Pathobiology 1992;60:246-251
Laboratory oflmmunoregulation, National Institute of Allergy and Infectious Diseases. National Institutes of Health, Bethesda, Md.. USA
Key Words Monocyte Macrophage Cytokines HIV Transcription
The Role of Monocyte/Macrophages and Cytokines in the Pathogenesis of HIV Infection Abstract The monocyte/macrophage system, which is characterized by a high degree of heterogeneity, is a target of the human immunodeficiency virus (HIV) in vitro as well as in vivo. Both bone-marrow-derived precursor elements and circulat ing monocytes are infectable in vitro and have also been found infected in seropositive individuals. However, terminally differentiated macrophages are the most commonly infected cells found in vivo in addition to the CD4+ T lymphocytes. Several immunoregulatory cytokines have been shown to either up-regulate or suppress virus replication/expression in vitro in cells belonging to the monocyte/macrophage lineage by affecting both transcriptional as well as posttranscriptional events. The observation that elevated levels of several of these cytokines are present in HIV-infected individuals suggests that they may play an important role as regulators of virus expression in vivo.
Introduction Shortly after the identification of the CD4+ T lympho cyte as the preferential target of infection with the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), monocyte/macrophages were also recognized to play an impor tant role in the pathogenesis of this new clinical entity [ 1]. Early studies showed that circulating monocytes were functionally defective in HIV-infected individuals [1], Furthermore, cells of monocyte/macrophage origin were found to be infectable in vitro [2-5] and infected in AIDS patients [5-7], The use of cytokines, the peptide regulatory hormones of the immune system, has been instrumental in the iden tification of the cellular targets for HIV. Interleukin-2 (IL2) allowed the in vitro growth and the infection of pri
Received: June 15.1991 Accepted: July 29,1991
mary T lymphocytes [1], Colony-stimulating factors (CSFs) were similarly indispensable to investigate the characteristics of monocyte/macrophage infection [3, 4], However, the role of cytokines in HIV infection soon became not only an important tissue culture tool, but also began to be appreciated as a potentially important net work of soluble factors which could modulate the state of viral latency and expression in infected individuals [1,8. 9],
The Monocyte/Macrophage System as a Target of HIV Infection A considerable level of heterogeneity characterizes the cells of the monocyte/macrophage system (more properly defined as mononuclear phagocytes). Following several
Dr. Guido Poli Laboratory oflmmunoregulation National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, MD 20892 (USA)
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Guido Poli Anthony S. Fauci
Table 1. Susceptibility to HIV infection among cells of the mvclomonocytic lineage Cell
In vitro In vivo References“
CD34+ stem cells Promyelocytic cells (HL-60) Promonocytic cells (U937) Monocytic cells (TH P-1) PBMs In vitro MDMs Lung alveolar macrophages Kupffcr cells Peritoneal macrophages Brain macrophages (microglial cells) Inflammatory brain macrophages
+ + + +
a
7
+ +
+ NA
+ ? 7 7 + NA + 7 7
11, A 12.13 14 7. 15 2-4. 16-18 5 19 18
7
20
+
6
12
NA = Nonapplicable. A = S.K. Stanley and A.S. Fauci, submitted.
cyte/macrophages (a phenomenon referred to as 'macro phage tropism’ [2-4]), the cell originally infected in vivo was the CD4+T cell in most of the cases [15], However, in a minority of individuals, in vivo infection of PBMs was also demonstrated [ 15], In contrast to PBMs, terminally differentiated macro phages represent an important target of HIV infection and replication in several tissues and organs (table 1). PBMs become more susceptible to HIV infection after in vitro maturation for several days into monocyte-derived macrophages (MDMs) [2-4, 16-18]. Furthermore, pri mary macrophages obtained from different anatomic sites, including the peritoneal cavity [18], lung alveolar macrophages [5] and brain microglial cells [20], can be efficiently infected in vitro. Inflammatory macrophages infiltrating the brain of AIDS patients express high levels of HIV [6] and secrete soluble toxic factors which may potentially explain the neurologic dysfunctions frequently observed in HIV-infected individuals [24], In certain anatomic sites, such as the skin and the lymph nodes, where macrophages, in the strict sense, are not usually present in physiologic conditions, the antigenpresenting function is carried on by dendritic cells. Wor thy of note, both epidermal Langerhans (dendritic) cells [25] and the follicular dendritic cells [26] of the lymph nodes have been shown to represent active sites of HIV expression in infected patients. Taken together these observations suggest that critical events in the differentiation process of mononuclear
