LTR (Hazan et al., 1990). This T-cell clone, in contrast to resting CD4 l y m p h o c y t e s , expresses detectable TNF-R. Indeed, addition of recombinant TNF to the culture medium resulted in clear translocation of p50/p65 NF-kB heterodimers into the cell nucleus. This NF~B activation did not result in HIV LTR transactivation. In contrast, PMA induced both NF-kB activation and HIV L T R activity. This indicated that nuclear translocation of NF-kB p e r se is necessary, but not sufficient, to activate HIV genome expression. Moreover, it was found that stimulation of the clone with antibody to CD3 induced NF-kB in a TNF- and lymphotoxin (LT)-independent way, as shown by the inability of neutralizing antibodies to these two lymphokines to suppress NF-kB induction (Hazan et al., 1990).
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IL2
NF-kB
CD4
50 / n 65 TCR
CD45
HIV TRANSCRIPTION /
T cell activation Im RESTING CD4 LYMPHOCYTES N.__oomembrane expression of TNF Receptors
Antibody to TNF / LT
TNF___~/ LT_T ANTIGEN-ACTIVATED CD4 T CELL CLONE Specific antigen, but not TNF stimulation activates HIV transcription, although both induce nuclear translocation of NF-kB
Fig. 1. Inability of TNF to activate HIV transcription in normal, non-transformed CD4 T lymphocytes.
Altogether, as depicted in figure 1, the above results indicate that integrated HIV proviruses use T lymphocytes for their transcriptional latency. The resting CD4 lymphocyte environment offers a niche where latency is profound or absolute. IL2-activated T cells proliferate, but do not show NF-kB activation. The latter ceils can reactivate HIV transcription only when multiple transduction signals provided by antigen recognition, but not TNF stimulation alone, induce both NF-kB and other molecular events (possibly SP1 phosphorylation (Vlach et al., in preparation) which induce HIV L T R activity. This is in keeping with results showing that isolation of virus from patient leukocytes is optimally achieved by stimulation with phytoaemagglutinin (PHA), a lymphokine mimicking full T-cell activation induced by antigen recognition.
Chronic infection of m o n o c y t i c cell lines activates NF-kB and H I V L T R activity in a T N F - i n d e p e n dent m a n n e r
It could be argued that normal T cells are not the appropriate e n v i r o n m e n t for T N F - i n d u c e d HIV
genome activation, whereas cells of the myelomonocyte lineage, due to their permanent expression of TNF-R, represent the cell environment in which TNF exerts its activating effects on HIV transcription. Indeed, macrophages are CD4 antigen-bearing cells, and harbour HIV DNA in patients. A subclone of the human monocytic cell line U937 (the U 1 line) chronically but latently infected with HIV is inducible for HIV replication by TNF (Folks et al., 1988). We reported that chronic HIV replication itself induces HIV LTR activation through permanent NFkB translocation in the U937 monocytic cell line ( B a c h e l e r i e e t al., 1991). T h i s p h e n o m e n o n , observed in monocytic but not lymphoblastoid Tcell lines, suggests that HIV replication is self-perpetuated in monocytes. The mechanism of the phenomenon is not yet elucidated, but is underlaid by NF-kB-dependent induction of the p105 gene, which produces the precursor protein of this p50 subunit of NF-kB (Paya et al., 1992). We could eliminate a role for autocrine secretion of TNF (and also IL I) in this cell system, since we did not detect secretion of these cytokines in HIV-infected U937 cells, and TNF neutralizing antibody did not interfere with the ability of chronic HIV replication to induce NF-kB
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(Bachelerie et al., 1991). W h a t e v e r the intimate mechanism(s) in cause, it appears that the phenomenon is endogenous to each infected monocyte and is independent of TNF secretion. Our in vitro results obviously do not rule out a role for TNF in activating HIV replication o f normal m o n o c y t e / m a c r o phages in patients, but they do suggest that such a role does not need to be postulated if normal monocytes, like U937 cells, activate NF-kB and self-perpetuate HIV genome transcription in a TNF-independent manner, upon induction by HIV itself.
