American Journal of Pathology, Vol. 138, No. 4, April 1991 Copyight © American Association of Pathologists

Simian Immunodeficiency Virus Infection of Macaque Bone Marrow Macrophages Correlates with Disease Progression In Vivo Masanobu Kitagawa,* Andrew A. Lackner,t Dale J. Martfeld, Murray B. Gardner,* and Satya Dandekart From the Departments of Medical Pathology and Internal Medicine,* and the California Regional Primate Research Center,t University of Califomnia, Davis, California

The pathogenesis of hematopoietic abnormalities associated with infection of susceptible hosts with either simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) is not fully understood To determine if bone marrow cells are infected with SIV and if the pattern of viral infection is correlated with the severity of disease and abnormalities in hematopoiesis 23 SNV-infected rhesus monkeys were examined by immunohistochemistry and in situ hybridization. By immunohistochemistry, only four monkeys werepositiveforSIVcore protein p27, while in situ hybridization revealed viral RNA in the bone marrow of 15 monkeys. Simian immunodeficiency virus RNA was consistently expressed in the bone marrow from monkeys with severe lymphoid depletion (11 of 11), but less so in monkeys with follicular hyperplasia (0 of 2) or mild lymphoid depletion (4 of 10). In animals with mild lymphoid depletion, bone marrow cells infected with SV were mainly mononuclear cells that appeared to be of myelomonocytic lineage. In contrast, monkeys with severe lymphoid depletion had SV RNA localized to larger mononuclear cells with abundant cytoplasm often located in small lucent areas of the stroma These SN RNA -positive mononuclear cells were positive for the macrophage determinant C068 as demonstrated by immunohistochemistry. Furthermore the stage of simian acquired immune deficiency syndrome, as indicated by lymphoid morphology, and SV localization in the bone marrow were correlated with the incidence of anemig bone marrow hyperplasig and abnormal distribution of macrophages in the bone marrow. These results indicate that, in common with other animal lentiviral infections, the

macrophage is a major target of SV infections in the bone marrow. (Am JPathol 1991, 138:921-930)

In addition to immune suppression, most patients infected with the human immunodeficiency virus (HIV) develop abnormalities in hematopoiesis with resulting peripheral blood cytopenias.1-3 The pathogenesis of these abnormalities is unclear and complicated by therapeutic agents intended to suppress HIV that often result in coincident marrow suppression.47 Relevant animal models, particularly in nonhuman primates, would help us to understand the pathogenesis of retrovirus infection of the bone marrow and viral-induced disturbances in hematopoiesis. Leukemogenic retroviruses of chickens, mice, and cats have set the precedents in nature for oncogenic retrovirus infections of the bone marrow and hematopoietic cells, lymphocytes, and megakaryocytes have been the major cell types infected.810 However none of these are optimal animal models of lentivirusassociated hematologic disorders. By contrast, lentiviral infection of sheep pointed to macrophages in the bone marrow as the reservoir of infection.1 1 Lentiviral infections of the bone marrow of nonhuman primates provide yet a better animal model for acquired immune deficiency syndrome (AIDS) due to the close evolutionary relationship between nonhuman primates and humans and their respective lentiviruses. Simian immunodeficiency virus (SIV) is a lentivirus similar to HIV in morphology, genetic sequence, cell tropism, and growth properties.12'13 Experimental infection Dr. Kitagawa's present address is Department of Pathology, Faculty of Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1chrome, Bunkyo-ku, Tokyo 113, Japan. Supported by USPHS grants R29-NS27338, RR00169, RO1A120573, N01-CA37467, and A125900. A. Lackner was supported by USPHS Special Emphasis Research Career Award in Laboratory Animal Science RR00039. Accepted for publication December 14, 1990. Address reprint requests to Dr. Satya Dandekar, Department of Intemal Medicine, School of Medicine, University of California, Davis, CA 95616.

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of rhesus monkeys with SIV from rhesus macaques (Macaca mulatta) (SlVmac) or sooty mangabeys (Cercocebus atys) (SlVSm) results in a uniformly fatal AIDS-like disease. The course of disease, from infection to death, varies from 3 months to more than 2 years, with a median life span of about 7 months.1420 To determine if the bone marrow of SIV-infected rhesus monkeys is infected with SIV in vivo and if viral expression correlates with stage of disease and abnormalities in hematopoiesis, we studied the bone marrow from 23 rhesus monkeys infected with SIV that covered a broad range of nodal pathologic findings progressing from follicular hyperplasia to mild lymphoid depletion to severe lymphoid depletion. We report here that the bone marrow macrophage is the major target cell of SIV infection and that the localization of SIV in the bone marrow correlates with the stage of simian AIDS as indicated by lymphoid morphology and abnormalities in hematopoiesis.

