Pathobiology 1992:60:213-218
Pulmonary and Critical Care Section. Department of Medicine, University of Pennsylvania. School of Medicine. Philadelphia, Pa., USA
Human Immunodeficiency Virus Type 1 Tropismfor Human Macrophages
Key Words
Abstract
HIV Monocyte Macrophage AIDS
Human immunodeficiency virus (HIV) infects cells of the monocyte/macrophage lineage in addition to lymphocytes, and infection of these cells may be responsible for viral persistence and dissemination, encephalopathy of the acquired immunodeficiency syndrome and other sequelae of HIV infection. We have developed an in vitro model utilizing peripheral-blood monocytederived macrophages to study HIV-1 infection of macrophages. HIV-1 iso lates vary' greatly in their ability to infect and replicate in macrophages, from highly restricted to highly productive infection. Productively infected macro phages undergo syncytium formation but remain viable in culture and support sustained levels of virus production for prolonged periods. Transformed monocytoid and lymphoid cell lines, however, show very different patterns of permissiveness for HIV-1 strains and do not reflect their corresponding pri mary cell types in studies of host cell tropism. Studies on viral entry show that the CD4 molecule, known to be the HIV receptor on lymphoid cells, is expressed at low levels on the surface of macrophages as well, where it func tions as the receptor for viral entry. Therefore, differential host cell tropism does not result from the use of an alternative macrophage-specific receptor instead of CD4.
Ronald Collman
Macrophage Infection in vivo
Received: June 15. 1991 Accepted: July 18. 1991
Dr. Ronald C oilman. Assistant Professor Pulmonary and Critical Care Section Department of Medicine, University o f Pennsylvania School of Medicine. Philadelphia. PA 19104-4283 (USA)
© 1992 S. Karger AG, Basel 10 15-2008/92/0604-0213 $2.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
In recent years there has been an explosion in the vari ety of cell types recognized as potential targets for infec tion by human immunodeficiency virus (HIV). Most prominent are cells of the monocyte/macrophage lineage, principally tissue macrophages. Brain macrophages (mi croglia) are the major infected cell type in the central ner vous system of HIV-infected individuals, and macro phages (or macrophage-related cells) express viral pro teins and/or nucleic acids in tissues from the lung, cervix.
skin and other organs [1-4]. In contrast, circulating monocytes probably carry only a small fraction of the viral burden in peripheral blood. Virus can be isolated from the monocyte subfraction of peripheral-blood leuko cytes of 30-100% of infected individuals [5-7], but poly merase chain reaction analysis of purified subpopulations indicates that the majority of circulating cells carrying proviral DNA are lymphocytes [8], Similarly, lymphoid precursor cells rather than monocyte/macrophage precur sors are the major bone marrow site of infection [7],
Visna virus, which causes encephalopathy and pneu monitis in sheep, and related ruminant lentiviruses are highly tropic for macrophages and share many biologic, genetic and pathogenic features with HIV [9], Infected macrophages are believed to be responsible for persis tence of visna infection, evasion of immune surveillance and viral dissemination, as well as immunopathologic host responses that lead to brain and lung disease. A simi lar role has been proposed for these cells in human HIV infection, including viral persistence, dissemination and evasion of immune surveillance [9], Recent reports have implicated infected macrophages as sources of soluble factors that are toxic for neural cells [10, 11], If con firmed. these studies would support a role for macrophage infection in HIV encephalopathy. In addition, infected macrophages may be targets for immunopathogenic re sponses in the lymphoid pneumonitis common in chil dren with the acquired immune deficiency syndrome (AIDS) [12]. It is not clear whether dysfunction of in fected macrophages has any part in the genesis of immune deficiency in AIDS, as both positive and negative effects on cellular immune function have been reported [ 13, 14],
HIV-1 Tropism for Macrophages in vitro
Early reports using standard, laboratory-passaged HIV-1 isolates showed a limited ability of these strains to infect human macrophages in vitro, resulting in mini mally productive infection [15, 16], Conversely, Gartner et al. [17], Koyanagi et al. [18] and others showed that some tissue-derived isolates showed a much greater tro pism for cultured macrophages, resulting in productive viral replication. Subsequently, a number of low-passage isolates derived from peripheral blood, cerebrospinal fluid and tissues have been described which infect and replicate in macrophages in vitro [5, 19], We have utilized primary human macrophages in an in vitro model to study HIV infection. Stringently purified peripheral-blood monocytes are maintained in culture to allow differentiation into monocyte-derived macro phages (MDM) in the presence of recombinant macro phage/ and granulocyte/macrophage colony-stimulating factors, which enhance cell survival in vitro [5, 20]. For comparison, peripheral-blood lymphocytes (PBL) are de pleted of monocytes, stimulated with mitogen and main tained with interleukin-2. We examined three prototype HIV-1 isolates, representing different points on the spec-
214
Coliman
Table 1. Replication of prototype HIV-l strains in primary cells and transformed cell lines
Cell type
Monocyte/macrophage Primary MDM U937 cell line T lymphocyte Primary PBL SUP-T1 cell line
Strain IIIB
DV
SF162
± +++++
++ +++++
+++
-H-H-
++ + +
++++
+++-H-
+++++
±
±
Titer of p24 antigen in supernatant: +++++ = > 1,000 ng/ml; ++++ = 100-1.000 ng/ml; +++ =10-100 ng/ml; ++ = 1-10 ng/ml; + = 0.1-1 ng/ml; ± = < 0.1 ng/ml.