Conclusion No definitive conclusion should be drawn from the data discussed above, since most of the evidence published is from in vitro work, and since in vivo data remain controversial. However, we have discussed evidence from our own work suggesting that the "appealing" concept of TNF as a cofactor in the pathogenesis of AIDS, through its ability to induce NF-kB and HIV replication, is not supported by molecular analysis of HIV genome activation in its two main, and possibly sole, target cells in the body, namely CD4 T lymphocytes and macrophages. The HIV transcriptional strategy appears to involve the use of normal lymphocytes for latency, an ideal manner to escape humoral and cell-mediated immunity, and also permanent, self-perpetuated, TNF-independent replication in macrophages, another manner for HIV to escape immune effector mechanisms if this replication occurs (as is well documented) in microglial cells of the brain, a well-known niche secluded from intervention by antibodies and T lymphocytes.
References Agosti, J.M., Coombs, R.W., Collier, A.C., Paradise, M.A., Jaffe, H.S. & Corey, L. (1992), A randomized, double-blind blind phase 1/1I trial of tumor necrosis factor and interferon gamma for treatment of AIDSrelated complex. AIDS Res. Hum. Retroviruses, 8, 581-587. Ayehunie, S., Sonnerborg, A., Yemane-Berhan, T., Zewdie, D.W., Britton, S. & Strannegard, O. (1993), Raised levels of tumor necrosis factor-alpha and neopterin, but not interferon-alpha, in serum of HIV1-infected patients from Ethiopa. Clin. Exp. lmmunol., 91, 37-42. Bachelerie, F., Alcami, J., Arenzana-Seisdedos, F., Hazan, U. & Virelizier, J.L. (1991), HIV enhancer activity perpetuated by NF-kb induction on infection of monocytes. Nature (Lond.), 350, 709-712. Bernton, E.W., Bryant, H.U., Decoster, M.A., Orenstein, J.M., Ribas, J.L., Meltzer, M.S. & Gendelman, H.E. (1992), No direct neuronotoxicity by HIV-1 virions or culture fluids from HIV-I infected T cells or monocytes. AIDS Res. Hum. Retroviruses, 8, 495-5031.
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Duh, E.J., Maury, W.J., Folks, T.M., Fauci, A.S. & Rabson, A.B. (1989), Tumor necrosis factor-~ activates human immunodeficiency virus through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat. Proc. Natl. Acad. Sci. USA, 86, 5974-5978. Ellaurie, M. & Rubinstein, A. (1992), Tumor necrosis factor-alpha in pediatric HIV-I infection. AIDS 6, 11 1265-1268. Folks, T.M., Justement, J., Kinter, A., Schnittman, S.M., Oreinstein, J., Poli, G. & Fauci, A.S. (1988), Characterization of a promonocyte clone chronically infected with HIV and inducible by 13-phorbol -12myristate acetate. J. Immunol., 140, 1117-1122. Hazan, U., Thomas, D., Alcami, J., Bachelerie, F., IsraSl, N., Yssel, H., Virelizer, J.L. & Arenzana-Seisdedos, F. (1990), Stimulation of a human T cell clone with anti-CD3 or tumor necrosis factor induces NF-kB translocation but not human immunodeficiency virus1 enhancer-dependent transcription. Proc. Natl. Acad. Sci. USA, 87, 7861-7865. IsraSl, N., Hazan, U., Alcami, J., Munnier, A., ArenzanaSeisdedos, F., Bachelerie, F., Israel, A. & Virelizier, J.L. (1989), Tumor necrosis factor stimulates transcription of HIV-I in human T lymphocytes, independently and synergistically with mitogens. J. Immunol., 143, 3956-3960. Jones, P.D., Shelley, L. & Wakefield, D. (1992), Tumor necrosis factor-alpha in advanced HIV infection in the absence of AIDS-related secondary infections. J. Acquir. Immune Defic. Syndr., 5, 1266-1271. Kehrl, J.H., Rieckmann, P., Koslow, E. & Fauci, A.S. (1992), Lymphokine production by B cells from normal and HIV-infected individuals. Ann. N. Y. Acad. Sci., 651,220-227. Lehmann, R., Joller, H., Jaagmans, B.L. & Lutz, H. (1992), Tumor necrosis factor alpha levels in cats experimentally infected with feline immunodeficiency virus: effects of immunization and feline leukemia virus infection. Vet. lmmunol. Immunopathol., 35, 61-91. Macchia, D., Amerigogna, F., Parronchi, P., Ravina, A., Maggi, E. & Romagnani, S. (1993), Membrane tumor necrosis factor-alpha is involved in the polyclonal B-cell activation induced by HIV-infected human T cells. Nature (Lond.), 363, 464-466. Osborne, L., Kunkel, S. & Nabel, G.J. (1989), Tumor necrosis factor and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear kB. Proc. Natl. Acad. Sci. USA, 86, 23362340. Paya, C.V., Ten, R.M., Bessia, C., Alcami, J., Hay, R.T. & Virelizier, J.L. (1992), NF-kB-dependent induction of the NF-kB p50 subunit underlies self-perpetuation of human immunodeficiency virus transcription in monocyte cells. Proc. Natl. Acad. Sci. USA, 89, 7826-7830. Scheurich, P.B., Thoma, B., Ucer, U. & Pfizenmaier (1987), Immunoregulatory activity of recombinant tumor necrosis factor (TNF)-0t: induction of TNF receptors on human T cells and TNF-mediated enhancement of T cell responses. J. Immunol., 138, 1786-1800. Valentin, A., Albert, J., Svenson, S.B. & Asjo, B. (1992), Blood-derived macrophages produce IL-1, but not TNF-alpha, after infection with HIV-1 isolates from patients at different stages of disease. Cytokine, 4, 185-191.