Materials and Methods

Animals, Tissues, and Virus Isolation Twenty-three rhesus macaques (Macaca mulatta) inoculated with SlVmac or SlVsm, prepared as previously

described,21'22 were examined and divided into groups based on peripheral lymph node morphology (Table 1). The classification scheme was modified from that described by Ringler et al.23 Briefly, three categories were used: 1) follicular hyperplasia-4ymph nodes with voluminous, round, or oval follicles, large, sometimes confluent, germinal centers, and thin mantle zones; 2) mild lymphoid depletion-lymph nodes with a paucity of follicles, involuting follicles, and a normal or expanded paracortex; and 3) severe lymphoid depletion-lymph nodes without recognizable follicles, depleted of lymphocytes, and containing multinucleated syncytial giant cells composed of fused macrophages in the sinusoids. By these criteria, 2 of 23 macaques (9%) were classified as follicular hyperplasia, 10 of 23 (43%) had mild lymphoid depletion, and 11 of 23 (48%) had severe lymphoid depletion. Lymphoid morphology previously was shown to correlate well with degree of immunologic abnormalities in HIV- and SIVinduced immunodeficiency syndromes.18'24'25 All the animals were inoculated intravenously with uncloned SIV (15 with SlVmac, 8 with SlVsm) and became persistently infected. SlVmac grown in human peripheral blood mononuclear cells was obtained from Ronald Desorsiers of the New England Primate Center. The source of SlVsm was a captive sooty mangabey in the United States.? The viral dose varied from 10 to 1 04 100% an-

Table 1. Rhesus Monkeys Infected with SIV Categorized by Lymnphoid Morphology atNecropsy Time after infection Age to death SIV (months)* strain and sex Animals (weeks) Morphology of lymph nodes Follicular hyperplasia SIVsM 47 M 47 MMU 21926 SIVMAC 32 13 F MMU 23475 Mild lymphoid depletion SIVSM 48 M 25 MMU 21831 SIVSM 22 48 M MMU 21866 SIVMAC 42 M 27 MMU 21891 SIVSM MMU 22659 20 F 79 SIVMAC MMU 22852 30 M 18 27 F MMU 23217 SIVMAC 37 11 M MMU 23582 SIVMAC 24 MMU 23613 SIVMAC 11 M 13 MMU 23631 SIVMAC 23 M 15 9F MMU 23834 SIVMAC 28 Severe lymphoid depletion including giant cell lymphadenitis SIVSM MMU 16417 144F 18 SIVSM MMU 22654 24 F 16 SIVSM MMU 22657 20 F 15 SIVSM MMU 22704 27 F 19 MMU 23064 SIVMACt 36 M 1 MMU 23091 20 M 37 SIVMAC MMU 23204 SIVMAC 28 M 28 MMU 23215 SIVMAC 18 M 31 MMU 23219 SIVMAC 39 M 36 MMU 23612 SIVMAC 11 F 14 10 M MMU 23664 SIVMAC 38 *

Age at time of inoculation.

t Coinfected with type D retrovirus serotype-1.

Length of viremia (weeks)

46 30 24 21 23 72 16 35 22 11 14 26

18 14 14 19 1 35 26 29 34 13 18

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imal infectious doses as described previously.27 Before inoculation, all animals were negative for type D retrovirus and simian T-lymphotropic virus type 1, except for MMU 23064, which was infected with type D retrovirus serotype-1 (formerly SRV-1 )28 At the time of inoculation, this animal was anemic, thrombocytopenic, and severely depleted of lymphoid cells. Viral isolation was performed as previously described.2122 Briefly, for viral isolation, stimulated rhesus peripheral blood mononuclear cells (PBMC) were cocultured with indicator cells (stimulated fresh human PBMCs or CEM x 174 cells). Production of virus in the culture supematant was monitored weekly by measurement of reverse transcriptase activity as