trum of macrophage tropism (fig. 1). Strain IIIB is a peripheral-blood isolate extensively passaged in T cell lines, which replicates minimally or not at all in MDM. SF162, isolated from cerebrospinal fluid, replicates to high levels in MDM, with production of both viral anti gen and infectious virus. DV shows an intermediate phe notype. producing modest levels of antigen but little if any infectious virus. In constrast, all three strains replicate well in PBL. To examine whether brain-derived macro phages are susceptible to infection in vitro, Watkins et al. [21 ] recently demonstrated that microglia in human brain cultures show a similar pattern of permissiveness for HIV isolates as MDM. Macrophages from different donors or from the same donor harvested at different times vary significantly in their ability to support viral replication. Although strain IIIB generally produces no antigen or only trace levels, titers of 2-4 ng/ml are occasionally seen (without infec tious virus production). Conversely, SF162 generally pro duces titers of 50-150 ng/ml, but on occasion range from as little as I ng/ml to as high as 1,000 ng/ml. Nonetheless, while absolute levels vary, the relative gradation among isolates remains consistent, from IIIB (least productive) to SF162 (most productive). The factors which underly this variability are not known. Because of the difficulty in working with primary cells in culture (especially human macrophages), a number of transformed human cell lines such as the monocytoid U937 and T lymphocytic SUP-T1 cells have been used for studying HIV infection. Surprisingly, both the U937 and SUP-T1 cells support replication of strains IIIB and DV, but do not support replication of the macrophage-tropic
HIV-1 Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
Macrophage Infection in HIV Pathogenesis
■
Days after infection
Fig. 1. Replication of three prototype strains of HIV-1 (o = SF162;n = DV: A = IIIB) in MDM (A), and PBL (B). Cells in culture were infected with equivalent amounts of virus and monitored for production of viral p24 (gag) antigen in supernatant. All three iso lates replicated well in PBL but showed a gradation in ability to repli cate in MDM. from IIIB (least productive) to SFI62 (most produc tive).
isolate SF162 (fig. 2). In fact, a large percentage of pri mary HIV-l isolates that replicate in PBL but have not been laboratory-passaged do not infect transformed CD4positive T cell lines; similarly, most isolates which infect and replicate in MDM do not productively; infect U937 cells. Therefore, despite many phenotypic characteristics in common with primary cells, these transformed lines do not resemble the corresponding primary cells in patterns of permissiveness for HIV-l replication and are not appropriate surrogates for primary cells in studies of host cell tropism (table l).
Days after infection
Fig. 2. Replication of prototype HIV-I isolates (o = SF162; □ = a = IIIB) in monocytoid U937 (A) and T4-lymphocyte SUP-Tl
DV;
(B) transformed cell lines. Cells were infected with equivalent
amounts of virus and monitored for production of viral p24 (gag) antigen in supernatant. Strain IIIB. which replicates in PBL but not MDM. replicates in both cell lines; strain SF162 replicates in both PBL and MDM but does not replicate in either cell line; strain DV. which replicates in PBL and to intermediate levels in MDM, repli cates to high titer in both cell lines. Thus, the transformed cell lines do not resemble their corresponding primary cells in patterns o f per missiveness for HIV-I isolates.
Characteristics of Macrophage Infection in vitro
The kinetics of infection in macrophages are generally slower than in lymphocytes, requiring up to 2 weeks or longer to reach maximum titer. However, infected macro phages maintain a sustained plateau of viral replication since, unlike lymphocytes, infection of these cells is noncytocidal. even though both result in cell fusion. Unin fected MDM in culture gradually undergo modest degrees of cell fusion and form mullinucleated cells containing up to 6-8 nuclei. HIV-infected cultures undergo much more extensive fusion and develop huge syncytia containing up to 100 nuclei (fig. 3). In contrast to T cell infection where cell fusion leads to cell death, these productively infected macrophage syncytia persist for prolonged periods in cul-
2 15
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
1,000
Fig. 3. Morphology of infected macrophages. MDM infected 3 weeks previously with HIV-1 isolate SF162 were stained by a modi fied Wright-Giemsa method. Infected cultures undergo extensive fusion and form very large syncytia, with dozens of nuclei in each cell (A), although the extent of cell fusion varies from culture to culture. Syncytia remain viable for weeks in culture and produce sustained levels ofinfectious virus. Uninfected MDM in culture gradually form small multinuclcated cells with up to 6-8 nuclei per cell (B). X 250. From (Tollman et al. [22], with permission of the publisher.
1,000
•
100
•
10
100
-
-
10 15 20 Days after infection D
Fig. 4. Viral antigen distribution in infected macrophages. MDM infected 3 weeks earlier with MIV-1 isolate SF162 were fixed and stained for viral antigens by immunofluorescence using polyclonal anti-HIV antiserum. Antigen is seen in single small polykaryons (A), but is most prominent at sites of cell-lo-cell contact (B) and in focal deposits throughout the cytoplasm of syncytia (C). Uninfected con trol culture is shown for comparison (D). X 250. From (Tollman et al. [22], with permission of the publisher.