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maturational steps in the bone marrow environment, my eloid precursor cells can proliferate and differentiate un der the influence of CSFs and cytokines. Unlike fully dif ferentiated circulating T lymphocytes, the peripheralblood monocyte (PBM) represents an intermediate stage of maturation and only after migration outside the blood stream can be considered a terminally differentiated mac rophage residing in different tissues and organs [10]. Of interest, cells belonging to many of these stages of differ entiation have been successfully infected in vitro with HIV, and for certain stages the demonstration that this event may occur in infected individuals has also been pro vided (table 1). Perhaps the earliest bone marrow precursor cell in fected in vitro with HIV has been identified by Folks et al. [11] as the CD34+ stem cell, although the experimental conditions of long-term culture may have played an important role in influencing the susceptibility of these cells to infection. Furthermore. CD34+ cells have been found to be infected in the bone marrow of a significant number of AIDS patients [S.K. Stanley and A.S. Fauci, submitted], in contrast with earlier negative reports [21], In addition, a variety of cell lines representative of differ ent stages of myelomonocytic differentiation express the CD4 molecule (the receptor for HIV) on their surface and are susceptible to virus infection (table 1). Finally, expres sion of virus in terminally differentiated macrophages residing in the bone marrow stroma has been demon strated in macaques infected with the simian immunode ficiency virus (SIV) [22], a retrovirus closely related to HIV which causes an AIDS-like syndrome in the infected animals. These findings also suggest that infection of pro genitor and mature cells in the bone marrow may repre sent an important viral reservoir and may contribute to several of the hematologic abnormalities frequently ob served in HIV-infected individuals, including cytopenias of different cell types [reviewed in ref. 9], Whether circulating monocytes represent an impor tant target of infection is still a matter of controversy. Cer tain studies have suggested that these cells may be infected in vivo at high frequency and in several individu als [7], However, polymerase chain reaction analysis of highly purified peripheral-blood cell subsets obtained from HIV-infcctcd individuals revealed that infection of circulating cells occurs at low frequency (approximately 1/1,000-1/10.000 circulating cells carry HIV proviral DNA), and that the infected cell type in this compartment is almost exclusively represented by the CD4+T lympho cyte [23]. Furthermore, where the isolated virus showed characteristics of preferential in vitro growth in mono-
Cytokine
Effect
References“
IL-I IL-3 IL-4 IL-6 TNF-a GM-CSF M-CSF IFN-a IFN-p IFN-y TGF-p
t t Î Î Î Î Î
A 4 28 16 16, 29. 30 4. 13. 16 3.4 31,32 B 4. C 17, 33
1 i u u
a A = F. Miedema. pers. communication: B = G. Poli, P. Biswas and A.S. Fauci, unpubl. observations; C = P. Biswas, G. Poli and A.S. Fauci, in preparation.
phagocytes are associated with an increased susceptibility of these cells to HIV infection. A similar correlation between viral expression and stage of differentiation of mononuclear phagocytes was previously described in ani mals infected with lentiviruses (a subclass of retroviruses to which HIV and SIV belong); lentivirus infection can occur in bone marrow precursor cells, but viral expression is predominantly observed in mature tissue macrophages [27], Several studies have emphasized the potential role of immunoregulatory cytokines (among other factors [re viewed in ref. I]) in leading to both HIV production and activation/differentiation of mononuclear phagocytes.
