406
8th F O R U M IN M I C R O B I O L O G Y
Cytokines, lymphokines, mycobacteria and AIDS G . A . W . R o o k IO and A. V y a k a r n a m is) ft; Department o f Medical Microbiology, University College and Middlesex School o f Medicine. 67-73 Riding House St., London W1P 7LD, and t2) Department o f Immunology, University College and Middlesex School o f Medicine, Arthur Stanley House. Tottenham St., London 14/1
Introduction
Comoined infection with tuberculosis and HIV has become a devastating problem, the consequences of which will be as serimls as those of the Black Death in many populations. In parts of Africa, as many as 90 o/0 of new tuberculosis eases are HIV-seropositive. There are three urgent problems to be addressed. 1) Why is suseepfibility to M. tuberculosis a very early consequence of HIV infection? 2) Why is suseeptibillty to M. avium seen only later in the syndrome? 3) Does established infectio~l with tuberculosis accelerate progression towards AIDS?
Immunity to myeobacteria in man
M. tuberculosis intelligent discussion of question (1) is inhibited by the fact that we do not understand the mechanism of intmunity to mycobacteria in man. The different effect of HIV on infection with M. tuberculosis and M. avium is nice evidence for the complexity of this question. Work on murine maerophages has been somewhat misleading. These cells are easily activated by IFN T (and to a lesser extent by several other cytokines) to inhibit M. tuberculosis (Rook et e ! . 1986a). This effect correlates with production of nitric ,oxide (NO) (J. Chan and B. Bloom, submitted, and personal communication) and can be blocked by the specific inhibitor of arginine deiminase (NC'-monomethyl-L-arginine; NMMA). Whatever the explanation for this correlation between antimyeobacterial activity and NO in marine cells (which is not necessarily due to a direct antimicrobial effect of the NO itself), it does not at present help us to understand the situation in man because human macrophages are unable to make tetrahydrobiopterin, which is an essential cofactor for this pathway. This is because the enzyme 6-pyruvol
tetrahydrobiopteria synthase is present at very low levels in human macrophages (Werner et aL, 1989). Lack of this enzyme may explain the failure of IFN'f to inhibit growth of M. tuberculosis in human cells (Rook et aL, 1986a). In fact, two groups have noted increased growth of M. tuberculosis in IFNy-exoosed macrophages (Rook et al., 1986a; Douvas et aL, 1985). A further important difference between murine and human macrophages is their use of vitamin D3 metabolites. Exposure to IFN~' causes human (but not murine) macrophages to express increased levels of a 1-hydroxylase which derives calcitriol from circulating 25(OH) vitamin D3 (Rook, 1988). This pathway may be o f crucial relevance in the H1V/tuberculosis interaction, and it is considered in that context later. The potential importance of this pathway in protection from tuberculosis lies in the autocrine effect of the caleitriol on the mac~ ophage itself. We observed that some inhibition of virulent M. tuberculosis is seen when human macrophages are exposed to both IFNT and ealcitriol (Rook et al., 1986b), and there is one unconfirmed report that effective killing of M. tuberculosis is seen if a combination of IFN,¢, caleitriol and TNF is used (Denis, 1991a). If this can be confirmed, it will be of great interest to know whether ealcitriol can upregulate 6-pyruvol tetrahydrobiopterin synthase in human macrophages. However, it remains possible that protection from M. tuberculosis in man is not only a nlatter of appropriate macrophage activation, but instead involves killing of infected cells, perhaps by eytotoxic cells. This event may be accompanied by unidentified microbicidal effects (Kumararame et at., 1990). M. avium As implied by the different consequences of HIV infection, different mechanisms may be able to control growth of M. avium in human maerophages. There are claims that exposure to TNF (Bermudez
MYCOBA CTERIA AND AIDS and Young, 1988), GM-CSF (Bermudez et al., 1990), IFN~' or calcitriol (Denis, 1991b) alone can reduce growth o f this organism, which ~,s not the case for M. tuberculosis. However I L l , M-CSF or 1L3 were all found to increase growth of M. avium (Denis, 1991b). Thus, for both mycobacteria, the blend of eytokines may be critical.
407
this dichotomy does exist in man. There is a report that the early proliferative defect associated with I-IIV infection of helper T-cen clones leaves antigen. specific recognition and IL4 secretion intact (Laurence et al., 1989). 3) T-cell dysfunction
lmmunopathology in tuberculosis There is considerable evidence that much of the tissue damage in tuberculosis is due to release of T N F , in sites rendered exquisitely sensitive to this cytokine (Rook et aL, 1991). This has implications for the interaction with HIV, discussed below.