216
Coliman
Fig. 5. Effect on macrophage infection of anti-CD4 monoclonal antibody (A) and recombinant soluble CD4 (rsCD4; B), A MDM were infected with HIV-I isolate SF162 in the presence of the antiCD4 monoclonal antibody Leu-3a (o), control monoclonal antibody B33.1 (D) or without antibody (A). B MDM were similary infected in the presence (o) or absence (o) of rsCD4. Infection was monitored for production of viral p24 (gag) antigen in supernatant. Both Leu-3a and rsCD4 inhibited MDM infection.
HIV-I Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
C
ture. consistent with the hypothesis that macrophages may be a reservoir for viral persistence in vivo. Within these syncytia, viral antigens are distributed at sites of cell-to-cell contact, suggesting a role in macrophage cell fusion (fig. 4). Antigen is also found in focal deposits throughout the cytoplasm of multinucleated cells, which is consistent with the observation by electron microscopy that maturation of virions in macrophages takes place into membrane-bound vesicles [23] rather than at the cell surface as is the case for lymphocytes.
nant soluble CD4 also blocks CD4-mediated infection by binding to viral envelope [22], As shown in figure 5, both Leu-3a and rsCD4 blocked infection of macrophages by prototype isolate SF162 as well as by two additional mac rophage-tropic strains. Therefore, CD4 is an essential component of the macrophage receptor for HIV-1 and the tropism of certain isolates for macrophages does not result from the use of an alternate macrophage receptor instead of CD4.
Immune Mechanisms of Viral Entry Macrophage Receptor for HIV
A number of studies have reported that, in the pres ence of nonneutralizing or subneutralizing concentrations of immune sera. HIV can enter and infect cellsexpressing receptors for the Fc portion of antibody and/or comple ment. It is controversial as to whether this can occur inde pendently of CD4 [26,27] or whether this pathway simply enhances the efficiency of CD4-dependent infection [28, 29], In addition, the levels of infection or enhancement achieved by this route are relatively low and of unclear significance. An intriguing possibility, however, is that immune enhancement might allow macrophage infection by isolates not inherently tropic for these cells [26],
Conclusions
Much information has been gained from the use of pri mary human MDM in in vitro studies, and this model appears to accurately reflect patterns of tropism for tissue macrophages. HIV isolates vary greatly in their ability to infect and replicate in monocyte/macrophage cells, and the characteristics of macrophage infection are distinct from infection of lymphocytes.
217
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
Numerous reports have shown CD4-independent in fection of a variety of cell types [24. 25], Since all HIV-1 isolates are competent to utilize the CD4 receptor yet only some are capable of productively infecting macrophages, we sought to determine what role the CD4 receptor played in HIV-1 entry into macrophages. By flow cytome try, 30-70% of freshly purified peripheral-blood mono cytes stain for surface CD4 at a level of fluorescence intensity approximately 10-fold lower than that on CD4positive lymphocytes, and immunofluorescence micros copy shows low levels of surface CD4 on approximately one third of mature adherent MDM [24], By Western blot, monocyte/macrophage CD4 is of the same SDSPAGE mobility as lymphocyte CD4. and total cellular levels remain approximately constant over the first 2 weeks in culture. In order to determine the functional role of CD4, we examined the effect on macrophage infection of two agents which interfere with the CD4-HIV interaction and are known to block infection of lymphocytes. Leu-3a is a monoclonal antibody directed against CD4 which com petes with the viral envelope gp 120 for binding. Recombi-
1 Chayal KJ. Harper ME. Marsellc EM. Lewin EB. Rose RM. Oleske JM. Epstein LG. WongStaal F, Gallo RC: Detection of HTLV-III RNA in lungs of patients with AIDS and pul monary involvement. JAMA 1986:256:2356— 2359. 2 Koenig S. Gendelman HE. Orcnstein JM. Dal Canto MC. Pezeshkpour GH. Yungbluth M. .lanotta F. Aksamit A, Martin MA. Fauci AS: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encepha lopathy. Science 1986:233:1089-1093. 3 Pomerantz RJ. Monte SM. Donega P. Rota TR. Vogt MW, Graven DE. Hirsch MS: Hu man immunodeficiency virus (HIV) infection of the uterine cervix. Ann Intern Med 1988: 108:321-327. 4 Stoler MH. Eskin TA, Beim S. Angerer R. Angerer LM: Human T-cell lymphotropic virus type III infection of the central nervous system. JAMA 1986:256:2360-2364. 5 Gendelman HE. Orenstein JM. Martin MA. Ferrua C. Mitra R. Phipps T, Wahl LA, Lane HC. Fauci AS. Burke DS. Skillman D. Meitzer MS: Efficient isolation and propagation of hu man immunodeficiency virus on recombinant colony-stimulating factor-1 treated monocytes. J Exp Med 1988;167:1428-1441. 6 Landay A. Kessler HA. Benson CA. Pottage JC Jr. Murphy R. Urbanski P. Kucilk S. Phair J: Isolation of HIV-I from monocytes of individ uals negative by conventional culture. J Infect Dis 1990:161:706-710. 7 McEIrath MJ. Pruett JE. Cohn ZA: Mononu clear phagocytes of blood and bone marrow: Comparative roles as viral reservoirs in human immunodeficiency virus type I infections. Proc Nall Acad Sei USA 1989:86:675-679. 8 Schnittman SM, Psallidopoulos MC. Lane HC, Thompson L. Baseler 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. 9 Narayan O. Zink MC, Huso D. Sheffer D. Crane S. Kennedy-Stoskopf S, Jolly PE. Cle ments JE: Lentiviruses of animals are biologi cal models of the human immunodeficiency viruses. Microb Pathog 1988:5:149-157. 10 Giulian D. Vaca K. Noonan CA: Secretion of neurotoxins by mononuclear phagocytes in fected with HIV-I. Science 1990:250:1593— 1596.
