Tissue-Resident Macrophages Are Productively Infec
http://www.100md.com
病菌学杂志 2005年第8期
Gladstone Institute of Virology and Immunology
Department of Pathology, San Francisco General Hospital, University of California San Francisco, San Francisco, California
Institute of Pathology
Department of Virology, University of Heidelberg, Heidelberg, Germany
ABSTRACT
Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.
TEXT
Most of our knowledge about human immunodeficiency virus type 1 (HIV-1) infection of macrophages has been inferred from experiments conducted with monocyte-derived macrophages (MDMs). Cultured MDMs express CD4, CCR5, and CXCR4 on their surface, albeit at low levels (5). Despite the presence of CXCR4, several reports have demonstrated that MDMs could be productively infected by R5 and R5X4, but not by X4 strains (1, 8, 11, 29, 30). On the basis of these observations, it was generally believed that the tropism of an HIV-1 strain for macrophages was linked to its ability to use CCR5 as a coreceptor. On the basis of an "R5-only" susceptibility of MDMs, a model was proposed in which selection for HIV-1 isolates with CCR5 coreceptor use may occur in vivo during transmission (33). In addition to their postulated role as a selective portal of entry, macrophages constitute a long-term reservoir for HIV-1 in infected individuals (24) and may be critical for central nervous system pathogenesis (21). Remarkably, in rhesus macaques infected with simian/human immunodeficiency virus DH12R, a strain that exclusively uses CXCR4, macrophages sustained high-level viremia in the absence of CD4 T lymphocytes (16-18).
Recently, the dogma of absolute restriction of X4 infection to macrophages has been challenged by the identification of primary isolates that can replicate to high levels in MDMs and that exclusively use CXCR4 (23, 34, 36). Earlier studies analyzed the susceptibility of MDMs to X4 infection by using T-cell line-adapted strains, including NL4-3 (31, 34, 37). Furthermore, it is unclear to what extent in vitro cultures of MDMs (4, 32, 36) reflect the characteristics of tissue macrophages in general terms and in particular in the context of an HIV-1 infection.
In the present study, we investigated whether primary X4 viruses can productively infect tissue macrophages. We conducted HIV-1 infections of explants of human tonsils, which harbor a wide variety of primary cell types including macrophages that, importantly, have terminally differentiated in vivo (12, 25). Primary isolates J130 and UG021 replicate in MDMs and have been shown to exclusively use CXCR4 (34, 36). First, we confirmed the coreceptor usage of expanded viral stocks (data not shown). Next, we demonstrated that J130 and UG021 efficiently replicated in tonsil histocultures as assessed by p24 concentrations in supernatants (data not shown). Infected tissue blocks were harvested at day 11 postchallenge, fixed in paraformaldehyde, and embedded in paraffin. For immunostaining of macrophages, either monoclonal antibody (MAb) PG-M1 to CD68 (15, 22, 26) or MAb HAM56, which detects an as yet undefined molecule (2, 13), were used. By analyses of native tissue sections, macrophages could also be identified by their cytomorphological appearance.
HIV-1 p24 and CD68 double immunohistochemistry Sections were stained with an anti-p24 MAb (Kal-1 at 1:10) and detected with the Envision (+) DAB kit. Subsequently, CD68 (PG-M1 at 1:100; DAKO) was visualized with the Envision alkaline phosphatase and the Vector Red alkaline phosphatase substrate kit.
The mock-infected tonsil sections showed intense brown staining for CD68 of cells with macrophage morphology (Fig. 1). In agreement with the replication data, all infected tonsils displayed robust cellular HIV-1 infection reflected by intense p24 staining. All viruses showed productive infection of cells of lymphocyte morphology, and infection of CD4 T cells was independently demonstrated by flow cytometry as previously described (7, 19, 20) (data not shown). For NL4-3, colocalization of immunohistochemical staining for CD68 and p24 could not be detected (Fig. 1E and F). This is in line with the absence of convincing data for productive NL4-3 infection of MDMs under standard cultivation and infection conditions (1, 8, 11, 30). In contrast, in tonsils infected with primary R5X4 isolate 7/86 and with primary X4 HIV-1 isolates J130 and UG021, colocalization of brown staining for CD68-positive macrophages and intense red p24 staining was frequently detected. These results suggested productive infection of tissue-resident macrophages in ex vivo tonsil histocultures by primary X4 viruses.
HIV-1 RNA in situ hybridization in conjunction with CD68 immunohistochemistry As an independent confirmation, tonsil sections were analyzed by HIV-1 RNA in situ hybridization (9, 10, 35) in conjunction with immunohistochemical identification of macrophages. Sections from NL4-3-infected tonsils hybridized strongly with the HIV-1 RNA probe (Fig. 2). However, there was no colocalization of the hybridization signal and CD68 staining. In contrast, sections infected with 7/86 and primary X4 isolates revealed intense HIV-1 RNA in situ hybridization signals also in CD68-positive macrophages. These findings supported the above-described finding that primary X4 isolates can productively infect tissue macrophages.