Increased susceptibility to mycobacterioses Although the pathway of immunity to mycobacteria in m a n is not elucidated, it is clear that the early increase in susceptibility to tuberculosis could be secondary to effects o f H I V on: 1) A ntigen presentation It is known that both macrophages and dendritic cells are susceptible to infection with HIV, and this can decrease their A P C function (Stingl et aL, 1990; Macatonia et oL, 1989). It is not clear that this dysfunction would be selectively permissive for growth o f M. tuberculosis rather than M. ovium, and this point needs to be clarified. 2) T-cell subsets The nature of the cytokines to which macrophages are exposed is clearly critical to the control of both M. tuberculosis and M. avium, as outlined above. Our own pilot experiments, and a much more extensive study by others (Haanau et al., 1991) indicate that normal BCG recipients, or donors who have successfully overcome subclinical tuberculosis in the past, secrete 1FN~- hut not detectable IL4 in response to myeobacterial antigens. Thus, it is probable that immunity to tuberculosis is associated with T H 1 helper cells, and these seem to be preferentially activated by mycobacterial antigens. Theoretically, therefore, activation of the disease could result if H1V infection were to result in a switch to a T H 2 response, and it may be significant that some patients with uncontrolled tubeJculosis can have eosinophilia and raised IgE levels, indicative o f IL4 release (Yong et al., 1989). Unfortunately there is little information on the effect o f early HIV infection on the T H I / T H 2 dichotomy, though there is no longer any doubt that
While it is not clear t hat H1V alters the T H I / T H 2 ratio, there is no doubt that it subtly upsets T-cell function, even before there is striking depletion of CD4 + T-cell numbers. For instance, H I V infection of T cells renders them less responsive to activation through the CD3 activation pathway (Pinching and Nye, 1990; Miedema et aL, 1990). These effects may be partly attributable to the excessive cytokine release described below. Moreover, H I V proliferates preferentially in m e m o r y T cells (Miedema el al. 1990; Vyakarnam et al., in press) and may therefore deplete T-cell memory of mycobaeterial antigens earlier than is apparent from peripheral CD4 + counts. 4) Direct triggering by H I V o f inappropriate cytokine release Recombinant g p l 2 0 has been shown to induce secretion of TNF, IL 1, IL6 and G M - C S F by monocytes (Clouse et al., 1991), and secretion of I ' N F by B cells (Rieckmann et al., 19~Cl). g p l 2 0 will also induce IFN~" production in CD4 ÷ T cells (Vyakarnam and Matear, unpublished). Since IFN~ used in the absence of the correct blend of other cytokines can enhance proliferation of M. tuberculosis (Rook et al., 1986a; Douvas et al., 1985), gpl20 could be directly responsible for upsetting the host/mycobacterium relationship. 5) Changed macrophage microbicidal function, and response to cytokines There are few studies of this aspect of HIVinfected macrophages, a,_td they are not relevant to the problem addressed here. The macrophages of H I V patients appeared to respond normally to activation by 1FN'r" in vitro or in vivo, in experiments involving production of hydrogen peroxide or killing of Leishmania donovani or Toxoplasma gondii (Murray et al., 1987; Eales et al., 1987). However. as explained above, IFNy used in this way is ineffective against mycobacterial infection, even in normal human macrophages, so this provides no indication of the effect of HIV inf~tion on growth of M. tuberculosis ,~r ll4. avium within these cells, or their control by the cytokiue/calcitriol c o m b i n a t i o n s discussed. In particular, there are no data on the ef-
408
8th F O R U M IN M I C R O B I O L O G Y
fect of HIV iafection on the activation of the I-hydroxylase required for the formation ofcalcitriol within maerophages, and we are at present looking at this point.
transcription). NF-KB (and therefore HIV) can also be induced in mouoeytes by TNF and by reactive oxygen intermediates (Schreek etal,, 1991). Clearly, the tendency of tuberculosis infection to lead to increased release of T N F and reactive oxygen intermediates could activate HIV by this mechanism.