11 Pulliam LB. Herndier G. Tang NM. McGrath MS: Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical altera tions in cultured human brains. J Clin Invest 1991:87:503-512. 12 Plata F. Autran B. Martins LP. Wain-Hobson S. Raphael M. Mayaud C. Denis M, Guillon JM: Debre P: AIDS virus-specific cytotoxic T lymphocytes in lung disorders. Nature 1987: 328:348-351. 13 Baldwin GC. Fleischmann J. Chung Y. Koyanagi Y. Chen ISY. Golde DW: Human immu nodeficiency virus causes mononuclear phago cyte dysfunction. Proc Nall Acad Sci USA 1990:87:3933-3937. 14 Molina J M. Schindler R, Ferriani R. Sakaguchi M. Vannier E. DinarelloC, Groopman JE: Pro duction of cytokines by peripheral blood monocytes/macrophages infected with human immunodeficiency virus type I (HIV-1). J In fect Dis 1990:161:888-893. 15 Ho D. Rota T, Hirsch MS: Infection of monocyte/macrophages by human T lymphotropic virus type III. J Clin Invest 1986:77:1712— 1715. 16 Nicholson JKA. Cross GD. Callaway CS, McDougal JS: in vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associate virus (HTLV-IIi/ l AV). J Immunol 1986;137:323329. 17 Gartner S. Markovits P. Markovitz DM. Ka plan MH, Gallo RC. Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV in fection. Science 1986;233:215-219. 18 Koyanagi Y. Miles S. Milsuyasu RT. Merrill JE. Vinters HV. Chen ISY: Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 1987: 236:819-822. 19 Cheng-Mayer C. Weiss C. Seto S. Levy JA: Iso lates of human immunodeficiency virus type I from the brain may constitute a special group of the AIDS virus. Proc Natl Acad Sci USA 1989;86:8575-8579. 20 Collman R. Hassan NF. Walker R. Godfrey B. Cutilli J. Hastings JC. Friedman H. Douglas SD. Nathanson N: Infection of monocyte-de rived macrophages with human immunodefi ciency virus type 1: Monocyte-tropic and lym phocyte-tropic strains of HIV-l show distinc tive patterns of replication in a panel of cell types. J Exp Med 1989; 170:1149-1163.
21 Watkins BA. Dorn HH. Kelly WB. Armstrong RC. Potts JB. Michaels F. Kufta CV. DuboisDalcq M: Specific tropism of HIV-I for micro glial cells in primary human brain cultures. Science 1990;249:549-553. 22 Collman R, Godfrey B. Cutilli J. Rhodes A. Hassan NF. Sweet R, Douglas SD. Friedman H, Nathanson N, Gonzalez-Scarano F: Macro phage-tropic strains of human immunodefi ciency virus type I utilize the CD4 receptor. J Virol 1990:64:4468-4476. 23 Orenstein JM. Meltzer MS. Phipps T. Gendelman HE: Cytoplasmic assembly and accumula tion of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulat ing factor-1 treated human monocytes: An ul trastructural study. J Virol 1988:62:2578— 2586. 24 Harouse JM. Kunsch C. Hartle HT. Ijughlin MA. Iloxie JA, Wigdahl B. Gonzalez-Scarano F: CD4-independent infection of human neu ral cells by human immunodeficiency virus type I.J Virol 1989:63:2527-2522. 25 Tateno M. Gonzalez-Scarano F. Levy JA: Hu man immunodeficiency virus can infect CD4negative human fibroblastoid cells. Proc Natl Acad Sci USA 1989:86:4287-4290. 26 Homsy J. Meyer M. Tateno M. Clarkson S, Levy JA: The Fc and not CD4 receptor me diates antibody enhancement of HIV infection in human cells. Science 1989:244:1357-1360. 27 McKeating JA. Griffiths PD. Weiss RA: HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Na ture 1990:343:659-661. 28 Robinson WE Jr, Montefiori DC, Mitchell WM: Complement-mediated antibody-depen dent enhancement of HIV-1 infection requires CD4 and complement receptors. Virology 1990:175:600-604. 29 Takeda A. Sweet RW. Ennis FA: Two receptors are required for antibody-dependent enhance ment of human immunodeficiency virus type 1 infection: CD4 and Fc-gamma. J Virol 1990; 64:5605-5610.