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SPECIFIC COMMENTARYON VIRELIZIER
By Poli, Kinter, Vicenzi and Fauci
By Kaplan and Moreira
Virelizier offers a critical reading of the literature concerning the role of TNF as an inducer of HIV replication in vitro and as a potential determinant of in vivo pathogenesis, as a consequence of its activating effect on the virus LTR. We fully agree with Virelizer's view that the above mentioned concepts have been likely too oversimplified by an abundant literature, and that the existing gaps are perhaps more important than the few acquired notions. However, a few remarks should be mentioned in this regard. First, NF-kB, the universally recognized mediator of TNF effect on virus replication, has rapidly evolved from being a heterodimeric transcription factor maintained in an inactivated from by the bound I-kB molecule to an extremely complex and still rapidly growing family of multiple related transcription factors. This fact per se suggests that some of the discrepancies described in the recent past by Virelizier's group and others may be the result of molecular interactions unknown at the time of the original studies. Second, we fully agree that the "TNF/NF-kB/HIV" connection has been taken too dogmatically as the modality through which cytokines in general and/or other cell activators induce HIV expression. For example, we have described how ILl and IL6, alone or in synergistic combination, can activate HIV production in U1 cells without activating TNFo~ secretion and/or NF-kB activation (see Poll et al., this Forum). Third, although the experiments conducted in resting T lymphocytes are certainly indicative of one possible modality of HIV to "evade" TNF-mediated virus production, it should be noted that the majority of infected T cells are present in the lymphoid organs, and not in the peripheral blood compartment (PBC), of HIV-infected individuals, and that a sizeable fraction of these cells appear to be activated, as judged by the constitutive expression of class II MHC molecules (see Graziosi et al., Forum). Thus, TNF as well as other factors leading to T-cell activation may act in the tissues and may not necessarily be detected in the PBC. Finally, as discussed by several authors in this issue, although autocrine/paracrine TNFo~-driven HIV expression can be demonstrated in several models of acute and chronic infection of both primary and immortalized T lymphocytic and monocytic cells, we fully agree that TNF-independent pathways of HIV expression certainly exist and should be an important focus of future research.
Dr Virelizier poses the question of how HIV might escape the effects of TNF, and then proceeds to challenge the importance of TNF in the regulation of HIV infection. One of the u n d e r l y i n g p r o b l e m s not addressed by this review is the difficulty many investigators have had in measuring TNF~ production in vivo and in vitro. This situation has resulted in extensive confusion concerning the importance of TNF(~ in HIV1 infection and other diseases. Indeed, activation of virus in vivo may not be exclusively dependent on TNF(z. However, evidence does exist for a role of this cytokine in HIV1 activation and disease progression. The author states that he would eliminate a role of autocrine secretion of the cytokine in the monocytic cell system infected with HIV, since he did not detect secretion of TNFo~ in U937 cells, and TNFo~ neutralizing antibodies did not interfere with chronic HIV replication. However TNF~ mRNA can be shown to be chronically produced in cell lines and primary cells infected with HIV (Makonkawkeyoon et aL (1993), Proc. Natl. Acad. ScL (USA) 90, 5974; and A. Moreira, manuscript in preparation). Also, it is possible that an endogenous TNFo~ regulatory pathway is utilized and TNFo~ does not need to be secreted in order to activate HIV1 replication. The author is aware of the possibility that there are differences in the regulatory pathway of HIV1 replication in T cells and monocytes, as well as differences in the regulation of TNFo~ gene expression in primary cells and cell lines (Griffin et al. (1989), Nature, 339, 70). Thus, the review seems a bit too negative as evidenced by the selection of publications which is clearly aimed at negating a role for TNFe~. For every paper referred to in this review, there are many other publications that provide evidence to the contrary.