Accelerated progrcssion to AIDS TNF and other cytokines such as IFN't which are potent inducers of TNF, can serve as autccrine growth factors for HIV replication. Thus, in vitro studies shov, that neutralizing antibodies to TNF can inhibit HIV replication in CIM + T cells (Vyakarnam et al., 1990) and monoeytes (Poll et al., 1990) and HIV as well as soluble gpl20 can trigger TNF and IFNT release in fresh CD4 ÷ T cells (Vyakaruam et aL, 1990; Matear, Weiler and Vyakarnam, unpublished). Furthermore, PBL taken from healthy HIVinfected individuals as well as from those who have progressed to AIDS, secrete high levels of T N F and IFN T compared to uninfected individuals (Vyakarham el al., 1991). The additional effects of M. tuberculosis both on cytokine release and function provide several potential reasons why established infection could accelerate virus production (Rook et al., 1991). 1) Cytokine-induced replication o f H l V : the role o f NF-KB Cytokines may regulate HIV replication at the cellular level by controlling cellular differentiation, or at the molecular level by directly activating the HIV long terminal repeat (LTR) which includes control elements, including KB, regulating virus transcription. KB genetic elements are enhancers present in many cellular and viral genes, including those encoding the kappa immunoglobulin light chain in B cells, from which the name KB was derived. The KB enhancer serves as a recognition site for members of a family of eukaryotic enhancer-binding proteins, known as nuclear factor kappa B (NF-KB), the binding of which leads to enhanced gene transcription. NF-KB are constitutively expressed in B cells, but expression in T cells can be induced by exposure to phorbol esters, ~he Tax protein of HTLV-I and TNFct (Greene et aL, 1989; Osborn et al., 1989). In vitro studies have shown that these same T-cell activation signals induce one or more NF-KB-Iike DNA-binding activities, which have been characterized by a combination of gel retardation assays and in situ DNAprotein cross-linking experiments (Molitor el ai., 1991). Four distinct but closely related polypeptide species between 50 kDa and 85 kDa have been identified. Cells infected with HIV1 from which the NF;~B-binding sites have been deleted (Osborn et aL, 1989), do not increase HIV transcription in the presence of TNF (though NF-KB may no: be the only DNA-binding protein through which TNF activates
2) Lipoarabinomannan M. tuberculosis releases lipoarabinomannan which is a phosphatidylinositol mannoslde (Hunter and Brennan, 1990). This is a potent trigger of cytokine release from maerophages (Moreno et al., 1989), but may inhibit cytokine production by T cells (D. Beret, personal communication) and can block protein k!nase C and also IFN'~-induced transcriptional activation of £enes (Chan et al., 1991). Irt pilot experiments performed in collaboration with Dr. Carlos Moreno, we have shown that LAM will increase production of HIV from normal human PBL (Vyakarnam, Moreno and Rook, unpublished), possibly via induction of NF-KB, as outlined in the previous paragraph. Mycobacteria also contain several other triggers of eytokine release, such as trehalose dlmycolate, and these also need to be tested in this type of experiment. 3) Calcitriol Release of this vitamin D3 metabolite (discussed above) accelerates maturation of monocyte precursors an:l renders them more susceptible to infection with HIV (Kitano et aL, 1990), perhaps partly by increasing expression of CD4 (Schlesinger et aL, 1989). There was a preliminary claim that ealcitriol may also limit productive infection of some monocytoid cell lines (Connor and Rigby, 1991), but an investigation using normal peripheral blood mononuclear cells has revealed that calcitriol at physiological levels can increase replication of monocyte- and lymphocytetropic strains in these cells by up to 10,000-fold (Skolnik et al., 1991). Disturbances in the regulation of calcitrlo.T production by monocytes could he fundamental to the replication of both HIV and TB. 4) Enhancement o f the toxicity o f T N F M. tuberc~dosis also releases a TNF-enhanelng activity (TEA) which greatly increases the toxicity of TNF for target cells (Filley and Rook, 1991), including normal human fibrublasts and endothelial cells (FiUey, Bull and Rook, submitted), and in synergy with TNF, it may contribute to cell damage in tuberculosis. HlV-infected cells tend to become sensitive to the cytotoxic properties of TNF and IFN T even in the
MYCOBACTERIA A N D AIDS absence of M. tuberculosis (Wong and Geeddel, 1986). This phenomenon may partly account for the cytopathie properties of HIV for CIM ÷ T ceils, and the CD4 depletion in HIV patients (Vyakarnam, Matear and Beverley, 1992). The additional enhancement of toxicity resulting from the presence of TEA may lead to very rapid elimination of infected effeetor lymphocytes in the mycobacterial lesion. We do not yet know whether TEA leads to increased levels of NF-KB, or will enhance the ability of T N F to cause transcriptional activation of HIV, hut we now know that T E A (or another unidentified component of M. tuberculosis) does enhance functions of TNF other than cytotoxicity (see next paragraph). 5) Enhanced expression o f adhesion molecules A component of M. tuberculosis, possibly identical to TEA, induces expression of ICAM-I on normal human ceils, and synergizes with TNF in the induction of very high levels of this adhesion molecule (Filley, Bull & Rook, submitted). ,Such phenomena could lead to enhanced syncytium formation.