218
Coliman
HIV-I Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
References
Pulmonary and Critical Care Section. Department of Medicine, University of Pennsylvania. School of Medicine. Philadelphia, Pa., USA
Human Immunodeficiency Virus Type 1 Tropismfor Human Macrophages
Key Words
Abstract
HIV Monocyte Macrophage AIDS
Human immunodeficiency virus (HIV) infects cells of the monocyte/macrophage lineage in addition to lymphocytes, and infection of these cells may be responsible for viral persistence and dissemination, encephalopathy of the acquired immunodeficiency syndrome and other sequelae of HIV infection. We have developed an in vitro model utilizing peripheral-blood monocytederived macrophages to study HIV-1 infection of macrophages. HIV-1 iso lates vary' greatly in their ability to infect and replicate in macrophages, from highly restricted to highly productive infection. Productively infected macro phages undergo syncytium formation but remain viable in culture and support sustained levels of virus production for prolonged periods. Transformed monocytoid and lymphoid cell lines, however, show very different patterns of permissiveness for HIV-1 strains and do not reflect their corresponding pri mary cell types in studies of host cell tropism. Studies on viral entry show that the CD4 molecule, known to be the HIV receptor on lymphoid cells, is expressed at low levels on the surface of macrophages as well, where it func tions as the receptor for viral entry. Therefore, differential host cell tropism does not result from the use of an alternative macrophage-specific receptor instead of CD4.
Ronald Collman
Macrophage Infection in vivo
Received: June 15. 1991 Accepted: July 18. 1991
Dr. Ronald C oilman. Assistant Professor Pulmonary and Critical Care Section Department of Medicine, University o f Pennsylvania School of Medicine. Philadelphia. PA 19104-4283 (USA)
© 1992 S. Karger AG, Basel 10 15-2008/92/0604-0213 $2.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
In recent years there has been an explosion in the vari ety of cell types recognized as potential targets for infec tion by human immunodeficiency virus (HIV). Most prominent are cells of the monocyte/macrophage lineage, principally tissue macrophages. Brain macrophages (mi croglia) are the major infected cell type in the central ner vous system of HIV-infected individuals, and macro phages (or macrophage-related cells) express viral pro teins and/or nucleic acids in tissues from the lung, cervix.
skin and other organs [1-4]. In contrast, circulating monocytes probably carry only a small fraction of the viral burden in peripheral blood. Virus can be isolated from the monocyte subfraction of peripheral-blood leuko cytes of 30-100% of infected individuals [5-7], but poly merase chain reaction analysis of purified subpopulations indicates that the majority of circulating cells carrying proviral DNA are lymphocytes [8], Similarly, lymphoid precursor cells rather than monocyte/macrophage precur sors are the major bone marrow site of infection [7],
Visna virus, which causes encephalopathy and pneu monitis in sheep, and related ruminant lentiviruses are highly tropic for macrophages and share many biologic, genetic and pathogenic features with HIV [9], Infected macrophages are believed to be responsible for persis tence of visna infection, evasion of immune surveillance and viral dissemination, as well as immunopathologic host responses that lead to brain and lung disease. A simi lar role has been proposed for these cells in human HIV infection, including viral persistence, dissemination and evasion of immune surveillance [9], Recent reports have implicated infected macrophages as sources of soluble factors that are toxic for neural cells [10, 11], If con firmed. these studies would support a role for macrophage infection in HIV encephalopathy. In addition, infected macrophages may be targets for immunopathogenic re sponses in the lymphoid pneumonitis common in chil dren with the acquired immune deficiency syndrome (AIDS) [12]. It is not clear whether dysfunction of in fected macrophages has any part in the genesis of immune deficiency in AIDS, as both positive and negative effects on cellular immune function have been reported [ 13, 14],
HIV-1 Tropism for Macrophages in vitro
Early reports using standard, laboratory-passaged HIV-1 isolates showed a limited ability of these strains to infect human macrophages in vitro, resulting in mini mally productive infection [15, 16], Conversely, Gartner et al. [17], Koyanagi et al. [18] and others showed that some tissue-derived isolates showed a much greater tro pism for cultured macrophages, resulting in productive viral replication. Subsequently, a number of low-passage isolates derived from peripheral blood, cerebrospinal fluid and tissues have been described which infect and replicate in macrophages in vitro [5, 19], We have utilized primary human macrophages in an in vitro model to study HIV infection. Stringently purified peripheral-blood monocytes are maintained in culture to allow differentiation into monocyte-derived macro phages (MDM) in the presence of recombinant macro phage/ and granulocyte/macrophage colony-stimulating factors, which enhance cell survival in vitro [5, 20]. For comparison, peripheral-blood lymphocytes (PBL) are de pleted of monocytes, stimulated with mitogen and main tained with interleukin-2. We examined three prototype HIV-1 isolates, representing different points on the spec-
214
Coliman
Table 1. Replication of prototype HIV-l strains in primary cells and transformed cell lines
Cell type
Monocyte/macrophage Primary MDM U937 cell line T lymphocyte Primary PBL SUP-T1 cell line
Strain IIIB
DV
SF162
± +++++
++ +++++
+++
-H-H-
++ + +
++++
+++-H-
+++++
±
±
Titer of p24 antigen in supernatant: +++++ = > 1,000 ng/ml; ++++ = 100-1.000 ng/ml; +++ =10-100 ng/ml; ++ = 1-10 ng/ml; + = 0.1-1 ng/ml; ± = < 0.1 ng/ml.