Detection and quantification of productively infected tissue macrophages by confocal double-immunofluorescence microscopy We then sought to further corroborate and quantify the productive X4 infection of tissue macrophages. Sections were stained for p24 and CD68 by indirect double immunofluorescence, which allowed rapid and unambiguous identification and quantification of productively infected macrophages (Fig. 3). Analyses were performed in principle as previously described (3). For immunostaining, a mixture of MAbs to p24 (Kal-1) and CD68 (PG-M1) was used. Subsequently, sections were incubated with biotinylated goat anti-mouse immunoglobulin G1 (1:100; Southern Biotech) and sheep anti-mouse immunoglobulin G3 (1:100; Serotec). A final incubation included Cy3-conjugated streptavidin (1:1,000) and Cy2-conjugated donkey anti-sheep antibodies (1:50; Dianova).
For both primary X4 isolates, a significant number of macrophages showed strong p24 staining, reflected by a yellow image in merged pictures. As specificity controls, p24-negative macrophages, as well as p24-positive, CD68-negative lymphocytes, could frequently be detected (Fig. 3). For infections with J130 and UG021, the frequency of p24 and CD68 double-positive macrophages was considerable, ranging from 9.4 to 12.1%. Remarkably, this percentage was in the same range as determined for 7/86 (12.2 to 12.5%) and higher than previously reported for R5 molecular clone 49-5 (6%) (14). The NL4-3-infected tissue showed a large number of p24-positive T lymphocytes that did not colocalize with CD68. Three macrophages in the field of view shown were scored positive (Fig. 3G to I, indicated by arrows and insets) on the basis of weak cytoplasmic staining for p24. In contrast to the primary isolates, strongly p24-positive macrophages could only very rarely be detected in the context of an NL4-3 infection. It is unclear whether this level of p24 staining reflects endocytosed virions or represents a low level of productive infection by NL4-3.
Macrophages in infected tonsils are CD3 negative To address whether the colocalization of p24 and CD68 could, in part, be due to phagocytosis of infected T cells, we performed costaining for CD68 and the T-cell marker CD3 (polyclonal rabbit antiserum [1:20; Diagnostic Biosystems] and Cy5-conjugated donkey anti-rabbit antibody [1:100; Jackson Immunotherapy]). In contrast to p24 staining (Fig. 3, middle row), CD3 staining showed virtually no colocalization with the CD68 marker (Fig. 4). A very rare event of a CD3 signal within a CD68-positive macrophage is highlighted in panel I, possibly reflecting the presence of T-cell fragments within a phagosome. However, the subcellular localization of this signal was quite different from that of p24 in Fig. 1 and 3. This indicates that the considerable level of strongly p24-positive macrophages in X4-infected tonsils is probably not a consequence of engulfment of infected T cells or fusion events with CD4 T cells but further supports the interpretation that these macrophages are productively infected.
Using several independent approaches, the present study establishes the quantitative nature and specificity of the productive infection of tissue macrophages by primary X4 HIV-1 isolates in ex vivo tonsil histocultures. This HIV-1 model system provided a convenient experimental platform for the analysis of macrophages that have differentiated in vivo and are embedded within a natural tissue.
The ability of certain primary X4 viruses and the relative inability of TCLA-X4 viruses to productively infect cells of the monocyte/macrophage lineage were previously found in in vitro MDM cultures (14, 29, 34, 36) and are now shown for tissue macrophages ex vivo. Primary isolates J130 and UG021 can be classified as X4 dualtropic (36) on the basis of their in vitro coreceptor usage and their ex vivo cytotropism. Productive infection of macrophages may be a relatively common (31, 34, 36), although not a universal (6, 27), feature of primary X4 isolates. According to our results, a model proposing in vivo selection for R5 strains on the basis of a postulated exclusive macrophage tropism appears unlikely. This is also supported by studies that identified CD4 T cells as the only simian immunodeficiency virus- or HIV-infected cell type during primary infection (28, 38). Taken together, our data demonstrate that primary X4 isolates productively infect a considerable fraction of macrophages residing within human lymphoid tissue. This shows that factors other than coreceptor usage and macrophage tropism determine the selective transmission of R5 strains in vivo.
ACKNOWLEDGMENTS
We thank Warner Greene and Hans-Georg Kr?usslich for encouragement and support. We thank the members of the surgical staff at Kaiser hospitals (San Rafael, San Francisco, and South San Francisco) for generous assistance in obtaining posttonsillectomy samples. Special thanks to John Carroll, Jack Hull, Chris Goodfellow, Stephen Ordway, and Garry Howard for assistance in the preparation of the manuscript. We are grateful to Cecil Fox and Roland Penzel for excellent technical assistance and Andreas Jekle, Jason Kreisberg, Marielle Cavrois, Christian Callebaut, Peggy Chin, Ann-Marie Roy, Lauren Eckstein, Nico Michel, and Oliver Fackler for valuable discussions.