Cytokine Up-Regulation of HIV Expression in Monocyte/Macrophages Several cytokines have been shown to induce HIV expression in primary monocyte/macrophages and/or myelomonocytic cell lines (table 2).CSFs such as granulo cyte-macrophage CSF (GM-CSF) [4] and macrophage CSF (M-CSF) [3] allowed the productive replication of HIV in primary long-term cultures of MDMs. Previous observations indicated that GM-CSF could also induce expression of latent HIV proviruses in the chronically infected promonocytic cell line U 1 [13]. Several features of cytokine up-regulation of virus pro duction in monocyte/macrophages mimic functional as pects exerted by these soluble molecules in controlling the
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homeostasis of the immune system. Synergistic induction of virus expression between tumor necrosis factor-a (TNF-a) and IL-6 has been described in the U 1 cell model [16], Furthermore, TNF-a has been shown to act in an autocrine/paracrine manner in the up-regulation of virus expression in both chronically infected cell lines [30] and primary MDM cultures [G. Poli, J.S. Justement and A.S. Fauci, unpubl. observations] (see fig. 1). At the biochemical level, activation of events mediated by protein kinase C seems to be crucial for virus expres sion in U1 cells stimulated with either phorbol myristate acetate (PMA) or TNF-a [34, 35], Methylation of proviral DNA has been suggested as a cause of HIV latency in chronically infected cell clones derived from the THP-1 monocytic cell line [14]. At the molecular level, two fundamental pathways of activation have been described. TNF-a stimulation of monocyte/macrophages, similar to T cells, leads to the activation of the pleiotropic cellular transcription factor NF-kB via uncoupling from its cytoplasmic inhibitor pro tein 1- kB. Activated NF-kB complexes then migrate into the nucleus and activate transcription of the HIV provirus as well as of several cellular genes via specific binding to consensus sequences present in their promoter/enhancer region [8. 29, 36]. In contrast to this well-defined molecu lar pathway of activation induced by TNF-a (or phorbol ester) in infected cells, limited information is available concerning the molecular mechanism(s) operative after stimulation with other cytokines. In addition to NF-kBdependent transcriptional mechanisms, the possibility that one or more posttranscriptional steps of HIV expres sion can be affected by certain cytokines has been recently proposed [16]. Stimulation of chronically infected U1 cells with IL-6 induced HIV protein and virion expression at levels comparable to those obtained with TNF-a. How ever, these IL-6-induced effects were not accounted for by significantly increased levels of newly transcribed HIV mRN A, as observed with TNF-a [ 16], Similar evidence of posttranscriptional regulation of HIV expression was ob served when U1 cells were stimulated with cytokines other than IL-6, such as GM-CSF and interferon-y (IFNy) [G. Poli and A.S. Fauci, unpubl. observations]. There fore, the U 1 cell line may represent a useful model for the identification of regulatory steps in the expression of HIV which are independent from viral transcription. On the other hand, more studies are required in order to establish the relative importance of cytokine-dependent transcrip tional and posttranscriptional levels of control of HIV expression in infected cells.
The Role of Monoeyte/Macrophages and Cytokines in the Pathogenesis of HIV Infection
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Table 2. Cytokines and lymphokines known to modulate HIV expression/replication in monocyte/macrophagcs
Fig. 1. Cytokine regulation of HIV ex pression in monocvte/macrophages. The re sults predominantly refer to the chronically infected U l cell model (see text). PKC = Pro tein kinase C.
Not only can cytokines up-regulate virus expression, but they have also been shown to down-regulate HIV pro duction in infected cells. The first class of cytokines which can exert such antiretroviral effects is represented by the IFNs. Recombinant IFN-a has been shown to effectively inhibit spreading of HIV in primary T cell blasts and MDMs acutely infected with HIV [31, 37], IFN-a also suppressed virus production in chronically infected T and promonocytic cell lines which were resistant to azidothymidine [32], the most widely used anti-HIV agent. In chronically infected cells, IFN-a inhibited the release of mature virions from the plasma membrane [32], presum ably the very last stage of retroviral expression, as pre viously observed in cells chronically infected with animal retroviruses. Similarly. IFN-J3 suppressed HIV infection of primary T cell blasts [38] and chronically infected cell lines (table 2). In contrast, complex dichotomous effects on HIV expression seem to be associated with IFN-y stim ulation of monocyte/macrophages. Koyanagi et al. [4] first reported that stimulation of primary MDMs with IFN-y could result in both up-regulation or suppression of viral replication. IFN-y directly induced HIV expression in U 1 cells, but, at the same time, significantly inhibited HIV production when these cells were stimulated with the phorbol ester PMA [P. Biswas, G. Poli and A.S. Fauci, in preparation].
Recently, another cytokine, transforming growth faclor-(3 (TGF-p). has been described to exert potent suppres sive effects on HIV expression in monocyte/macrophages [ 17], TGF-P inhibited virus expression in U 1 cells stimu lated with either PMA or cytokines such as IL-6. Unlike the IFNs, TGF-P suppressed HIV protein synthesis as well as viral transcription and accumulation of viral RNA in chronically infected cells [17], In addition. TGF-P inhibited virus production in primary MDMs previously infected with HIV [ 17], However, up-regulatory effects on virus replication were also observed in primary MDMs if cell cultures were stimulated with TGF-P prior to HIV infection [17, 33], In addition, a dose-dependent dichot omy of TGF-p effects on HIV infection of primary T cell blasts has been recently described by Peterson et al. [39]. Therefore, it is conceivable that cell stimulation with cer tain cytokines such as IFN-y and TGF-P may result in opposite regulatory effects of different steps of the virus life cycle.
