Receptor CCR5 - Europe PMC

American Journal of Pathology, Vol. 151, No. 5, November 1997 Copyright American Society for Investigative Pathology

Cellular Localization of the Chemokine Receptor CCR5 Correlation to Cellular Targets of HIV- 1 Infection

James B. Rottman,* Kenneth P. Ganley,* Kenneth Williams,t Lijun Wu,* Charles R. Mackay,* and Douglas J. Ringler* From LeukoSite Inc, Cambridge, and the Division of Comparative Pathology,t New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts *

The chemokine receptor CCR5 has recently been described as a co-receptor for macrophage-tropic strains of human immunodeficiency virus (HIV)-l. In this study, using a panel of monoclonal antibodies specific for human CCR5, we show by immunohistochemistry and flow cytometry that CCR5 is expressed by bone-marrow-derived cells known to be targets for HV-1 infection, including a subpopulation of lymphocytes and monocyte/macrophages in blood, primary and secondary lymphoid organs, and noninflamed tissues. In the central nervous system, CCR5 is expressed on neurons, astrocytes, and microglla In other tissues, CCR5 is expressed on epithelium, endothelium, vascular smooth muscle, and fibroblasts. Chronically Inflamed tissues contain an increased number of CCR5+ mononuclear cells, and the number of immunoreactive cells is directly associated with a histopathological correlate of Inflamtory severity. Collectively, these results suggest that CCR5+ cells are recruited to inflammatory sites and, as such, may facilitate transmission of macrophagetropic strains of HIV-1. (Am J Pathol 1997, 151:1341-1351)

Chemokines are a family of structurally related, low molecular weight proteins that play a role in leukocyte chemotaxis, emigration, and activation (For review, see Springer1 or Furie et a12). There are two families of chemokines currently recognized, each designated by the presence or absence of an amino acid separating a cysteine pair within the primary structure. These families include the C-X-C chemokines, which primarily act on neutrophils and include the prototypic chemokines IL-8 and GRO-a, and the C-C family, including chemokines primarily acting on mononuclear cells and eosinophils,

such as monocyte chemoattractant protein (MCP)-1, RANTES, and eotaxin.34 The ability of a leukocyte to respond to a given chemokine is dependent on surface expression of the appropriate receptor (CXCR and CCR, respectively). Chemokine receptors are G-protein coupled, seven-transmembrane-spanning proteins.5 Genes encoding chemokine receptors are often closely linked and share significant nucleotide sequence homology. For example, CCR1, CCR2, and the recently cloned CCR5 are all located on chromosome 3p21l6 In addition, members of the CCR family share >48% amino acid homology with other members of the CCR group.7 Yet, despite this sequence homology, functional divergence has been demonstrated between closely related CCR members. As an example, although CCR2b and CCR5 share 75% amino acid homology, they bind a different spectrum of ligands, including MCP-1 and MCP-3 for CCR2b versus RANTES, MIP-1la, and MIP-1,B for CCR5.6 Recently, it has been shown that the CCR5 ligands RANTES, MIP-la, and MIP-1,B are potent inhibitors of human immunodeficiency virus (HIV)-1 infection in vitro.6 Subsequent to this observation, several groups have demonstrated that CCR5 is a major co-receptor for macrophage-tropic, non-syncytium-inducing (non-SI) HIV-1 primary isolates.9-12 In contrast, laboratory-cell-lineadapted, syncytium-inducing (SI) strains of HIV-1 use a different co-receptor, CXCR4 (also known as fusin, HUMSTR, or LESTR).13 Of the aforementioned receptors, CCR5 may play a particularly important role during the early stages of HIV-1 infection, because macrophagetropic, non-SI strains appear to be preferentially transmitted by sexual contact,14 and individuals with a homozygous 32-bp deletion in the CCR5 gene appear to be more resistant to HIV-1 infection.15 The experimental infection of rhesus macaques with the simian immunodeficiency virus (SIV) is a relevant animal model of human AIDS and, like HIV-1, macrophage-tropic SIV isolates use CCR5 as a co-receptor.16-18 Analysis of the structural requirements for CCR5 utilization by macrophage-tropic and T-cell-tropic SIV Supported in part by Public Health Service grants RR00168 and NS35732 and a grant from the National Multiple Sclerosis Society (RG 2856-A-1). Accepted for publication July 28, 1997. Address reprint requests to Dr. James B. Rottman, LeukoSite Inc., 215 First Street, Cambridge, MA 02142.