This work was supported by NIH grants (CA86814 and AI43695) to M.A.G., by the J. David Gladstone Institutes, and by the University of Heidelberg. P.J. is a graduate student in the Biomedical Sciences Program at the University of California, San Francisco.
Present address: Genencor International, Inc., Palo Alto, CA 94304.
REFERENCES
Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284-291.
Adams, C. W., and R. N. Poston. 1990. Macrophage histology in paraffin-embedded multiple sclerosis plaques is demonstrated by the monoclonal pan-macrophage marker HAM-56: correlation with chronicity of the lesion. Acta Neuropathol. 80:208-211.
Autschbach, F., E. Palou, G. Mechtersheimer, C. Rohr, F. Pirotto, N. Gassler, H. F. Otto, B. Schraven, and A. Gaya. 1999. Expression of the membrane protein tyrosine phosphatase CD148 in human tissues. Tissue Antigens 54:485-498.
Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson. 1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types. J. Exp. Med. 170:1149-1163.
Collman, R. G., and Y. Yi. 1999. Cofactors for human immunodeficiency virus entry into primary macrophages. J. Infect. Dis. 179:S422-S426.
Connor, R. I., W. A. Paxton, K. E. Sheridan, and R. A. Koup. 1996. Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1. J. Virol. 70:8758-8764.
Eckstein, D. A., M. P. Sherman, M. L. Penn, P. S. Chin, C. M. De Noronha, W. C. Greene, and M. A. Goldsmith. 2001. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells. J. Exp. Med. 194:1407-1419.
Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Nature 316:262-265.
Fox, C. H., and M. Cottler-Fox. 1993. In situ hybridization for the detection of HIV RNA in cells and tissues, p. 12.8.1-12.8.21. In J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, New York, N.Y.
Fox, C. H., and M. Cottler-Fox. 1993. In situ hybridization in HIV research. Microsc. Res. Tech. 25:78-84.
Gendelman, H. E., J. M. Orenstein, M. A. Martin, C. Ferrua, R. Mitra, T. Phipps, L. A. Wahl, H. C. Lane, A. S. Fauci, D. S. Burke, et al. 1988. Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J. Exp. Med. 167:1428-1441.
Glushakova, S., B. Baibakov, L. B. Margolis, and J. Zimmerberg. 1995. Infection of human tonsil histocultures: a model for HIV pathogenesis. Nat. Med. 1:1320-1322.
Gown, A. M., T. Tsukada, and R. Ross. 1986. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am. J. Pathol. 125:191-207.
Grivel, J. C., M. L. Penn, D. A. Eckstein, B. Schramm, R. F. Speck, N. W. Abbey, B. Herndier, L. Margolis, and M. A. Goldsmith. 2000. Human immunodeficiency virus type 1 coreceptor preferences determine target T-cell depletion and cellular tropism in human lymphoid tissue. J. Virol. 74:5347-5351.
Holness, C. L., and D. L. Simmons. 1993. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81:1607-1613.
Igarashi, T., C. R. Brown, R. A. Byrum, Y. Nishimura, Y. Endo, R. J. Plishka, C. Buckler, A. Buckler-White, G. Miller, V. M. Hirsch, and M. A. Martin. 2002. Rapid and irreversible CD4+ T-cell depletion induced by the highly pathogenic simian/human immunodeficiency virus SHIVDH12R is systemic and synchronous. J. Virol. 76:379-391.
Igarashi, T., C. R. Brown, Y. Endo, A. Buckler-White, R. Plishka, N. Bischofberger, V. Hirsch, and M. A. Martin. 2001. Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): implications for HIV-1 infections of humans. Proc. Natl. Acad. Sci. USA 98:658-663.
Igarashi, T., O. K. Donau, H. Imamichi, M. J. Dumaurier, R. Sadjadpour, R. J. Plishka, A. Buckler-White, C. Buckler, A. F. Suffredini, H. C. Lane, J. P. Moore, and M. A. Martin. 2003. Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages. J. Virol. 77:13042-13052.
Jekle, A., O. T. Keppler, E. De Clercq, D. Schols, M. Weinstein, and M. A. Goldsmith. 2003. In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells. J. Virol. 77:5846-5854.
Jekle, A., B. Schramm, P. Jayakumar, V. Trautner, D. Schols, E. De Clercq, J. Mills, S. M. Crowe, and M. A. Goldsmith. 2002. Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo, leading to increased virulence. J. Virol. 76:6966-6973.
Kaul, M., G. A. Garden, and S. A. Lipton. 2001. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410:988-994.