Cytokines as Potential in vivo Regulators of HIV Expression Although direct proof of a correlation between secre tion of certain cytokines and modulation of HIV expres sion in infected individuals has not yet been provided, several observations support this hypothesis. Increased levels of several cytokines capable of regulating virus
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Cytokine Suppression of HIV Expression in Monocyte/Macrophages
expression in vitro such as TNF-a. IL-6 and IFN-y have been detected in the plasma and/or cerebrospinal fluid of infected individuals [40-42], Some of these cytokines have been demonstrated either by immunocytochcmistry or by in situ hybridization techniques to be increased in the tissues such as the gastrointestinal tract [D. Cotier, pers. communication] and lymph nodes [43] in close proximity to areas where expression of virus was demon strable. In addition, increased in vitro production of TNF-a [44-46], IL-6 [45. 46] and TGF-|3 [47. 48] from either monocytes or B lymphocytes from infected individ uals has been documented. In experiments where B lym phocytes secreting increased levels of TNF-a and IL-6 obtained from lymph nodes of HIV-infected individuals were cocultivated with their autologous T cells, increased production of virus was observed in the absence of exoge nous stimuli [45], It is conceivable that cytokine secretion was responsible at least in part for the observed effect and that similar results could be obtained if monocyte/macrophages instead of B cells were used as the 'inducer' popu lation.
Conclusions Regulation of HIV expression is likely to be the result of a complex network of extracellular signals, such as those provided by certain cytokines, together with tran scriptional and posttranscriptional effects mediated en dogenously by regulatory viral proteins such as tat and rev [8], The monocyte/macrophage system is characterized by a high degree of heterogeneity associated with the dif ferent stages of cell differentiation (bone marrow precur sor cells, circulating monocytes, tissue macrophages). HIV infection of both CD4+ T cells and mononuclear phagocytes results in the profound state of immune sup pression which underlies the clinical symptoms of AIDS. Further definition of the different endogenous and exoge nous mechanisms of regulation of virus expression in these target cells may become of crucial importance for the design of more effective therapeutical strategies against HIV infection.
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8 Cullen BR. Green WC: Regulatory pathways governing HIV-1 replication. Cell 1989:58: 423-426. 9 McCune JM: HIV-1: The infective process in vivo. Cell 1991:64:351-363. 10 Metcalf D: The molecular control of cell divi sion, differentiation commitment and matura tion in hemopoietic cells. Nature 1989:339:27— 30. 11 Folks TM. Kessler SW. Orenstein JM. Juste ment JS. Jaffe ES. Fauci AS: Infection and replication of HIV-1 in purified progenitor cells of normal human bone marrow. Science 1988:242:919-922. 12 Larcher C. Schulz TF, Hofbauer J. Hengster P. Romani N. Wächter H. Dierich MP: Expres sion of the C3d/EBV receptor and of other cell membrane surface markers is altered upon HIV-1 infection o f myeloid. T and B cells. J AIDS 1990:3:103-108’. 13 Folks TM, Justement J, Kinter A. Dinarello CA, Fauci AS: Cvtokine-induced expression of HIV-1 in a chronically infected promonocytic cell line. Science 1987;238:800-802. 14 Mikovits J. Raziuddin A. Gonda M. Ruta M. Lohrey NC. Kung HF. Ruscetti FW: Negative regulation of human immune deficiency virus replication in monocytes: Distinctions between restricted and latent expression in THP-I cells. J Exp Med 1990;171:1705-1720.
15 Massari FE. Poli G, Schnittman SM. Psallidopoulos MC. Davey V. Fauci AS: In vivoT lym phocyte origin of macrophage-tropic srains of HIV. J Immunol 1990:144:4628-4632. 16 Poli G. Bressler P. Kinter A. Duh E. Timmer WC. Rabson A. Justement JS. Stanley S. Fauci AS: Interleukin-6 induces human immunodefi ciency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor a by transcriptional and post-transcrip tional mechanisms. J Exp Med 1990; 172:151 — 158. 17 Poli G. Kinter AL. Justement JS. Bressler P. Kehrl JH. l-'auci AS: Transforming growth fac tor p suppresses human immunodeficiency vi rus expression and replication in infected cells of the monocyte/macrophage lineage. J Exp Med 1991;173:589-597. 18 Olafsson K. Smith MS. Marshburn P. Carter SG. Haskill S: Variation of HIV infectivity of macrophages as a function of donor, stage of differentiation, and site of origin. J AIDS 1990: 4:154-164. 19 Schmitt MP. Gendrault JL. Schweitzer C. Sleffan AM. Beyer C. Royer C, Jaeck D. Pasquali JL. Kirn A. Aubertin AM: Pcrmissivity of pri mary cultures of human Kupfler cells of HI V-1. AIDS Res Hum Retroviruses 1991:6:987-991.