described.' Seventeen of the twenty-three animals were killed when moribund; the exceptions were two animals that were killed while still healthy (MMU 21926, MMU 23215): one animal (MMU 23064), infected with type D retrovirus serotype-1, was killed 1 week after inoculation with SIV and three animals died unexpectedly (MMU 22654, MMU 23613, and MMU 23631). All necropsies were performed within 4 hours of death. The mean time from inoculation with SIV to death was 27 weeks (1 week to 79 weeks). Bone marrow and lymph nodes were collected immediately after death and were fixed in 10% buffered formalin. Formalin-fixed tissues were embedded in paraffin, sectioned at 4 to 5 ,lm, and examined by light microscopy and processed for immunohistochemistry and in situ hybridization to detect viral expression. Matched tissues from two normal rhesus monkeys served as uninfected controls. An additional control consisted of matched tissues from an SlV-negative rhesus macaque that died of simian AIDS subsequent to infection with type D retrovirus.28

Clinical Hematology Laboratory values were examined once or twice a month from the time of viral inoculation until death. Hemoglobin, hematocrit, mean corpuscular volume, white blood cell count with differential based on blood smear, and platelet count were obtained. Anemia was defined as a hemo-

globin concentration of less than 10 g/dl; leukopenia was defined as peripheral blood white blood cell count of less than 4000/mm3; lymphopenia was defined as lymphoid cell count of less than 2000/mm3; and thrombocytopenia was defined as platelet count of less than 1 50,000/mm3. Hematologic abnormalities occurred at variable times after inoculation and for varying periods of time. Only hematologic abnormalities that existed at the time of death were described in this retrospective study.

Immunohistochemistry To detect viral antigens, formalin-fixed paraffinembedded tissues from the 23 rhesus monkeys were examined using a monoclonal antibody against the major core protein of SIV (p27).30 Another monoclonal antibody KP1 (DAKO, Carpinteria, CA), specific for CD68, a monocyte/macrophage determinant, also was applied on paraffin sections from all monkeys. Immunohistochemical staining was performed using an avidin-biotinylated horseradish peroxidase complex (ABC) technique31 with diaminobenzidine as the chromogen. Control procedures included the substitution of equivalent concentrations of heavy chain-matched monoclonal antibodies of irrelevant specificity and matched tissues from uninfected rhesus monkeys.

In Situ Hybridization In situ hybridization procedures were modifications of those described by Haase et al.32 Paraffin-embedded bone marrow tissues were cut in 4- to 5-,um sections and placed on Histostik (Accurate Chemical and Scientific Corp., Westbury, NY)-coated glass slides. Slides were deparaffinized, hydrated through graded alcohols, and treated with proteinase K (1 mg/ml in 50 mmol/I [millimolar] ethylenediamino tetraacetic acid [EDTA] and 100 mmol/l TRIS-HCI, pH 7.4) for 15 minutes at 370C, rinsed in phosphate-buffered saline (PBS), placed in 0.2 mol/l (molar) TRIS/0.1 mol/l glycine for 15 minutes, dehydrated,

Table 2. Detection of SIVAntigen and RNA in Bone Marrow of RhesusMonkeys Lymphoid morphology of SIV-infected monkeys Follicular Mild Severe Uninfected hyperplasia depletion depletion Immunohistochemistry for SIV p27 0/2* 0/2 0/10 4/11 In situ hybridization for SIV RNA 0/2 0/2 4/10 11/lit localization MN Mac, MN

(MNGC, Mgk, Ly) MN, mononuclear cells in the hematopoietic compartment; Mac, macrophages with abundant cytoplasm in lucent areas of the stromal space; MNGC, multinucleated giant cell; Mgk, megakaryocyte; Ly, lymphoid cell; (, only observed in MMU 23064, a type D retrovirus and SIV coinfected monkey. * Number positive/number tested. t Statistically significant compared to the incidence in monkeys with follicular hyperplasia and mild depletion (P < 0.005 by Chi-square test).