Micklem, K., E. Rigney, J. Cordell, D. Simmons, P. Stross, H. Turley, B. Seed, and D. Mason. 1989. A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies. Br. J. Haematol. 73:6-11.
Naif, H. M., A. L. Cunningham, M. Alali, S. Li, N. Nasr, M. M. Buhler, D. Schols, E. de Clercq, and G. Stewart. 2002. A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR532 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4. J. Virol. 76:3114-3124.
Orenstein, J. M., C. Fox, and S. M. Wahl. 1997. Macrophages as a source of HIV during opportunistic infections. Science 276:1857-1861.
Penn, M. L., J. C. Grivel, B. Schramm, M. A. Goldsmith, and L. Margolis. 1999. CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue. Proc. Natl. Acad. Sci. USA 96:663-668.
Pulford, K. A., A. Sipos, J. L. Cordell, W. P. Stross, and D. Y. Mason. 1990. Distribution of the CD68 macrophage/myeloid associated antigen. Int. Immunol. 2:973-980.
Rana, S., G. Besson, D. G. Cook, J. Rucker, R. J. Smyth, Y. Yi, J. D. Turner, H. H. Guo, J. G. Du, S. C. Peiper, E. Lavi, M. Samson, F. Libert, C. Liesnard, G. Vassart, R. W. Doms, M. Parmentier, and R. G. Collman. 1997. Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the ccr5 mutation. J. Virol. 71:3219-3227.
Schacker, T., S. Little, E. Connick, K. Gebhard, Z. Q. Zhang, J. Krieger, J. Pryor, D. Havlir, J. K. Wong, R. T. Schooley, D. Richman, L. Corey, and A. T. Haase. 2001. Productive infection of T cells in lymphoid tissues during primary and early human immunodeficiency virus infection. J. Infect. Dis. 183:555-562.
Schmidtmayerova, H., M. Alfano, G. Nuovo, and M. Bukrinsky. 1998. Human immunodeficiency virus type 1 T-lymphotropic strains enter macrophages via a CD4- and CXCR4-mediated pathway: replication is restricted at a postentry level. J. Virol. 72:4633-4642.
Schuitemaker, H., N. A. Kootstra, R. E. de Goede, F. de Wolf, F. Miedema, and M. Tersmette. 1991. Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J. Virol. 65:356-363.
Simmons, G., J. D. Reeves, A. McKnight, N. Dejucq, S. Hibbitts, C. A. Power, E. Aarons, D. Schols, E. De Clercq, A. E. Proudfoot, and P. R. Clapham. 1998. CXCR4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages. J. Virol. 72:8453-8457.
Stent, G., G. B. Joo, P. Kierulf, and B. Asjo. 1997. Macrophage tropism: fact or fiction? J. Leukoc. Biol. 62:4-11.
van't Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, and H. Schuitemaker. 1994. Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J. Clin. Investig. 94:2060-2067.
Verani, A., E. Pesenti, S. Polo, E. Tresoldi, G. Scarlatti, P. Lusso, A. G. Siccardi, and D. Vercelli. 1998. CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates. J. Immunol. 161:2084-2088.
Ward, J. M. 2000. Pathology of genetically engineered mice. Iowa State University Press, Ames.
Yi, Y., S. N. Isaacs, D. A. Williams, I. Frank, D. Schols, E. De Clercq, D. L. Kolson, and R. G. Collman. 1999. Role of CXCR4 in cell-cell fusion and infection of monocyte-derived macrophages by primary human immunodeficiency virus type 1 (HIV-1) strains: two distinct mechanisms of HIV-1 dual tropism. J. Virol. 73:7117-7125.
Yi, Y., S. Rana, J. D. Turner, N. Gaddis, and R. G. Collman. 1998. CXCR-4 is expressed by primary macrophages and supports CCR5-independent infection by dual-tropic but not T-tropic isolates of human immunodeficiency virus type 1. J. Virol. 72:772-777.
Zhang, Z., T. Schuler, M. Zupancic, S. Wietgrefe, K. A. Staskus, K. A. Reimann, T. A. Reinhart, M. Rogan, W. Cavert, C. J. Miller, R. S. Veazey, D. Notermans, S. Little, S. A. Danner, D. D. Richman, D. Havlir, J. Wong, H. L. Jordan, T. W. Schacker, P. Racz, K. Tenner-Racz, N. L. Letvin, S. Wolinsky, and A. T. Haase. 1999. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286:1353-1357.(Prerana Jayakumar, Irina )
Department of Pathology, San Francisco General Hospital, University of California San Francisco, San Francisco, California
Institute of Pathology
Department of Virology, University of Heidelberg, Heidelberg, Germany
ABSTRACT
Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.