Poli/Fauci
The Role of Monocyte/,Macrophages and Cytokines in the Pathogenesis of HIV Infection
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References
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20 Walkins BA, Dorn HH. Kelly WB. Armstrong RC, Potts BJ, Michaels F. Kufta CV, DuboisDalcq M: Specific tropism of HIV-1 for micro glial cells in primary human brain cultures. Science 1990:249:547-553. 21 von Laer D. Hufert TE, Fenner S. Schwander S. Dietrich M. Schmitz H. Kern P: CD34* hematopoietic progenitor cells arc not a major reservoir of the human immunodeficiency vi rus. Blood 1990;76:1281-1286. 22 Watanabe M, Ringlcr DJ. Nakamura M. Del.ong PA. Letvin NL: Simian immunodefi ciency virus inhibits bone marrow hematopoctic progenitor cell growth. J Virol 1990:64: 656-663. 23 Schnittman SM. Psallidopoulos MC. Lane HC. Thompson L. Basclcr M. Massari F, Fox CH. Salzman NP. Fauci AS: The reservoir for HI VI in human peripheral blood is a T cell that maintains expression of CD4. Science 1989: 245:305-308. 24 Giulian D. Vaca K. Noonan CA: Secretion of neurotoxins by mononuclear phagocytes in fected with HIV-1. Science 1990:250:1593— 1596. 25 Tschachler E, Groh V. Popovic M. Mann DL. Konrad K. Safai B. Eron L. diMarzo Veronese F. Wolff K, Stingl G: Epidermal Langerhans cells - A target for HTLV-lll/LAV infection. J Invest Dermatol 1987:88:233—237. 26 Tenner-Racz K. Racz R, Bofill M, SchulzMeyer A. Dietrich M. Kern P. Weber J. Pinch ing AJ. Veronese-Dimarzo F, Popovic M. Klatzmann D, Gluckmann JC, Janossy G: HTLV-III/LAV viral antigens in lymph nodes of homosexual men with persistent generalized lymphadenopathy with AIDS. Am J Pathol 1986;123:9-15. ’ 27 Gendelman HE. Narayan O. Molineaux S. Clements JE, Ghotbi Z: Slow, persistent repli cation of lentiviruses: Role of tissue macro phages and macrophage precursors in bone marrow. Proc Natl Acad Sci USA 1985:82: 7086-7090. 28 Novak RM. HolzerTJ. Kennedy MM. Hcynen CA. Dawson G: The effect of interleukin-4 (BSF-I)on infection of peripheral blood mono cyte-derived macrophages with HIV-I. AIDS Res Hum Retroviruses 1990:6:973-976. 29 Griffin GE. Leung K. Folks TM. Kunkcl S. Nabel GJ, Activation of HIV gene expression during monocyte differentiation by induction of NF-kB. Nature 1989;339:70-73.
Laboratory oflmmunoregulation, National Institute of Allergy and Infectious Diseases. National Institutes of Health, Bethesda, Md.. USA
Key Words Monocyte Macrophage Cytokines HIV Transcription
The Role of Monocyte/Macrophages and Cytokines in the Pathogenesis of HIV Infection Abstract The monocyte/macrophage system, which is characterized by a high degree of heterogeneity, is a target of the human immunodeficiency virus (HIV) in vitro as well as in vivo. Both bone-marrow-derived precursor elements and circulat ing monocytes are infectable in vitro and have also been found infected in seropositive individuals. However, terminally differentiated macrophages are the most commonly infected cells found in vivo in addition to the CD4+ T lymphocytes. Several immunoregulatory cytokines have been shown to either up-regulate or suppress virus replication/expression in vitro in cells belonging to the monocyte/macrophage lineage by affecting both transcriptional as well as posttranscriptional events. The observation that elevated levels of several of these cytokines are present in HIV-infected individuals suggests that they may play an important role as regulators of virus expression in vivo.