924 Kitagawa et al AJPApril 1991, Vol. 138, No. 4

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and air dried. The hybridization solution contained 50% deionized formamide, 10% dextran sulfate, 50 mmol/l NaH2PO4, 0.6 mol/A NaCI, 0.5 mmol/I EDTA, lx Denhardt's solution, 75 ug/rml Escherichia Coli tRNA, 100 ,ug/ml salmon sperm DNA, and 20 mmol/l dithiothreitol (DTT). A 9-Kb SIVMAC genomic DNA fragment consisting of entire gag, pol, and env regions was obtained.33 It was labeled by nick-translation reaction to obtain a 35S_ labeled DNA probe (specific activity 108 to 109 cpm/,ug) and was added at 1 x 106 cpmI/20 ,u of hybridization solution. Before the addition of salts and DTT, the mixture was heated at 950C for 10 minutes, chilled on ice, and dispensed onto each specimen, which then was placed in a humidification chamber to hybridize overnight at 420C. After removal of coverslips and washing with 50% deionized formamide/2x standard saline citrate (SSC) at 420C, followed by three washes with 2x SSC at room temperature, the slides were dehydrated in 70% and 95% ethanols containing 0.3 mol/l ammonium acetate and air dried. Autoradiography was performed with NTB2 emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 with 0.6 mol/l ammonium acetate. The slides were exposed at 40C for 7 to 21 days, developed with Kodak D-19, and counterstained with hematoxylin. Controls included 1) SIVinfected and uninfected cultured human T cells (HUT 78); 2) matched tissues from uninfected and type D retrovirusinfected rhesus monkeys; 3) pretreatment of sections with RNase to demonstrate that hybridization was to RNA and; 4) hybridization with a nick-translated probe containing only the vector.

Results Immunohistochemical Localization of SIV The major core protein (p27) of SIV was identified in scattered individual cells in the bone marrow of 4 of 23 monkeys (Table 2), all of which had severe lymphoid depletion. The p27-positive cells in the bone marrow had a

round to oval nucleus that sometimes was located eccentrically in cells with abundant cytoplasm (Figure 1a, b).

In Situ Hybridization for SIV RNA In situ hybridization revealed SIV RNA in bone marrow cells of 4 of 10 animals with mild depletion of lymph nodes and in all 11 of the animals with severe depletion (Table 2). In marked contrast to a scattered background of 1 to 2 grains per cell, discrete, focal collections of grains were observed over and around nuclei of scattered large mononuclear cells. In monkeys with mild depletion, hybridization was limited to a few mononuclear cells that appeared to be of myelomonocytic lineage (Figure 2a). In monkeys with severe lymphoid depletion, virus also was present in these cells, but more commonly was observed in mononuclear cells with abundant cytoplasm often located in small lucent areas of the stromal space (Figure 2b). Monkeys with severe depletion had larger numbers of cells with significant hybridization than did monkeys with mild lymphoid depletion. The level of expression in each cell, as indicated by the number of detectable silver grains, however, was not significantly different between monkeys with mild lymphoid depletion and severe lymphoid depletion. In MMU 23064, which had severe lymphoid depletion and was coinfected with type D retrovirus serotype-1 and SIV, hybridization in the bone marrow was evident in individual cells within lymphoid aggregates and scattered cells with abundant cytoplasm and multilobulated nuclei or multiple tightly packed nuclei. The cells with multiple closely packed nuclei were present within lymphoid nodules in the bone marrow and were interpreted to be multinucleated giant cells (Figure 2c). The cells with multilobulated nuclei were present in the hematopoeitic compartment and were interpreted to be megakaryocytes (Figure 2d). No significant collection of grains were observed in immature or mature erythroid cells, myeloid cells, or endothelial cells

Figure 1. Immunohistochemical detection ofthe major coreprotein of SIV(p27) in mononuclear cells with abundant cytopklsm in the bone marrow of (A) an SNV-infected animal with severe lymphoid depletion (MMU 22657; magnification, x625) and; (B) an animal coinfected with type D retrovirus serotype-1 and SIV (MMU 23064; magnification, x625). Figure 2. Detection ofSlVby in situ hybridization in the bone marrow showing (A) hybridization in a mononuclear cell in the hematopoietic compartment in an animal with mild lymphoid depletion (MMU 21891; magnification, x625); (B) hybridization over large mononuclear cells in lucent areas of the stroma in a macaque with severe lymphoid depletion (MMU 23219; magnification, x 160); (C) hybridization in cells with multiple closely packed nuclei, probably a multinucleated giant cell within a lymphoid aggregate (magnification, X250) and (D) multilobulated cells, probably a megakaryocyte, in the hematopoietic compartment (magnification, x250) in MMU 23064, the macaque that was coinfected with type D retrovirus and SIV Figure 3. Two pairs ofserial sections of bone marrow demonstrating localization of SIV nucleic acid by in situ hybridization (a and c) and monocyte/macrophages by immunohistochemistry with KP-1, a CD68-specifc monoclonal antibody (b and d). Figures a and b show an SIV-positive, CD68-positive cell in the hematopoietic compartment (MMU 22657; magnification, x 625). Figures c and d show an SNV-positive, CD68-positive cell associated with the luminal surface of a vessel in the bone marrow (MMU 22654; magniflcation, x625). Arrowheads outline the vessel margins and many unstained erythrocytes can be seen filling its lumen. Figure 4. Immunohistochemistry for the monocyte/macrophage marker CD68 demonstrating the unique localization of monocytel macrophages in the bone marrow of animals that had SIVRNA detected in their bone marrow. Note the presence of CD68-positive cells in lucent areas of the stroma (MMU 22657; magnification, x625).