References Bermudez, L.E. & Young, L.S. 0988), TNF alone or in combination with [L-2, but not IFN'~, is associated with maerophage killing of ~/. avium complex. J. lmmunoL, 140, 3006-3013. Bermudez, L.E., Young, L.S. & Gupta, S. (1990), 1,25 Dihydroxyvitamin D3-dependent inhibition of growth or killing of M. avium complex in human macrophages is mediated by TNF and GM-CSF. Cell. lmmunoL, 127, 432-441. Chan, J., Fan, X., Hunter, S., Brennan, P.J. & Bloom, B.R. (1991), Lipoarabinomannan, a possible virulence factor involved in persistence of M. tuberculosis within macrophages. Infect. lmmun., 59, 1755-1761. Clouse, K., Cosentino, L.M., Weih, K., Pyle, S., Robbins, P., Hochstein, H.D., Natarajan, V. & Farrar, W. (1991), The HIV-I gp-120 envelope protein has the intrinsic capacily In stimulate monokine secretion. J. ImmunoL, 147, 2892-2901. ConRor, R.I. & Rigby, W.F. (1991), l~t,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-I. Biochern. biophvs. Res, Cornman., 176, 852-859. Denis, M. (1991a), Killing of M. tuberculosis within human monocytes: activation by cytokines and calcitriol. Clin. exp. hnmunoL, 84, 200-206. Denis, M. (1991b), Growth of Mycobacterium avium in human monocytes: identification of cytokines which reduce and enhance intracellular microbial growth. Europ. J. hnmunoL, 21, 391-395. Douvas, G.S., Looker, D.L, Vatter, A.E. & Crowlc, A.J. (1985), Ganmra interferon activates human macrophages to become tumoricidal and leishmanicidal, bu~
409
enhances replication of macrophage-associated mycohacteria. Infect. /mmun., 50, 1-8. Eales, L.J., Moshtael, O, & Pinching, A.J. (1987), Microbicidal activity of monocyte-derived macrophages in AIDS and related disorders. C/in. exp. hnraunoL, 67, 227-235. FilMy, E. & Rook, G.A.W. (1991), The effec~ of mycobacteria on sensitivity to the cytotoxic effects of TNF. Infect. Immun., 59, 2567-2572. Greene, W.C., Bohnlein, E. & Ballard, D.W. (1989), H[V-|, HTLV-I and normal T cell growth: tran~ciiptional strategies and surprises. Immunol. Today, 10, 272-278. Haanan, J., de Want Malefijt, R., Res, P., Kraakman, M., Ottenhoff, T., de Vries, R. & Spits, H. (1991). Selection of a human T helper type I-like T cell subset by myeobacteria, .L exp. &led., 174, 583-592. Hunter, S.W, & Brennav., P.J. (1990), Evidence for the presence of a phosphatidylinositol anchor on the [ipoarabinomannan and lipomanr,an of Mycobacterium tuberculosis. J. biol. Chem., 265, 9272-9279. Kitano, K., Baldwin, G.C., Raines, M.A. & Golde, D.W. (1990), Differentiating agents facilitate infection of myeloid leukemia cell lines by monocytotropic HIV-I strains. Blood., 76, 1980-1988. Kumararatne, D.S., Pith]e, A.S., Drysdale, P., Gaston, J.S.. Kiessling, R., lies, P.B., Elhs, C,J., lnnes, J. & Wise, R. (1990), Specific iy,is of mycobacterial antigen-bearing macrophages by class II-restric~ed polyclonal T celt lines from healthy dtmors or patients with tuberculosis. Clin. exp. Imrnunok. 80, 314-323. Laurence, J., Friedman, S.M., Chartash, E.K., Crow, M.K. & Posnett, D.N. (1989), HIX infection of helper T cell clones. Early proliferative defects despite intact antigen-specific recognition and IL-4 secretion. £ clin. Invest., 83, 1843-1848. Macatonia, S.E., Patterson, S. & Knight, S.C. (1989), Suppression of immune responses by dendritic ceils infected with HIV. hmnunology, 67, 285-289. Miedema, F., Tersmette, M. & Van Lint, R.A.W. (1990), AIDS pathogenesis: a dynamic interaction between HIV and the immune system. ImmunoL Today, l I, 293-297. Molitor, J., Walker, W., Doerre, S., BaUard, D. & Greene, W.C. (1991), NF KB: A family of inducible and differentially expressed enhancer-binding proteins in human T cells. Proc. nat. Acad. Sci. (Wash.), 87, 10028-10032. Moreno. C., Taverne, J., Mehlert, A., P2te, C., Brealey, R., Meager, A., Rook, G.A.W. & Playfair, J.H. (1989), Lipoarabinoma~man from M. tuberculosis induces the production of IN;- /tom human alld tflurine macrophages. Clin. pap. lramunol., 76, 240-245. Murray, H.W., Seavuzzo, D., Jacobs, J.L., Kaplan, M.H., Libby, D.M., gchhldler, J. & Roberts, R.B. (1987), In vitro and in vivo activation of human mononudear phagocytes by IFN'f. Studies with normal and AIDS mt~nocytes. J. ImtnunoL. 138. 2a57-2462. Osborn, L., Kunkel, S. & Nabel, G.L. (1989), TNF and IL I stimulate HIV enhancer by activation of the nuclear factor B. Proc. nat. Acad. Sci. (Wash.), 86, 2336-2340. Pinching, A.J. & Nye, K.E. (1990), Defective signal transduct]on -- a common pathway for cellular dysfunction in HI 1~"infection, lmmunol. Today, 7, 256259.