Introduction Shortly after the identification of the CD4+ T lympho cyte as the preferential target of infection with the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome (AIDS), monocyte/macrophages were also recognized to play an impor tant role in the pathogenesis of this new clinical entity [ 1]. Early studies showed that circulating monocytes were functionally defective in HIV-infected individuals [1], Furthermore, cells of monocyte/macrophage origin were found to be infectable in vitro [2-5] and infected in AIDS patients [5-7], The use of cytokines, the peptide regulatory hormones of the immune system, has been instrumental in the iden tification of the cellular targets for HIV. Interleukin-2 (IL2) allowed the in vitro growth and the infection of pri
Received: June 15.1991 Accepted: July 29,1991
mary T lymphocytes [1], Colony-stimulating factors (CSFs) were similarly indispensable to investigate the characteristics of monocyte/macrophage infection [3, 4], However, the role of cytokines in HIV infection soon became not only an important tissue culture tool, but also began to be appreciated as a potentially important net work of soluble factors which could modulate the state of viral latency and expression in infected individuals [1,8. 9],
The Monocyte/Macrophage System as a Target of HIV Infection A considerable level of heterogeneity characterizes the cells of the monocyte/macrophage system (more properly defined as mononuclear phagocytes). Following several
Dr. Guido Poli Laboratory oflmmunoregulation National Institute of Allergy and Infectious Diseases National Institutes of Health, Bethesda, MD 20892 (USA)
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Guido Poli Anthony S. Fauci
Table 1. Susceptibility to HIV infection among cells of the mvclomonocytic lineage Cell
In vitro In vivo References“
CD34+ stem cells Promyelocytic cells (HL-60) Promonocytic cells (U937) Monocytic cells (TH P-1) PBMs In vitro MDMs Lung alveolar macrophages Kupffcr cells Peritoneal macrophages Brain macrophages (microglial cells) Inflammatory brain macrophages
+ + + +
a
7
+ +
+ NA
+ ? 7 7 + NA + 7 7
11, A 12.13 14 7. 15 2-4. 16-18 5 19 18
7
20
+
6
12
NA = Nonapplicable. A = S.K. Stanley and A.S. Fauci, submitted.
cyte/macrophages (a phenomenon referred to as 'macro phage tropism’ [2-4]), the cell originally infected in vivo was the CD4+T cell in most of the cases [15], However, in a minority of individuals, in vivo infection of PBMs was also demonstrated [ 15], In contrast to PBMs, terminally differentiated macro phages represent an important target of HIV infection and replication in several tissues and organs (table 1). PBMs become more susceptible to HIV infection after in vitro maturation for several days into monocyte-derived macrophages (MDMs) [2-4, 16-18]. Furthermore, pri mary macrophages obtained from different anatomic sites, including the peritoneal cavity [18], lung alveolar macrophages [5] and brain microglial cells [20], can be efficiently infected in vitro. Inflammatory macrophages infiltrating the brain of AIDS patients express high levels of HIV [6] and secrete soluble toxic factors which may potentially explain the neurologic dysfunctions frequently observed in HIV-infected individuals [24], In certain anatomic sites, such as the skin and the lymph nodes, where macrophages, in the strict sense, are not usually present in physiologic conditions, the antigenpresenting function is carried on by dendritic cells. Wor thy of note, both epidermal Langerhans (dendritic) cells [25] and the follicular dendritic cells [26] of the lymph nodes have been shown to represent active sites of HIV expression in infected patients. Taken together these observations suggest that critical events in the differentiation process of mononuclear
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maturational steps in the bone marrow environment, my eloid precursor cells can proliferate and differentiate un der the influence of CSFs and cytokines. Unlike fully dif ferentiated circulating T lymphocytes, the peripheralblood monocyte (PBM) represents an intermediate stage of maturation and only after migration outside the blood stream can be considered a terminally differentiated mac rophage residing in different tissues and organs [10]. Of interest, cells belonging to many of these stages of differ entiation have been successfully infected in vitro with HIV, and for certain stages the demonstration that this event may occur in infected individuals has also been pro vided (table 1). Perhaps the earliest bone marrow precursor cell in fected in vitro with HIV has been identified by Folks et al. [11] as the CD34+ stem cell, although the experimental conditions of long-term culture may have played an important role in influencing the susceptibility of these cells to infection. Furthermore. CD34+ cells have been found to be infected in the bone marrow of a significant number of AIDS patients [S.K. Stanley and A.S. Fauci, submitted], in contrast with earlier negative reports [21], In addition, a variety of cell lines representative of differ ent stages of myelomonocytic differentiation express the CD4 molecule (the receptor for HIV) on their surface and are susceptible to virus infection (table 1). Finally, expres sion of virus in terminally differentiated macrophages residing in the bone marrow stroma has been demon strated in macaques infected with the simian immunode ficiency virus (SIV) [22], a retrovirus closely related to HIV which causes an AIDS-like syndrome in the infected animals. These findings also suggest that infection of pro genitor and mature cells in the bone marrow may repre sent an important viral reservoir and may contribute to several of the hematologic abnormalities frequently ob served in HIV-infected individuals, including cytopenias of different cell types [reviewed in ref. 9], Whether circulating monocytes represent an impor tant target of infection is still a matter of controversy. Cer tain studies have suggested that these cells may be infected in vivo at high frequency and in several individu als [7], However, polymerase chain reaction analysis of highly purified peripheral-blood cell subsets obtained from HIV-infcctcd individuals revealed that infection of circulating cells occurs at low frequency (approximately 1/1,000-1/10.000 circulating cells carry HIV proviral DNA), and that the infected cell type in this compartment is almost exclusively represented by the CD4+T lympho cyte [23]. Furthermore, where the isolated virus showed characteristics of preferential in vitro growth in mono-
Cytokine
Effect
References“
IL-I IL-3 IL-4 IL-6 TNF-a GM-CSF M-CSF IFN-a IFN-p IFN-y TGF-p
t t Î Î Î Î Î
A 4 28 16 16, 29. 30 4. 13. 16 3.4 31,32 B 4. C 17, 33
1 i u u
a A = F. Miedema. pers. communication: B = G. Poli, P. Biswas and A.S. Fauci, unpubl. observations; C = P. Biswas, G. Poli and A.S. Fauci, in preparation.