trum of macrophage tropism (fig. 1). Strain IIIB is a peripheral-blood isolate extensively passaged in T cell lines, which replicates minimally or not at all in MDM. SF162, isolated from cerebrospinal fluid, replicates to high levels in MDM, with production of both viral anti gen and infectious virus. DV shows an intermediate phe notype. producing modest levels of antigen but little if any infectious virus. In constrast, all three strains replicate well in PBL. To examine whether brain-derived macro phages are susceptible to infection in vitro, Watkins et al. [21 ] recently demonstrated that microglia in human brain cultures show a similar pattern of permissiveness for HIV isolates as MDM. Macrophages from different donors or from the same donor harvested at different times vary significantly in their ability to support viral replication. Although strain IIIB generally produces no antigen or only trace levels, titers of 2-4 ng/ml are occasionally seen (without infec tious virus production). Conversely, SF162 generally pro duces titers of 50-150 ng/ml, but on occasion range from as little as I ng/ml to as high as 1,000 ng/ml. Nonetheless, while absolute levels vary, the relative gradation among isolates remains consistent, from IIIB (least productive) to SF162 (most productive). The factors which underly this variability are not known. Because of the difficulty in working with primary cells in culture (especially human macrophages), a number of transformed human cell lines such as the monocytoid U937 and T lymphocytic SUP-T1 cells have been used for studying HIV infection. Surprisingly, both the U937 and SUP-T1 cells support replication of strains IIIB and DV, but do not support replication of the macrophage-tropic
HIV-1 Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
Macrophage Infection in HIV Pathogenesis
■
Days after infection
Fig. 1. Replication of three prototype strains of HIV-1 (o = SF162;n = DV: A = IIIB) in MDM (A), and PBL (B). Cells in culture were infected with equivalent amounts of virus and monitored for production of viral p24 (gag) antigen in supernatant. All three iso lates replicated well in PBL but showed a gradation in ability to repli cate in MDM. from IIIB (least productive) to SFI62 (most produc tive).
isolate SF162 (fig. 2). In fact, a large percentage of pri mary HIV-l isolates that replicate in PBL but have not been laboratory-passaged do not infect transformed CD4positive T cell lines; similarly, most isolates which infect and replicate in MDM do not productively; infect U937 cells. Therefore, despite many phenotypic characteristics in common with primary cells, these transformed lines do not resemble the corresponding primary cells in patterns of permissiveness for HIV-l replication and are not appropriate surrogates for primary cells in studies of host cell tropism (table l).
Days after infection
Fig. 2. Replication of prototype HIV-I isolates (o = SF162; □ = a = IIIB) in monocytoid U937 (A) and T4-lymphocyte SUP-Tl
DV;
(B) transformed cell lines. Cells were infected with equivalent
amounts of virus and monitored for production of viral p24 (gag) antigen in supernatant. Strain IIIB. which replicates in PBL but not MDM. replicates in both cell lines; strain SF162 replicates in both PBL and MDM but does not replicate in either cell line; strain DV. which replicates in PBL and to intermediate levels in MDM, repli cates to high titer in both cell lines. Thus, the transformed cell lines do not resemble their corresponding primary cells in patterns o f per missiveness for HIV-I isolates.
Characteristics of Macrophage Infection in vitro
The kinetics of infection in macrophages are generally slower than in lymphocytes, requiring up to 2 weeks or longer to reach maximum titer. However, infected macro phages maintain a sustained plateau of viral replication since, unlike lymphocytes, infection of these cells is noncytocidal. even though both result in cell fusion. Unin fected MDM in culture gradually undergo modest degrees of cell fusion and form mullinucleated cells containing up to 6-8 nuclei. HIV-infected cultures undergo much more extensive fusion and develop huge syncytia containing up to 100 nuclei (fig. 3). In contrast to T cell infection where cell fusion leads to cell death, these productively infected macrophage syncytia persist for prolonged periods in cul-
2 15
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
1,000
Fig. 3. Morphology of infected macrophages. MDM infected 3 weeks previously with HIV-1 isolate SF162 were stained by a modi fied Wright-Giemsa method. Infected cultures undergo extensive fusion and form very large syncytia, with dozens of nuclei in each cell (A), although the extent of cell fusion varies from culture to culture. Syncytia remain viable for weeks in culture and produce sustained levels ofinfectious virus. Uninfected MDM in culture gradually form small multinuclcated cells with up to 6-8 nuclei per cell (B). X 250. From (Tollman et al. [22], with permission of the publisher.
1,000
•
100
•
10
100
-
-
10 15 20 Days after infection D
Fig. 4. Viral antigen distribution in infected macrophages. MDM infected 3 weeks earlier with MIV-1 isolate SF162 were fixed and stained for viral antigens by immunofluorescence using polyclonal anti-HIV antiserum. Antigen is seen in single small polykaryons (A), but is most prominent at sites of cell-lo-cell contact (B) and in focal deposits throughout the cytoplasm of syncytia (C). Uninfected con trol culture is shown for comparison (D). X 250. From (Tollman et al. [22], with permission of the publisher.