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8lh F O R U M I N MICROB,'OLOG Y
Poll, G.. Rimer, A., Juslment, J., Kehrl, J.H., Bressler, A., Stanley, S, & Fauci, A. (1990), Tumour necrosis factor ~ functions in an autocrine manner in the induction of human immunodefieiency virus expression. Proc. nat. Acad. Scl. fWa~h.), 87. 7a2-785. Rieckr~ann, p., Poli, G.. Fox, C., Kehrl, J. & Fauci, A. ([991), Recombinant gpl20 specifically enhances TNF-~ production and lg secl~tion in B lymphocytes from HIV-infectcd individuals, but not front seronegative donors. J. Immunof., 147~ 2922-2927. Book, G.A.W. (1988), The role of vitamin D in Tuberculosis. Amer. Rev. Resp. D&., 138, 768-770. Rook, G.A.W., Ai Attiyah, R. & Filley, E. (1991), New insights into the immunopa~hology of tuberculosis. Pathobiology, 59, 148-152. Rook, G.A.W., Steele, J., Ainsworth, M. & Champion, B.R. (1986a), Activation of macrophages to inhibit proliferation of M. tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages. Immunology, 59, 333-338. Rook, G.A.W., Steele, J., Fraher, L., Barker, S., Karmali, R., O'Riordan, J. & Stanford, J. {1986b), Vitamin D3, gamma interferon, arid control of proliferation of M. tuberculosis by human monocytes. Immunology, 57, 159-163. Sehlesinger, M., Bar-Shavit, Z., Hadar, R. & Rabinowitz, R. (1989), Modulation of the expression of CD4 on HL 60 ceils by exposure to 1,25-dihydroxy vitamin D3. tmmunol. Letters, 22, 307-311. Schreck, R., Rieher, P. & Bauerle, P.A. (1991), Reactive oxygen intermediates as apparently widely used messengers in the acnvation of NF-kappa B transcription factor and HIV-I. EMBO Z, 10, 2247-2258.
Skolnik, P.R., John, B., Wang, M.Z., Rota, T.R., Hirsch, M.S. & Krane, S M. (1991), Enhancement of human immunodefieiency virus I replication in monocytes by 1,25-dihydroxycholecaleiferoL Proc. nat. Acad. $ci. (Wash.). 88, 6632-6636. Stingl, G., Rappersberger, K , Tschachler, E., Gartner, S., Groh, V., Mann, D., Wolff, K. & Popovic, M. 0990), Langerhans cells in HIV-I infe,~tion, J. Amer. Acad. DermatoL, 22, 1210-1217. V',akarnam, A., Matear, P.M, & Beverley, P.C.L. (1992), HIV-TNF interactions in virgin and memory CD4 ~ T-cells, in "Cytotoxic T cells in HIV and other retroviral infections" {P. Racz and N.L. Letvln). S. Karger, Basel (in press). V)akarnam, A., Matear, P.M., Meager, A., KelIy, G. & Weller, I.V. (1991), Altered prodmticn of tumour necrosis factors ~ and [3 and IFN~" by HlV-infected individuals. Clin. exp. ImmunoL, r~4, 109-115. Vyakarnam, A.. McKeating, J., Meager, A. & Beverley, P.C.L. (1990) TNF ~ and [3 indttced by HIV in peripheral bloc,d mononuelear cells potentiate virus replication. AIDS, 4, 21-27. • Wcrner, E.R., Werner-Felmayer, G., F Jchs, D.. Hausen, A.. Reibnegger, G. & Wachter, tl. (1989), Parallel induction of tetrahydrobiopterin [,iosynthesis and indoleamine 2,3-dioxygen~e in human cells and cell lines. Biochem. J., 262, 861-86(,. Wong, G.H.W. & Goeddel, D.V. (1986), Turnout necrosi~ factors ¢(and [3inhibit virus replication and synergize with interferons. Nature (L,,-,nd.). 323, 819-822. Yong, A.J., Grange, .1.M., Tee. R.D., Beck, J.S., Bot hornley, G.H., Kemeny, D.M. & Kar djito, T. (1989), Total and antimy¢ohacterial IgE !evels in serum from patients with tuberculosi~ and I/:prosy. Tubercle, 70, 273-279.