TEXT
Most of our knowledge about human immunodeficiency virus type 1 (HIV-1) infection of macrophages has been inferred from experiments conducted with monocyte-derived macrophages (MDMs). Cultured MDMs express CD4, CCR5, and CXCR4 on their surface, albeit at low levels (5). Despite the presence of CXCR4, several reports have demonstrated that MDMs could be productively infected by R5 and R5X4, but not by X4 strains (1, 8, 11, 29, 30). On the basis of these observations, it was generally believed that the tropism of an HIV-1 strain for macrophages was linked to its ability to use CCR5 as a coreceptor. On the basis of an "R5-only" susceptibility of MDMs, a model was proposed in which selection for HIV-1 isolates with CCR5 coreceptor use may occur in vivo during transmission (33). In addition to their postulated role as a selective portal of entry, macrophages constitute a long-term reservoir for HIV-1 in infected individuals (24) and may be critical for central nervous system pathogenesis (21). Remarkably, in rhesus macaques infected with simian/human immunodeficiency virus DH12R, a strain that exclusively uses CXCR4, macrophages sustained high-level viremia in the absence of CD4 T lymphocytes (16-18).
Recently, the dogma of absolute restriction of X4 infection to macrophages has been challenged by the identification of primary isolates that can replicate to high levels in MDMs and that exclusively use CXCR4 (23, 34, 36). Earlier studies analyzed the susceptibility of MDMs to X4 infection by using T-cell line-adapted strains, including NL4-3 (31, 34, 37). Furthermore, it is unclear to what extent in vitro cultures of MDMs (4, 32, 36) reflect the characteristics of tissue macrophages in general terms and in particular in the context of an HIV-1 infection.
In the present study, we investigated whether primary X4 viruses can productively infect tissue macrophages. We conducted HIV-1 infections of explants of human tonsils, which harbor a wide variety of primary cell types including macrophages that, importantly, have terminally differentiated in vivo (12, 25). Primary isolates J130 and UG021 replicate in MDMs and have been shown to exclusively use CXCR4 (34, 36). First, we confirmed the coreceptor usage of expanded viral stocks (data not shown). Next, we demonstrated that J130 and UG021 efficiently replicated in tonsil histocultures as assessed by p24 concentrations in supernatants (data not shown). Infected tissue blocks were harvested at day 11 postchallenge, fixed in paraformaldehyde, and embedded in paraffin. For immunostaining of macrophages, either monoclonal antibody (MAb) PG-M1 to CD68 (15, 22, 26) or MAb HAM56, which detects an as yet undefined molecule (2, 13), were used. By analyses of native tissue sections, macrophages could also be identified by their cytomorphological appearance.
HIV-1 p24 and CD68 double immunohistochemistry Sections were stained with an anti-p24 MAb (Kal-1 at 1:10) and detected with the Envision (+) DAB kit. Subsequently, CD68 (PG-M1 at 1:100; DAKO) was visualized with the Envision alkaline phosphatase and the Vector Red alkaline phosphatase substrate kit.
The mock-infected tonsil sections showed intense brown staining for CD68 of cells with macrophage morphology (Fig. 1). In agreement with the replication data, all infected tonsils displayed robust cellular HIV-1 infection reflected by intense p24 staining. All viruses showed productive infection of cells of lymphocyte morphology, and infection of CD4 T cells was independently demonstrated by flow cytometry as previously described (7, 19, 20) (data not shown). For NL4-3, colocalization of immunohistochemical staining for CD68 and p24 could not be detected (Fig. 1E and F). This is in line with the absence of convincing data for productive NL4-3 infection of MDMs under standard cultivation and infection conditions (1, 8, 11, 30). In contrast, in tonsils infected with primary R5X4 isolate 7/86 and with primary X4 HIV-1 isolates J130 and UG021, colocalization of brown staining for CD68-positive macrophages and intense red p24 staining was frequently detected. These results suggested productive infection of tissue-resident macrophages in ex vivo tonsil histocultures by primary X4 viruses.
HIV-1 RNA in situ hybridization in conjunction with CD68 immunohistochemistry As an independent confirmation, tonsil sections were analyzed by HIV-1 RNA in situ hybridization (9, 10, 35) in conjunction with immunohistochemical identification of macrophages. Sections from NL4-3-infected tonsils hybridized strongly with the HIV-1 RNA probe (Fig. 2). However, there was no colocalization of the hybridization signal and CD68 staining. In contrast, sections infected with 7/86 and primary X4 isolates revealed intense HIV-1 RNA in situ hybridization signals also in CD68-positive macrophages. These findings supported the above-described finding that primary X4 isolates can productively infect tissue macrophages.
Detection and quantification of productively infected tissue macrophages by confocal double-immunofluorescence microscopy We then sought to further corroborate and quantify the productive X4 infection of tissue macrophages. Sections were stained for p24 and CD68 by indirect double immunofluorescence, which allowed rapid and unambiguous identification and quantification of productively infected macrophages (Fig. 3). Analyses were performed in principle as previously described (3). For immunostaining, a mixture of MAbs to p24 (Kal-1) and CD68 (PG-M1) was used. Subsequently, sections were incubated with biotinylated goat anti-mouse immunoglobulin G1 (1:100; Southern Biotech) and sheep anti-mouse immunoglobulin G3 (1:100; Serotec). A final incubation included Cy3-conjugated streptavidin (1:1,000) and Cy2-conjugated donkey anti-sheep antibodies (1:50; Dianova).