216
Coliman
Fig. 5. Effect on macrophage infection of anti-CD4 monoclonal antibody (A) and recombinant soluble CD4 (rsCD4; B), A MDM were infected with HIV-I isolate SF162 in the presence of the antiCD4 monoclonal antibody Leu-3a (o), control monoclonal antibody B33.1 (D) or without antibody (A). B MDM were similary infected in the presence (o) or absence (o) of rsCD4. Infection was monitored for production of viral p24 (gag) antigen in supernatant. Both Leu-3a and rsCD4 inhibited MDM infection.
HIV-I Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
C
ture. consistent with the hypothesis that macrophages may be a reservoir for viral persistence in vivo. Within these syncytia, viral antigens are distributed at sites of cell-to-cell contact, suggesting a role in macrophage cell fusion (fig. 4). Antigen is also found in focal deposits throughout the cytoplasm of multinucleated cells, which is consistent with the observation by electron microscopy that maturation of virions in macrophages takes place into membrane-bound vesicles [23] rather than at the cell surface as is the case for lymphocytes.
nant soluble CD4 also blocks CD4-mediated infection by binding to viral envelope [22], As shown in figure 5, both Leu-3a and rsCD4 blocked infection of macrophages by prototype isolate SF162 as well as by two additional mac rophage-tropic strains. Therefore, CD4 is an essential component of the macrophage receptor for HIV-1 and the tropism of certain isolates for macrophages does not result from the use of an alternate macrophage receptor instead of CD4.
Immune Mechanisms of Viral Entry Macrophage Receptor for HIV
A number of studies have reported that, in the pres ence of nonneutralizing or subneutralizing concentrations of immune sera. HIV can enter and infect cellsexpressing receptors for the Fc portion of antibody and/or comple ment. It is controversial as to whether this can occur inde pendently of CD4 [26,27] or whether this pathway simply enhances the efficiency of CD4-dependent infection [28, 29], In addition, the levels of infection or enhancement achieved by this route are relatively low and of unclear significance. An intriguing possibility, however, is that immune enhancement might allow macrophage infection by isolates not inherently tropic for these cells [26],
Conclusions
Much information has been gained from the use of pri mary human MDM in in vitro studies, and this model appears to accurately reflect patterns of tropism for tissue macrophages. HIV isolates vary greatly in their ability to infect and replicate in monocyte/macrophage cells, and the characteristics of macrophage infection are distinct from infection of lymphocytes.
217
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
Numerous reports have shown CD4-independent in fection of a variety of cell types [24. 25], Since all HIV-1 isolates are competent to utilize the CD4 receptor yet only some are capable of productively infecting macrophages, we sought to determine what role the CD4 receptor played in HIV-1 entry into macrophages. By flow cytome try, 30-70% of freshly purified peripheral-blood mono cytes stain for surface CD4 at a level of fluorescence intensity approximately 10-fold lower than that on CD4positive lymphocytes, and immunofluorescence micros copy shows low levels of surface CD4 on approximately one third of mature adherent MDM [24], By Western blot, monocyte/macrophage CD4 is of the same SDSPAGE mobility as lymphocyte CD4. and total cellular levels remain approximately constant over the first 2 weeks in culture. In order to determine the functional role of CD4, we examined the effect on macrophage infection of two agents which interfere with the CD4-HIV interaction and are known to block infection of lymphocytes. Leu-3a is a monoclonal antibody directed against CD4 which com petes with the viral envelope gp 120 for binding. Recombi-
1 Chayal KJ. Harper ME. Marsellc EM. Lewin EB. Rose RM. Oleske JM. Epstein LG. WongStaal F, Gallo RC: Detection of HTLV-III RNA in lungs of patients with AIDS and pul monary involvement. JAMA 1986:256:2356— 2359. 2 Koenig S. Gendelman HE. Orcnstein JM. Dal Canto MC. Pezeshkpour GH. Yungbluth M. .lanotta F. Aksamit A, Martin MA. Fauci AS: Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encepha lopathy. Science 1986:233:1089-1093. 3 Pomerantz RJ. Monte SM. Donega P. Rota TR. Vogt MW, Graven DE. Hirsch MS: Hu man immunodeficiency virus (HIV) infection of the uterine cervix. Ann Intern Med 1988: 108:321-327. 4 Stoler MH. Eskin TA, Beim S. Angerer R. Angerer LM: Human T-cell lymphotropic virus type III infection of the central nervous system. JAMA 1986:256:2360-2364. 5 Gendelman HE. Orenstein JM. Martin MA. Ferrua C. Mitra R. Phipps T, Wahl LA, Lane HC. Fauci AS. Burke DS. Skillman D. Meitzer MS: Efficient isolation and propagation of hu man immunodeficiency virus on recombinant colony-stimulating factor-1 treated monocytes. J Exp Med 1988;167:1428-1441. 6 Landay A. Kessler HA. Benson CA. Pottage JC Jr. Murphy R. Urbanski P. Kucilk S. Phair J: Isolation of HIV-I from monocytes of individ uals negative by conventional culture. J Infect Dis 1990:161:706-710. 7 McEIrath MJ. Pruett JE. Cohn ZA: Mononu clear phagocytes of blood and bone marrow: Comparative roles as viral reservoirs in human immunodeficiency virus type I infections. Proc Nall Acad Sei USA 1989:86:675-679. 8 Schnittman SM, Psallidopoulos MC. Lane HC, Thompson L. Baseler 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. 9 Narayan O. Zink MC, Huso D. Sheffer D. Crane S. Kennedy-Stoskopf S, Jolly PE. Cle ments JE: Lentiviruses of animals are biologi cal models of the human immunodeficiency viruses. Microb Pathog 1988:5:149-157. 10 Giulian D. Vaca K. Noonan CA: Secretion of neurotoxins by mononuclear phagocytes in fected with HIV-I. Science 1990:250:1593— 1596.