By Torbett and Mosier: How important is TNF in the pathogenesis of AIDS? It is still difficult to know, and this review correctly points out the confusion in the literature. I am not sure that anything (good or bad) can be concluded from the 25-patient study of Agosti et al., since low doses of TNF (10 lig/m 2) were administered at the same time as IFNI,, and the patients were followed for only 16 weeks. A
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negative effect of TNF could have been masked by a positive benefit of IFN?. The cited paper of Macchia et al. may be the most important contribution to this literature, particularly if polyclonal activation of B cells contributes to HIV pathogenesis, as might be the case if VH3 immunoglobulins target virus to macrophages, for example (Berberian et al., 1993).
References Agosti, J.M., Coombs, R.W., Collier, A.C., Paradise, M.A., Benedetti, J.K., Jaffe, H.S. & Corey, L. (1992), A randomized, double-blind, phase 1/11trial of tumor necrosis factor and interferon gamma for treatment of AIDS-related complex. AIDS Res. Hum. Retroviruses, 8, 581-587. Berberian, L., Goodglick, L., Kipps, T. & Braun, J. (1993), Immunoglobulin VH3 gene products: natural ligands for HIV gp120. Science, 261, 15881591. Macchia, D., Almerigogna, F., Parronchi, P., Ravina, A., Maggi, E. & Romagnani, S. (1993), Membrane tumour necrosis factor-alpha is involved in the polyclonal B-cell activation induced by HIVinfected human T cells. Nature, 363, 464-466.
By A. Lau: This paper reviews some of the controversies surrounding the role of TNFo~ in HIV infection. The arguments are well stipulated by the author. However, we wish to point out some underlying reasons that may explain these controversies. Studying cytokine levels in body fluids or tissues can be confusing. Different investigators used different assay techniques including ELISA, radioimmunoassays, bioassays with different indicator cell lines. Northern analysis for mRNA, or RT-PCR with or without semi-quantification. In addition, the patients studied were far from being homogenous with variations in sampling time at different stages of HIV infection, opportunistic infections, or associated cancers. The kind of primary cells or tissues, or tumour cell lines also used added another layer of confusion. Moreover, the biological system can be more complicated, for instance, simultaneous induction of soluble TNF receptors (sTNF-R) in vivo would neutralize the biological activity of TNF(~ and yet the TNF~ = sTNF-R complex may still be detectable by ELISA. With regard to cytokine mRNA, detection of messages does not necessarily mean that there w i l l be s u b s e q u e n t translation and release of the cytokine. TNF(~, a prototype of proinflammatory cytokines, is cons t i t u t i v e l y transcribed at low levels; but its mRNA are rapidly degraded due to the presence
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of UA sequence in its 3' untranslated end of the message. With respect to the role of NF-kB in activating HIV-LTR, it remains possible that other transcription factors are involved. For instance, it has been postulated that oncogenes, jun/fos, may be involved in the mediation of HIV activation in addition to NF-kB.