For both primary X4 isolates, a significant number of macrophages showed strong p24 staining, reflected by a yellow image in merged pictures. As specificity controls, p24-negative macrophages, as well as p24-positive, CD68-negative lymphocytes, could frequently be detected (Fig. 3). For infections with J130 and UG021, the frequency of p24 and CD68 double-positive macrophages was considerable, ranging from 9.4 to 12.1%. Remarkably, this percentage was in the same range as determined for 7/86 (12.2 to 12.5%) and higher than previously reported for R5 molecular clone 49-5 (6%) (14). The NL4-3-infected tissue showed a large number of p24-positive T lymphocytes that did not colocalize with CD68. Three macrophages in the field of view shown were scored positive (Fig. 3G to I, indicated by arrows and insets) on the basis of weak cytoplasmic staining for p24. In contrast to the primary isolates, strongly p24-positive macrophages could only very rarely be detected in the context of an NL4-3 infection. It is unclear whether this level of p24 staining reflects endocytosed virions or represents a low level of productive infection by NL4-3.
Macrophages in infected tonsils are CD3 negative To address whether the colocalization of p24 and CD68 could, in part, be due to phagocytosis of infected T cells, we performed costaining for CD68 and the T-cell marker CD3 (polyclonal rabbit antiserum [1:20; Diagnostic Biosystems] and Cy5-conjugated donkey anti-rabbit antibody [1:100; Jackson Immunotherapy]). In contrast to p24 staining (Fig. 3, middle row), CD3 staining showed virtually no colocalization with the CD68 marker (Fig. 4). A very rare event of a CD3 signal within a CD68-positive macrophage is highlighted in panel I, possibly reflecting the presence of T-cell fragments within a phagosome. However, the subcellular localization of this signal was quite different from that of p24 in Fig. 1 and 3. This indicates that the considerable level of strongly p24-positive macrophages in X4-infected tonsils is probably not a consequence of engulfment of infected T cells or fusion events with CD4 T cells but further supports the interpretation that these macrophages are productively infected.
Using several independent approaches, the present study establishes the quantitative nature and specificity of the productive infection of tissue macrophages by primary X4 HIV-1 isolates in ex vivo tonsil histocultures. This HIV-1 model system provided a convenient experimental platform for the analysis of macrophages that have differentiated in vivo and are embedded within a natural tissue.
The ability of certain primary X4 viruses and the relative inability of TCLA-X4 viruses to productively infect cells of the monocyte/macrophage lineage were previously found in in vitro MDM cultures (14, 29, 34, 36) and are now shown for tissue macrophages ex vivo. Primary isolates J130 and UG021 can be classified as X4 dualtropic (36) on the basis of their in vitro coreceptor usage and their ex vivo cytotropism. Productive infection of macrophages may be a relatively common (31, 34, 36), although not a universal (6, 27), feature of primary X4 isolates. According to our results, a model proposing in vivo selection for R5 strains on the basis of a postulated exclusive macrophage tropism appears unlikely. This is also supported by studies that identified CD4 T cells as the only simian immunodeficiency virus- or HIV-infected cell type during primary infection (28, 38). Taken together, our data demonstrate that primary X4 isolates productively infect a considerable fraction of macrophages residing within human lymphoid tissue. This shows that factors other than coreceptor usage and macrophage tropism determine the selective transmission of R5 strains in vivo.
ACKNOWLEDGMENTS
We thank Warner Greene and Hans-Georg Kr?usslich for encouragement and support. We thank the members of the surgical staff at Kaiser hospitals (San Rafael, San Francisco, and South San Francisco) for generous assistance in obtaining posttonsillectomy samples. Special thanks to John Carroll, Jack Hull, Chris Goodfellow, Stephen Ordway, and Garry Howard for assistance in the preparation of the manuscript. We are grateful to Cecil Fox and Roland Penzel for excellent technical assistance and Andreas Jekle, Jason Kreisberg, Marielle Cavrois, Christian Callebaut, Peggy Chin, Ann-Marie Roy, Lauren Eckstein, Nico Michel, and Oliver Fackler for valuable discussions.
This work was supported by NIH grants (CA86814 and AI43695) to M.A.G., by the J. David Gladstone Institutes, and by the University of Heidelberg. P.J. is a graduate student in the Biomedical Sciences Program at the University of California, San Francisco.
Present address: Genencor International, Inc., Palo Alto, CA 94304.
REFERENCES
Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J. Virol. 59:284-291.