11 Pulliam LB. Herndier G. Tang NM. McGrath MS: Human immunodeficiency virus-infected macrophages produce soluble factors that cause histological and neurochemical altera tions in cultured human brains. J Clin Invest 1991:87:503-512. 12 Plata F. Autran B. Martins LP. Wain-Hobson S. Raphael M. Mayaud C. Denis M, Guillon JM: Debre P: AIDS virus-specific cytotoxic T lymphocytes in lung disorders. Nature 1987: 328:348-351. 13 Baldwin GC. Fleischmann J. Chung Y. Koyanagi Y. Chen ISY. Golde DW: Human immu nodeficiency virus causes mononuclear phago cyte dysfunction. Proc Nall Acad Sci USA 1990:87:3933-3937. 14 Molina J M. Schindler R, Ferriani R. Sakaguchi M. Vannier E. DinarelloC, Groopman JE: Pro duction of cytokines by peripheral blood monocytes/macrophages infected with human immunodeficiency virus type I (HIV-1). J In fect Dis 1990:161:888-893. 15 Ho D. Rota T, Hirsch MS: Infection of monocyte/macrophages by human T lymphotropic virus type III. J Clin Invest 1986:77:1712— 1715. 16 Nicholson JKA. Cross GD. Callaway CS, McDougal JS: in vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associate virus (HTLV-IIi/ l AV). J Immunol 1986;137:323329. 17 Gartner S. Markovits P. Markovitz DM. Ka plan MH, Gallo RC. Popovic M: The role of mononuclear phagocytes in HTLV-III/LAV in fection. Science 1986;233:215-219. 18 Koyanagi Y. Miles S. Milsuyasu RT. Merrill JE. Vinters HV. Chen ISY: Dual infection of the central nervous system by AIDS viruses with distinct cellular tropisms. Science 1987: 236:819-822. 19 Cheng-Mayer C. Weiss C. Seto S. Levy JA: Iso lates of human immunodeficiency virus type I from the brain may constitute a special group of the AIDS virus. Proc Natl Acad Sci USA 1989;86:8575-8579. 20 Collman R. Hassan NF. Walker R. Godfrey B. Cutilli J. Hastings JC. Friedman H. Douglas SD. Nathanson N: Infection of monocyte-de rived macrophages with human immunodefi ciency virus type 1: Monocyte-tropic and lym phocyte-tropic strains of HIV-l show distinc tive patterns of replication in a panel of cell types. J Exp Med 1989; 170:1149-1163.
21 Watkins BA. Dorn HH. Kelly WB. Armstrong RC. Potts JB. Michaels F. Kufta CV. DuboisDalcq M: Specific tropism of HIV-I for micro glial cells in primary human brain cultures. Science 1990;249:549-553. 22 Collman R, Godfrey B. Cutilli J. Rhodes A. Hassan NF. Sweet R, Douglas SD. Friedman H, Nathanson N, Gonzalez-Scarano F: Macro phage-tropic strains of human immunodefi ciency virus type I utilize the CD4 receptor. J Virol 1990:64:4468-4476. 23 Orenstein JM. Meltzer MS. Phipps T. Gendelman HE: Cytoplasmic assembly and accumula tion of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulat ing factor-1 treated human monocytes: An ul trastructural study. J Virol 1988:62:2578— 2586. 24 Harouse JM. Kunsch C. Hartle HT. Ijughlin MA. Iloxie JA, Wigdahl B. Gonzalez-Scarano F: CD4-independent infection of human neu ral cells by human immunodeficiency virus type I.J Virol 1989:63:2527-2522. 25 Tateno M. Gonzalez-Scarano F. Levy JA: Hu man immunodeficiency virus can infect CD4negative human fibroblastoid cells. Proc Natl Acad Sci USA 1989:86:4287-4290. 26 Homsy J. Meyer M. Tateno M. Clarkson S, Levy JA: The Fc and not CD4 receptor me diates antibody enhancement of HIV infection in human cells. Science 1989:244:1357-1360. 27 McKeating JA. Griffiths PD. Weiss RA: HIV susceptibility conferred to human fibroblasts by cytomegalovirus-induced Fc receptor. Na ture 1990:343:659-661. 28 Robinson WE Jr, Montefiori DC, Mitchell WM: Complement-mediated antibody-depen dent enhancement of HIV-1 infection requires CD4 and complement receptors. Virology 1990:175:600-604. 29 Takeda A. Sweet RW. Ennis FA: Two receptors are required for antibody-dependent enhance ment of human immunodeficiency virus type 1 infection: CD4 and Fc-gamma. J Virol 1990; 64:5605-5610.
218
Coliman
HIV-I Tropism for Human Macrophages
Downloaded by: Stockholm University Library 130.237.165.40 - 12/18/2018 7:28:33 AM
References