8th F O R U M IN M I C R O B I O L O G Y
Cytokines, lymphokines, mycobacteria and AIDS G . A . W . R o o k IO and A. V y a k a r n a m is) ft; Department o f Medical Microbiology, University College and Middlesex School o f Medicine. 67-73 Riding House St., London W1P 7LD, and t2) Department o f Immunology, University College and Middlesex School o f Medicine, Arthur Stanley House. Tottenham St., London 14/1
Introduction
Comoined infection with tuberculosis and HIV has become a devastating problem, the consequences of which will be as serimls as those of the Black Death in many populations. In parts of Africa, as many as 90 o/0 of new tuberculosis eases are HIV-seropositive. There are three urgent problems to be addressed. 1) Why is suseepfibility to M. tuberculosis a very early consequence of HIV infection? 2) Why is suseeptibillty to M. avium seen only later in the syndrome? 3) Does established infectio~l with tuberculosis accelerate progression towards AIDS?
Immunity to myeobacteria in man
M. tuberculosis intelligent discussion of question (1) is inhibited by the fact that we do not understand the mechanism of intmunity to mycobacteria in man. The different effect of HIV on infection with M. tuberculosis and M. avium is nice evidence for the complexity of this question. Work on murine maerophages has been somewhat misleading. These cells are easily activated by IFN T (and to a lesser extent by several other cytokines) to inhibit M. tuberculosis (Rook et e ! . 1986a). This effect correlates with production of nitric ,oxide (NO) (J. Chan and B. Bloom, submitted, and personal communication) and can be blocked by the specific inhibitor of arginine deiminase (NC'-monomethyl-L-arginine; NMMA). Whatever the explanation for this correlation between antimyeobacterial activity and NO in marine cells (which is not necessarily due to a direct antimicrobial effect of the NO itself), it does not at present help us to understand the situation in man because human macrophages are unable to make tetrahydrobiopterin, which is an essential cofactor for this pathway. This is because the enzyme 6-pyruvol
tetrahydrobiopteria synthase is present at very low levels in human macrophages (Werner et aL, 1989). Lack of this enzyme may explain the failure of IFN'f to inhibit growth of M. tuberculosis in human cells (Rook et aL, 1986a). In fact, two groups have noted increased growth of M. tuberculosis in IFNy-exoosed macrophages (Rook et al., 1986a; Douvas et aL, 1985). A further important difference between murine and human macrophages is their use of vitamin D3 metabolites. Exposure to IFN~' causes human (but not murine) macrophages to express increased levels of a 1-hydroxylase which derives calcitriol from circulating 25(OH) vitamin D3 (Rook, 1988). This pathway may be o f crucial relevance in the H1V/tuberculosis interaction, and it is considered in that context later. The potential importance of this pathway in protection from tuberculosis lies in the autocrine effect of the caleitriol on the mac~ ophage itself. We observed that some inhibition of virulent M. tuberculosis is seen when human macrophages are exposed to both IFNT and ealcitriol (Rook et al., 1986b), and there is one unconfirmed report that effective killing of M. tuberculosis is seen if a combination of IFN,¢, caleitriol and TNF is used (Denis, 1991a). If this can be confirmed, it will be of great interest to know whether ealcitriol can upregulate 6-pyruvol tetrahydrobiopterin synthase in human macrophages. However, it remains possible that protection from M. tuberculosis in man is not only a nlatter of appropriate macrophage activation, but instead involves killing of infected cells, perhaps by eytotoxic cells. This event may be accompanied by unidentified microbicidal effects (Kumararame et at., 1990). M. avium As implied by the different consequences of HIV infection, different mechanisms may be able to control growth of M. avium in human maerophages. There are claims that exposure to TNF (Bermudez
MYCOBA CTERIA AND AIDS and Young, 1988), GM-CSF (Bermudez et al., 1990), IFN~' or calcitriol (Denis, 1991b) alone can reduce growth o f this organism, which ~,s not the case for M. tuberculosis. However I L l , M-CSF or 1L3 were all found to increase growth of M. avium (Denis, 1991b). Thus, for both mycobacteria, the blend of eytokines may be critical.
407
this dichotomy does exist in man. There is a report that the early proliferative defect associated with I-IIV infection of helper T-cen clones leaves antigen. specific recognition and IL4 secretion intact (Laurence et al., 1989). 3) T-cell dysfunction
lmmunopathology in tuberculosis There is considerable evidence that much of the tissue damage in tuberculosis is due to release of T N F , in sites rendered exquisitely sensitive to this cytokine (Rook et aL, 1991). This has implications for the interaction with HIV, discussed below.