Adams, C. W., and R. N. Poston. 1990. Macrophage histology in paraffin-embedded multiple sclerosis plaques is demonstrated by the monoclonal pan-macrophage marker HAM-56: correlation with chronicity of the lesion. Acta Neuropathol. 80:208-211.
Autschbach, F., E. Palou, G. Mechtersheimer, C. Rohr, F. Pirotto, N. Gassler, H. F. Otto, B. Schraven, and A. Gaya. 1999. Expression of the membrane protein tyrosine phosphatase CD148 in human tissues. Tissue Antigens 54:485-498.
Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson. 1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types. J. Exp. Med. 170:1149-1163.
Collman, R. G., and Y. Yi. 1999. Cofactors for human immunodeficiency virus entry into primary macrophages. J. Infect. Dis. 179:S422-S426.
Connor, R. I., W. A. Paxton, K. E. Sheridan, and R. A. Koup. 1996. Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1. J. Virol. 70:8758-8764.
Eckstein, D. A., M. P. Sherman, M. L. Penn, P. S. Chin, C. M. De Noronha, W. C. Greene, and M. A. Goldsmith. 2001. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells. J. Exp. Med. 194:1407-1419.
Fisher, A. G., E. Collalti, L. Ratner, R. C. Gallo, and F. Wong-Staal. 1985. A molecular clone of HTLV-III with biological activity. Nature 316:262-265.
Fox, C. H., and M. Cottler-Fox. 1993. In situ hybridization for the detection of HIV RNA in cells and tissues, p. 12.8.1-12.8.21. In J. Coligan, A. Kruisbeek, D. Margulies, E. Shevach, and W. Strober (ed.), Current protocols in immunology. John Wiley & Sons, New York, N.Y.
Fox, C. H., and M. Cottler-Fox. 1993. In situ hybridization in HIV research. Microsc. Res. Tech. 25:78-84.
Gendelman, H. E., J. M. Orenstein, M. A. Martin, C. Ferrua, R. Mitra, T. Phipps, L. A. Wahl, H. C. Lane, A. S. Fauci, D. S. Burke, et al. 1988. Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. J. Exp. Med. 167:1428-1441.
Glushakova, S., B. Baibakov, L. B. Margolis, and J. Zimmerberg. 1995. Infection of human tonsil histocultures: a model for HIV pathogenesis. Nat. Med. 1:1320-1322.
Gown, A. M., T. Tsukada, and R. Ross. 1986. Human atherosclerosis. II. Immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am. J. Pathol. 125:191-207.
Grivel, J. C., M. L. Penn, D. A. Eckstein, B. Schramm, R. F. Speck, N. W. Abbey, B. Herndier, L. Margolis, and M. A. Goldsmith. 2000. Human immunodeficiency virus type 1 coreceptor preferences determine target T-cell depletion and cellular tropism in human lymphoid tissue. J. Virol. 74:5347-5351.
Holness, C. L., and D. L. Simmons. 1993. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81:1607-1613.
Igarashi, T., C. R. Brown, R. A. Byrum, Y. Nishimura, Y. Endo, R. J. Plishka, C. Buckler, A. Buckler-White, G. Miller, V. M. Hirsch, and M. A. Martin. 2002. Rapid and irreversible CD4+ T-cell depletion induced by the highly pathogenic simian/human immunodeficiency virus SHIVDH12R is systemic and synchronous. J. Virol. 76:379-391.
Igarashi, T., C. R. Brown, Y. Endo, A. Buckler-White, R. Plishka, N. Bischofberger, V. Hirsch, and M. A. Martin. 2001. Macrophage are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): implications for HIV-1 infections of humans. Proc. Natl. Acad. Sci. USA 98:658-663.
Igarashi, T., O. K. Donau, H. Imamichi, M. J. Dumaurier, R. Sadjadpour, R. J. Plishka, A. Buckler-White, C. Buckler, A. F. Suffredini, H. C. Lane, J. P. Moore, and M. A. Martin. 2003. Macrophage-tropic simian/human immunodeficiency virus chimeras use CXCR4, not CCR5, for infections of rhesus macaque peripheral blood mononuclear cells and alveolar macrophages. J. Virol. 77:13042-13052.
Jekle, A., O. T. Keppler, E. De Clercq, D. Schols, M. Weinstein, and M. A. Goldsmith. 2003. In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells. J. Virol. 77:5846-5854.
Jekle, A., B. Schramm, P. Jayakumar, V. Trautner, D. Schols, E. De Clercq, J. Mills, S. M. Crowe, and M. A. Goldsmith. 2002. Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo, leading to increased virulence. J. Virol. 76:6966-6973.
Kaul, M., G. A. Garden, and S. A. Lipton. 2001. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410:988-994.
Micklem, K., E. Rigney, J. Cordell, D. Simmons, P. Stross, H. Turley, B. Seed, and D. Mason. 1989. A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies. Br. J. Haematol. 73:6-11.