phagocytes are associated with an increased susceptibility of these cells to HIV infection. A similar correlation between viral expression and stage of differentiation of mononuclear phagocytes was previously described in ani mals infected with lentiviruses (a subclass of retroviruses to which HIV and SIV belong); lentivirus infection can occur in bone marrow precursor cells, but viral expression is predominantly observed in mature tissue macrophages [27], Several studies have emphasized the potential role of immunoregulatory cytokines (among other factors [re viewed in ref. I]) in leading to both HIV production and activation/differentiation of mononuclear phagocytes.
Cytokine Up-Regulation of HIV Expression in Monocyte/Macrophages Several cytokines have been shown to induce HIV expression in primary monocyte/macrophages and/or myelomonocytic cell lines (table 2).CSFs such as granulo cyte-macrophage CSF (GM-CSF) [4] and macrophage CSF (M-CSF) [3] allowed the productive replication of HIV in primary long-term cultures of MDMs. Previous observations indicated that GM-CSF could also induce expression of latent HIV proviruses in the chronically infected promonocytic cell line U 1 [13]. Several features of cytokine up-regulation of virus pro duction in monocyte/macrophages mimic functional as pects exerted by these soluble molecules in controlling the
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homeostasis of the immune system. Synergistic induction of virus expression between tumor necrosis factor-a (TNF-a) and IL-6 has been described in the U 1 cell model [16], Furthermore, TNF-a has been shown to act in an autocrine/paracrine manner in the up-regulation of virus expression in both chronically infected cell lines [30] and primary MDM cultures [G. Poli, J.S. Justement and A.S. Fauci, unpubl. observations] (see fig. 1). At the biochemical level, activation of events mediated by protein kinase C seems to be crucial for virus expres sion in U1 cells stimulated with either phorbol myristate acetate (PMA) or TNF-a [34, 35], Methylation of proviral DNA has been suggested as a cause of HIV latency in chronically infected cell clones derived from the THP-1 monocytic cell line [14]. At the molecular level, two fundamental pathways of activation have been described. TNF-a stimulation of monocyte/macrophages, similar to T cells, leads to the activation of the pleiotropic cellular transcription factor NF-kB via uncoupling from its cytoplasmic inhibitor pro tein 1- kB. Activated NF-kB complexes then migrate into the nucleus and activate transcription of the HIV provirus as well as of several cellular genes via specific binding to consensus sequences present in their promoter/enhancer region [8. 29, 36]. In contrast to this well-defined molecu lar pathway of activation induced by TNF-a (or phorbol ester) in infected cells, limited information is available concerning the molecular mechanism(s) operative after stimulation with other cytokines. In addition to NF-kBdependent transcriptional mechanisms, the possibility that one or more posttranscriptional steps of HIV expres sion can be affected by certain cytokines has been recently proposed [16]. Stimulation of chronically infected U1 cells with IL-6 induced HIV protein and virion expression at levels comparable to those obtained with TNF-a. How ever, these IL-6-induced effects were not accounted for by significantly increased levels of newly transcribed HIV mRN A, as observed with TNF-a [ 16], Similar evidence of posttranscriptional regulation of HIV expression was ob served when U1 cells were stimulated with cytokines other than IL-6, such as GM-CSF and interferon-y (IFNy) [G. Poli and A.S. Fauci, unpubl. observations]. There fore, the U 1 cell line may represent a useful model for the identification of regulatory steps in the expression of HIV which are independent from viral transcription. On the other hand, more studies are required in order to establish the relative importance of cytokine-dependent transcrip tional and posttranscriptional levels of control of HIV expression in infected cells.