Increased susceptibility to mycobacterioses Although the pathway of immunity to mycobacteria in m a n is not elucidated, it is clear that the early increase in susceptibility to tuberculosis could be secondary to effects o f H I V on: 1) A ntigen presentation It is known that both macrophages and dendritic cells are susceptible to infection with HIV, and this can decrease their A P C function (Stingl et aL, 1990; Macatonia et oL, 1989). It is not clear that this dysfunction would be selectively permissive for growth o f M. tuberculosis rather than M. ovium, and this point needs to be clarified. 2) T-cell subsets The nature of the cytokines to which macrophages are exposed is clearly critical to the control of both M. tuberculosis and M. avium, as outlined above. Our own pilot experiments, and a much more extensive study by others (Haanau et al., 1991) indicate that normal BCG recipients, or donors who have successfully overcome subclinical tuberculosis in the past, secrete 1FN~- hut not detectable IL4 in response to myeobacterial antigens. Thus, it is probable that immunity to tuberculosis is associated with T H 1 helper cells, and these seem to be preferentially activated by mycobacterial antigens. Theoretically, therefore, activation of the disease could result if H1V infection were to result in a switch to a T H 2 response, and it may be significant that some patients with uncontrolled tubeJculosis can have eosinophilia and raised IgE levels, indicative o f IL4 release (Yong et al., 1989). Unfortunately there is little information on the effect o f early HIV infection on the T H I / T H 2 dichotomy, though there is no longer any doubt that
While it is not clear t hat H1V alters the T H I / T H 2 ratio, there is no doubt that it subtly upsets T-cell function, even before there is striking depletion of CD4 + T-cell numbers. For instance, H I V infection of T cells renders them less responsive to activation through the CD3 activation pathway (Pinching and Nye, 1990; Miedema et aL, 1990). These effects may be partly attributable to the excessive cytokine release described below. Moreover, H I V proliferates preferentially in m e m o r y T cells (Miedema el al. 1990; Vyakarnam et al., in press) and may therefore deplete T-cell memory of mycobaeterial antigens earlier than is apparent from peripheral CD4 + counts. 4) Direct triggering by H I V o f inappropriate cytokine release Recombinant g p l 2 0 has been shown to induce secretion of TNF, IL 1, IL6 and G M - C S F by monocytes (Clouse et al., 1991), and secretion of I ' N F by B cells (Rieckmann et al., 19~Cl). g p l 2 0 will also induce IFN~" production in CD4 ÷ T cells (Vyakarnam and Matear, unpublished). Since IFN~ used in the absence of the correct blend of other cytokines can enhance proliferation of M. tuberculosis (Rook et al., 1986a; Douvas et al., 1985), gpl20 could be directly responsible for upsetting the host/mycobacterium relationship. 5) Changed macrophage microbicidal function, and response to cytokines There are few studies of this aspect of HIVinfected macrophages, a,_td they are not relevant to the problem addressed here. The macrophages of H I V patients appeared to respond normally to activation by 1FN'r" in vitro or in vivo, in experiments involving production of hydrogen peroxide or killing of Leishmania donovani or Toxoplasma gondii (Murray et al., 1987; Eales et al., 1987). However. as explained above, IFNy used in this way is ineffective against mycobacterial infection, even in normal human macrophages, so this provides no indication of the effect of HIV inf~tion on growth of M. tuberculosis ,~r ll4. avium within these cells, or their control by the cytokiue/calcitriol c o m b i n a t i o n s discussed. In particular, there are no data on the ef-
408
8th F O R U M IN M I C R O B I O L O G Y
fect of HIV iafection on the activation of the I-hydroxylase required for the formation ofcalcitriol within maerophages, and we are at present looking at this point.
transcription). NF-KB (and therefore HIV) can also be induced in mouoeytes by TNF and by reactive oxygen intermediates (Schreek etal,, 1991). Clearly, the tendency of tuberculosis infection to lead to increased release of T N F and reactive oxygen intermediates could activate HIV by this mechanism.