Naif, H. M., A. L. Cunningham, M. Alali, S. Li, N. Nasr, M. M. Buhler, D. Schols, E. de Clercq, and G. Stewart. 2002. A human immunodeficiency virus type 1 isolate from an infected person homozygous for CCR532 exhibits dual tropism by infecting macrophages and MT2 cells via CXCR4. J. Virol. 76:3114-3124.
Orenstein, J. M., C. Fox, and S. M. Wahl. 1997. Macrophages as a source of HIV during opportunistic infections. Science 276:1857-1861.
Penn, M. L., J. C. Grivel, B. Schramm, M. A. Goldsmith, and L. Margolis. 1999. CXCR4 utilization is sufficient to trigger CD4+ T cell depletion in HIV-1-infected human lymphoid tissue. Proc. Natl. Acad. Sci. USA 96:663-668.
Pulford, K. A., A. Sipos, J. L. Cordell, W. P. Stross, and D. Y. Mason. 1990. Distribution of the CD68 macrophage/myeloid associated antigen. Int. Immunol. 2:973-980.
Rana, S., G. Besson, D. G. Cook, J. Rucker, R. J. Smyth, Y. Yi, J. D. Turner, H. H. Guo, J. G. Du, S. C. Peiper, E. Lavi, M. Samson, F. Libert, C. Liesnard, G. Vassart, R. W. Doms, M. Parmentier, and R. G. Collman. 1997. Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the ccr5 mutation. J. Virol. 71:3219-3227.
Schacker, T., S. Little, E. Connick, K. Gebhard, Z. Q. Zhang, J. Krieger, J. Pryor, D. Havlir, J. K. Wong, R. T. Schooley, D. Richman, L. Corey, and A. T. Haase. 2001. Productive infection of T cells in lymphoid tissues during primary and early human immunodeficiency virus infection. J. Infect. Dis. 183:555-562.
Schmidtmayerova, H., M. Alfano, G. Nuovo, and M. Bukrinsky. 1998. Human immunodeficiency virus type 1 T-lymphotropic strains enter macrophages via a CD4- and CXCR4-mediated pathway: replication is restricted at a postentry level. J. Virol. 72:4633-4642.
Schuitemaker, H., N. A. Kootstra, R. E. de Goede, F. de Wolf, F. Miedema, and M. Tersmette. 1991. Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J. Virol. 65:356-363.
Simmons, G., J. D. Reeves, A. McKnight, N. Dejucq, S. Hibbitts, C. A. Power, E. Aarons, D. Schols, E. De Clercq, A. E. Proudfoot, and P. R. Clapham. 1998. CXCR4 as a functional coreceptor for human immunodeficiency virus type 1 infection of primary macrophages. J. Virol. 72:8453-8457.
Stent, G., G. B. Joo, P. Kierulf, and B. Asjo. 1997. Macrophage tropism: fact or fiction? J. Leukoc. Biol. 62:4-11.
van't Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, and H. Schuitemaker. 1994. Macrophage-tropic variants initiate human immunodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J. Clin. Investig. 94:2060-2067.
Verani, A., E. Pesenti, S. Polo, E. Tresoldi, G. Scarlatti, P. Lusso, A. G. Siccardi, and D. Vercelli. 1998. CXCR4 is a functional coreceptor for infection of human macrophages by CXCR4-dependent primary HIV-1 isolates. J. Immunol. 161:2084-2088.
Ward, J. M. 2000. Pathology of genetically engineered mice. Iowa State University Press, Ames.
Yi, Y., S. N. Isaacs, D. A. Williams, I. Frank, D. Schols, E. De Clercq, D. L. Kolson, and R. G. Collman. 1999. Role of CXCR4 in cell-cell fusion and infection of monocyte-derived macrophages by primary human immunodeficiency virus type 1 (HIV-1) strains: two distinct mechanisms of HIV-1 dual tropism. J. Virol. 73:7117-7125.
Yi, Y., S. Rana, J. D. Turner, N. Gaddis, and R. G. Collman. 1998. CXCR-4 is expressed by primary macrophages and supports CCR5-independent infection by dual-tropic but not T-tropic isolates of human immunodeficiency virus type 1. J. Virol. 72:772-777.
Zhang, Z., T. Schuler, M. Zupancic, S. Wietgrefe, K. A. Staskus, K. A. Reimann, T. A. Reinhart, M. Rogan, W. Cavert, C. J. Miller, R. S. Veazey, D. Notermans, S. Little, S. A. Danner, D. D. Richman, D. Havlir, J. Wong, H. L. Jordan, T. W. Schacker, P. Racz, K. Tenner-Racz, N. L. Letvin, S. Wolinsky, and A. T. Haase. 1999. Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286:1353-1357.(Prerana Jayakumar, Irina )