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in any of the animals. All negative controls, including bone marrow sections from uninfected animals, were negative.

Immunohistochemistry for Macrophages To determine if the cells that expressed SIV-RNA were monocyte/macrophages as they appeared to be morphologically, we performed immunohistochemistry on serial sections of bone marrow adjacent to those used for in situ hybridization. KP-1, a monoclonal antibody specific for the monocyte/macrophage marker CD68, stained most cells that expressed SIV RNA. Two examples are presented in Figure 3. Labeling for CD68 also revealed that hybridization for SIV on the luminal surface of capillary endothelium actually was associated with monocytes (Figure 3c and d). A unique distribution of bone marrow monocyte/ macrophages was seen in association with SIV-RNA in the bone marrow and severe lymphoid depletion. In these animals, CD68-positive monocyte/macrophages were present in lucent areas of the stroma (Figure 4).

Hematology and Bone Marrow Morphology To determine the relation of SIV infection of the bone marrow with abnormalities in hematopoiesis, peripheral blood cell data and morphology of the bone marrow of SIV-infected monkeys were analyzed. Table 3 shows the

frequency of hematologic abnormalities and histopathologic changes in the bone marrow of SIV-infected rhesus monkeys classified according to lymphoid morphology and SIV localization in the bone marrow as determined by in situ hybridization. As seen in this table, the prevalence of anemia, thrombocytopenia, and lymphopenia was correlated with the degree of lymphoid depletion and the extent of SIV in the bone marrow. Leukopenia, however, was not a common abnormality in SIV-infected monkeys. The anemia was normocytic and normochromic at the onset, although, terminally, erythrocytes were microcytic and hypochromic regardless of the development of anemia.

The most common histologic abnormality in the bone marrow of these monkeys was hyperplasia, observed in 15 of 23 monkeys. The bone marrow hyperplasia occurred in the absence of peripheral leukocytosis or any evidence of secondary bacterial infection. All the monkeys with severe lymphoid depletion had hyperplastic bone marrow and yet were anemic and frequently showed lymphopenia, thrombocytopenia, or both. Increased myeloid:erythroid cell ratio, increased megakaryocyte count, and lymphoid aggregates were observed in several monkeys but these features were not correlated with the presence of SIV in the bone marrow. In most monkeys with abundant hybridization for SIV in the bone marrow and in two monkeys without detectable SIV, an abnormal distribution of individual adipocytes was present in the bone marrow. This was characterized by the separation of adipocytes by hematopoietic cells in contrast to normal bone marrow in which small numbers of adipocytes are closely apposed without intervening hematopoietic cells.

Table 3. Frequency of Hemwtologic Abnormalities and Histopathologic Changes of Bone Marrow in SIV-infected Rbesus Monkeys at the Time of Death SIV RNA (+) Bone marrow SIV RNA (-) Mild lymphoid Severe lymphoid Bone marrow depletion depletion Hematologic abnormalities Anemia(Hb < 10) 1/8 0/4 6/11* (Hb < 11) 1/8 2/4 11/lit Leukopenia 1/8 1/4 2/11 Thrombocytopenia 2/8 0/4 4/11 Lymphopenia 3/8 1/4 7/11 Histopathologic changes of bone marrow BM hyperplasia 2/8 2/4 11/11t Increased M:E ratio 3/8 1/4 4/11 Increased Mgk count 2/8 3/4 5/11 Lymphoid aggregation 1/8 1/4 3/11 Abnormal distribution of macrophages 0/8 1/4 11/11§ Abnormal distribution of adipocytes 1/8 1/4 10/111" Anemia, hemoglobin