By A. Vyakarnam: This paper deals with the question of the potential importance of the cytokine TNF in the pathogenesis of AIDS and concludes that the cytokine is unlikely to play an important role due to the author's data s h o w i n g the failure of recombinant TNF to activate the HIV1 LTR in Tcell clones or in macrophage cell lines. Furthermore, the author sites the example of cellular data showing a correlation between excess TNF production and disease to be equivocal due to contradictory observations, some showing this cytokine to be elevated in HIV infection whilst some others have shown the opposite. The rationale for believing that TNF may be important in the pathogenesis of AIDS is based on three sets of observations: (i) in vitro cellular data showing that HIV replication is enhanced or induced in PBMC (T cells and macrophages) as well as various cell lines cultured with recombinant TNF, an observation that is not reviewed in this manuscript (see Poli et al., this Forum); (ii) data showing excess TNF production in HIV by some groups; (iii) data showing that the HIV1 LTR can be directly activated by recombinant TNF (see Poli et aL, this Forum). In this context, studies of HIV replication in cell lines can only prove to be of limited value as tumour cell lines which are self-perpetuating cannot accurately represent the differentiation stage of a primary cell, e.g. resting T cells or macrophages. A more representative model would be the use of PBMC infected in vitro or derived from HIV patients. An extension would be to carry out ex vivo experiments on tissuederived cells from HIV patients. There are not many studies of this nature, but a few do show TNF to be a potent inducer of HIV replication in PBMC infected in v i t r o (see Poli et al., this Forum). In this context, the author's observations that TNF alone failed to activate the HIV1LTR in a T-cell clone despite inducing the translocation of NF-kB are interesting and suggest
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that the effects of TNF on HIV in T cells may be indirect. However, in the absence of parallel experiments on the effects of TNF directly on virus production in such clones, it is difficult to establish the implications of the HIV1-LTR-CAT studies. The contradictory observation of excess TNF production in HIV have to be viewed in the context of many observations in the past showing that the failure to detect this cytokine in chronic diseases is associated with high levels of soluble TNF receptors (also observed in HIV: Godfried et al. (1993) AIDS, 7, 93) to which the cytokine becomes bound, thereby escaping detection. Secondly, recent o b s e r v a t i o n s of p o l y m o r phisms in the TNF gene governing TNF protein levels (Wilson et aL (1994) Br. J. Rheumatol., 33, 89) might have important implications in HIV (see for recent discussion in malaria: McGuire et
al., 1994). Variation in the TNFo~ promoter is associated w i t h s u s c e p t i b i l i t y to cerebral malaria. Nature (in press). In summary, experiments to decide the relative importance of any cytokine in the pathogenesis of a given disease is a complex process and could be facilitated by appropriate animal models in which a given gene is deleted or in transgenic animals which overproduce a cytokine in tissues. In the case of HIV, data from in vitro experimental systems which are likely to represent the complex in in vivo situations consisting of defined cell populations mixed together and natural HIV isolates are still awaited. In the absence of such data, results of clinical trials with TNF inhibitors in HIV could serve as important markers. On the whole, therefore, the role of TNF in HIV pathogenesis is still a question mark.
Does preferential Th subset activation contribute to the murine acquired immunodeficiency disease (MAIDS)? B.E. T o r b e t t and D.E. M o s i e r
Department of lmmunology-IMM7, The Scripps Research Institute, La Jolla, CA 92037 (USA)
Introduction Is has recently been proposed that imbalances between the CD4 T h l and Th2 subsets and the unique cytokines they produce are important in the pathogenesis of AIDS and also in a related murine retrovirus-induced disease named MAIDS (Clerici and Shearer, 1993, Gazzinelli et al., 1992). We review here the relevance of the MAIDS model for studying HIV pathogenesis, and the data supporting the importance o f selective loss of T h l responses and increases in Th2 cytokines in allowing disease progression. We conclude that the cytokine changes seen by most groups in MAIDS are not compelling evidence for the hypothesized Thl to Th2 shift, and that MAIDS has sufficient differences from AIDS to necessitate caution in extrapolating results from this mouse disease to HIV infection of humans. Since the changes of cytokines in HIV infection are discussed elsewhere in this Forum, we will focus on the role of Thl and Th2 cells in MAIDS.
Characteristics o f both MAIDS and AIDS are summarized in table I. One of the arguments cited in support of a T h l to Th2 shift in HIV infection is the importance of a similar shift in MAIDS. Even if one were convinced that an imbalance of T h l and Th2 cells is important in the pathogenesis of MAIDS (a conclusion we will challenge), on would also have to argue that MAIDS is a relevant animal model for AIDS. As table I illustrates, there are some superficial similarities (e.g., hypergammaglobulinaemia, Tcell based immunodeficiency) but many more differences, both in the disease process and the viruses involved. MAIDS is a disease where B-cell proliferation is critical and is directly linked to virus infection ( H u a n g et al., 1989, 1991, 1992). B cells become unresponsive to stimulation with agents like lipopolysaccharide (Mosier et al., 1985), and B-cell lymphomas harbouring integrated provirus appear late in disease (Jolictxur, 1991 ; Morse et al., 1992). The critical open question in MAIDS is why CD4 and CD8 T cells exposed to virus-infected B cells