The Role of Monoeyte/Macrophages and Cytokines in the Pathogenesis of HIV Infection
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Table 2. Cytokines and lymphokines known to modulate HIV expression/replication in monocyte/macrophagcs
Fig. 1. Cytokine regulation of HIV ex pression in monocvte/macrophages. The re sults predominantly refer to the chronically infected U l cell model (see text). PKC = Pro tein kinase C.
Not only can cytokines up-regulate virus expression, but they have also been shown to down-regulate HIV pro duction in infected cells. The first class of cytokines which can exert such antiretroviral effects is represented by the IFNs. Recombinant IFN-a has been shown to effectively inhibit spreading of HIV in primary T cell blasts and MDMs acutely infected with HIV [31, 37], IFN-a also suppressed virus production in chronically infected T and promonocytic cell lines which were resistant to azidothymidine [32], the most widely used anti-HIV agent. In chronically infected cells, IFN-a inhibited the release of mature virions from the plasma membrane [32], presum ably the very last stage of retroviral expression, as pre viously observed in cells chronically infected with animal retroviruses. Similarly. IFN-J3 suppressed HIV infection of primary T cell blasts [38] and chronically infected cell lines (table 2). In contrast, complex dichotomous effects on HIV expression seem to be associated with IFN-y stim ulation of monocyte/macrophages. Koyanagi et al. [4] first reported that stimulation of primary MDMs with IFN-y could result in both up-regulation or suppression of viral replication. IFN-y directly induced HIV expression in U 1 cells, but, at the same time, significantly inhibited HIV production when these cells were stimulated with the phorbol ester PMA [P. Biswas, G. Poli and A.S. Fauci, in preparation].
Recently, another cytokine, transforming growth faclor-(3 (TGF-p). has been described to exert potent suppres sive effects on HIV expression in monocyte/macrophages [ 17], TGF-P inhibited virus expression in U 1 cells stimu lated with either PMA or cytokines such as IL-6. Unlike the IFNs, TGF-P suppressed HIV protein synthesis as well as viral transcription and accumulation of viral RNA in chronically infected cells [17], In addition. TGF-P inhibited virus production in primary MDMs previously infected with HIV [ 17], However, up-regulatory effects on virus replication were also observed in primary MDMs if cell cultures were stimulated with TGF-P prior to HIV infection [17, 33], In addition, a dose-dependent dichot omy of TGF-p effects on HIV infection of primary T cell blasts has been recently described by Peterson et al. [39]. Therefore, it is conceivable that cell stimulation with cer tain cytokines such as IFN-y and TGF-P may result in opposite regulatory effects of different steps of the virus life cycle.
Cytokines as Potential in vivo Regulators of HIV Expression Although direct proof of a correlation between secre tion of certain cytokines and modulation of HIV expres sion in infected individuals has not yet been provided, several observations support this hypothesis. Increased levels of several cytokines capable of regulating virus
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Cytokine Suppression of HIV Expression in Monocyte/Macrophages
expression in vitro such as TNF-a. IL-6 and IFN-y have been detected in the plasma and/or cerebrospinal fluid of infected individuals [40-42], Some of these cytokines have been demonstrated either by immunocytochcmistry or by in situ hybridization techniques to be increased in the tissues such as the gastrointestinal tract [D. Cotier, pers. communication] and lymph nodes [43] in close proximity to areas where expression of virus was demon strable. In addition, increased in vitro production of TNF-a [44-46], IL-6 [45. 46] and TGF-|3 [47. 48] from either monocytes or B lymphocytes from infected individ uals has been documented. In experiments where B lym phocytes secreting increased levels of TNF-a and IL-6 obtained from lymph nodes of HIV-infected individuals were cocultivated with their autologous T cells, increased production of virus was observed in the absence of exoge nous stimuli [45], It is conceivable that cytokine secretion was responsible at least in part for the observed effect and that similar results could be obtained if monocyte/macrophages instead of B cells were used as the 'inducer' popu lation.
Conclusions Regulation of HIV expression is likely to be the result of a complex network of extracellular signals, such as those provided by certain cytokines, together with tran scriptional and posttranscriptional effects mediated en dogenously by regulatory viral proteins such as tat and rev [8], The monocyte/macrophage system is characterized by a high degree of heterogeneity associated with the dif ferent stages of cell differentiation (bone marrow precur sor cells, circulating monocytes, tissue macrophages). HIV infection of both CD4+ T cells and mononuclear phagocytes results in the profound state of immune sup pression which underlies the clinical symptoms of AIDS. Further definition of the different endogenous and exoge nous mechanisms of regulation of virus expression in these target cells may become of crucial importance for the design of more effective therapeutical strategies against HIV infection.
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