Accelerated progrcssion to AIDS TNF and other cytokines such as IFN't which are potent inducers of TNF, can serve as autccrine growth factors for HIV replication. Thus, in vitro studies shov, that neutralizing antibodies to TNF can inhibit HIV replication in CIM + T cells (Vyakarnam et al., 1990) and monoeytes (Poll et al., 1990) and HIV as well as soluble gpl20 can trigger TNF and IFNT release in fresh CD4 ÷ T cells (Vyakaruam et aL, 1990; Matear, Weiler and Vyakarnam, unpublished). Furthermore, PBL taken from healthy HIVinfected individuals as well as from those who have progressed to AIDS, secrete high levels of T N F and IFN T compared to uninfected individuals (Vyakarham el al., 1991). The additional effects of M. tuberculosis both on cytokine release and function provide several potential reasons why established infection could accelerate virus production (Rook et al., 1991). 1) Cytokine-induced replication o f H l V : the role o f NF-KB Cytokines may regulate HIV replication at the cellular level by controlling cellular differentiation, or at the molecular level by directly activating the HIV long terminal repeat (LTR) which includes control elements, including KB, regulating virus transcription. KB genetic elements are enhancers present in many cellular and viral genes, including those encoding the kappa immunoglobulin light chain in B cells, from which the name KB was derived. The KB enhancer serves as a recognition site for members of a family of eukaryotic enhancer-binding proteins, known as nuclear factor kappa B (NF-KB), the binding of which leads to enhanced gene transcription. NF-KB are constitutively expressed in B cells, but expression in T cells can be induced by exposure to phorbol esters, ~he Tax protein of HTLV-I and TNFct (Greene et aL, 1989; Osborn et al., 1989). In vitro studies have shown that these same T-cell activation signals induce one or more NF-KB-Iike DNA-binding activities, which have been characterized by a combination of gel retardation assays and in situ DNAprotein cross-linking experiments (Molitor el ai., 1991). Four distinct but closely related polypeptide species between 50 kDa and 85 kDa have been identified. Cells infected with HIV1 from which the NF;~B-binding sites have been deleted (Osborn et aL, 1989), do not increase HIV transcription in the presence of TNF (though NF-KB may no: be the only DNA-binding protein through which TNF activates
2) Lipoarabinomannan M. tuberculosis releases lipoarabinomannan which is a phosphatidylinositol mannoslde (Hunter and Brennan, 1990). This is a potent trigger of cytokine release from maerophages (Moreno et al., 1989), but may inhibit cytokine production by T cells (D. Beret, personal communication) and can block protein k!nase C and also IFN'~-induced transcriptional activation of £enes (Chan et al., 1991). Irt pilot experiments performed in collaboration with Dr. Carlos Moreno, we have shown that LAM will increase production of HIV from normal human PBL (Vyakarnam, Moreno and Rook, unpublished), possibly via induction of NF-KB, as outlined in the previous paragraph. Mycobacteria also contain several other triggers of eytokine release, such as trehalose dlmycolate, and these also need to be tested in this type of experiment. 3) Calcitriol Release of this vitamin D3 metabolite (discussed above) accelerates maturation of monocyte precursors an:l renders them more susceptible to infection with HIV (Kitano et aL, 1990), perhaps partly by increasing expression of CD4 (Schlesinger et aL, 1989). There was a preliminary claim that ealcitriol may also limit productive infection of some monocytoid cell lines (Connor and Rigby, 1991), but an investigation using normal peripheral blood mononuclear cells has revealed that calcitriol at physiological levels can increase replication of monocyte- and lymphocytetropic strains in these cells by up to 10,000-fold (Skolnik et al., 1991). Disturbances in the regulation of calcitrlo.T production by monocytes could he fundamental to the replication of both HIV and TB. 4) Enhancement o f the toxicity o f T N F M. tuberc~dosis also releases a TNF-enhanelng activity (TEA) which greatly increases the toxicity of TNF for target cells (Filley and Rook, 1991), including normal human fibrublasts and endothelial cells (FiUey, Bull and Rook, submitted), and in synergy with TNF, it may contribute to cell damage in tuberculosis. HlV-infected cells tend to become sensitive to the cytotoxic properties of TNF and IFN T even in the
MYCOBACTERIA A N D AIDS absence of M. tuberculosis (Wong and Geeddel, 1986). This phenomenon may partly account for the cytopathie properties of HIV for CIM ÷ T ceils, and the CD4 depletion in HIV patients (Vyakarnam, Matear and Beverley, 1992). The additional enhancement of toxicity resulting from the presence of TEA may lead to very rapid elimination of infected effeetor lymphocytes in the mycobacterial lesion. We do not yet know whether TEA leads to increased levels of NF-KB, or will enhance the ability of T N F to cause transcriptional activation of HIV, hut we now know that T E A (or another unidentified component of M. tuberculosis) does enhance functions of TNF other than cytotoxicity (see next paragraph). 5) Enhanced expression o f adhesion molecules A component of M. tuberculosis, possibly identical to TEA, induces expression of ICAM-I on normal human ceils, and synergizes with TNF in the induction of very high levels of this adhesion molecule (Filley, Bull & Rook, submitted). ,Such phenomena could lead to enhanced syncytium formation.
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