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AJP November 1997, Vol. 151, No. 5

isolates has revealed that env (gpl2O) of macrophagetropic SIV strains binds the CCR5 amino terminus, and env of T-cell-tropic strains binds the second extracellular loop of CCR5.17 Thus, HIV-1 and SIV tropism may be determined by the repertoire of co-receptors used by a given virus strain as well as cofactor domains that interact with env.17 Expression of CCR5 in vivo has been incompletely defined. CCR5 mRNA expression has been demonstrated in a variety of cells including granulocyte precur-

gradient and centrifuged at 15,000 rpm for 30 minutes. Dissociated cells at the interface were removed, washed twice with PBS, and adjusted to a concentration of 106/ ml, and 100 p.l was used to prepare cytospin preparations. The cytospin preparations of freshly isolated cells were dried, wrapped individually in aluminum foil, and then frozen at -80°C until used for immunohistochemical staining.

sors,19 primary monocyte/macrophages,6 9 10 20 primary

Immunohistochemistry and Monoclonal Antibodies

T cells,10 and CD4+ and CD8+ lymphocytes.620 CCR5 mRNA has also been detected in the thymus, spleen, and small intestine.6 However, expression of CCR5 by specific parenchymal cells in tissues has not, to our knowledge, been reported. In this study, using a panel of specific monoclonal antibodies raised against human CCR5, we examined various normal and inflamed tissues from both humans and macaques by immunohistochemistry and flow cytometry to define the cellular expression of CCR5. In both species, CCR5 was expressed by small numbers of lymphocytes and monocyte/macrophages in blood, primary and secondary lymphoid organs, and noninflamed tissues. Within the central nervous system, CCR5 expression was detected on neurons, astrocytes, and microglia. Endothelium and vascular smooth muscle were variably immunoreactive for CCR5 in all tissues examined. Using immunohistochemical staining and morphometric analysis on sections of normal and inflamed human colon, there were increased numbers of CCR5+ mononuclear leukocytes in inflamed mucosa compared with normal mucosa. Collectively, these results demonstrate that CCR5 is expressed by specific bone-marrow-derived and resident cells within various tissues and that sites of chronic inflammation have increased numbers of potential cellular targets for HIV-1 infection.

Materials and Methods Tissues Human tissues (normal and inflamed) were obtained from the National Disease Research Institute, a service organization funded by the National Institutes of Health. Normal rhesus macaque tissues were obtained from the New England Regional Primate Research Center.

Isolation of Macaque Glial Cells A detailed description of this protocol has been previously published.21 Briefly, macaque brains were collected at necropsy immediately after euthanasia and prepared by removing the dura and arachnoid, mechanically dissociating the tissue with scalpel blades, and then treating the tissue with trypsin (0.25%) and DNAse (50 ,ug/ml) at 370C for 45 minutes. Dissociated tissue was then filtered through a 130-,um nylon mesh and washed twice in phosphate-buffered saline (PBS). The cell suspension was then layered over a discontinuous Percoll

Monoclonal antibodies specific for CCR5 were generated and determined to be specific for CCR5 by flow cytometric analysis of CCR5 transfectants and transfectant cells expressing other chemokine receptors.20 Immunohistochemical analysis for CCR5 was performed on frozen tissue samples. Briefly, tissue was sectioned at a thickness of 4 ,um, desiccated, and then fixed in 2% paraformaldehyde/0.5X PBS for 10 minutes at 4°C. After PBS washing, nonspecific antibody binding sites were blocked with 10% normal goat serum/5% human AB serum/PBS for 30 minutes at room temperature. Next the purified CCR5 murine antibodies (2F9, 3A9, or 5C7, all isotype IgG2a) or anti-cytokeratin 19 (clone RCK108, Dako, Carpinteria, CA) were diluted to a concentration of 10 ,ug/ml in 0.3% Triton X-100/0.2% Tween 20/1% fetal calf serum/5% human AB serum/0. 1% sodium azide and applied to tissue sections overnight at 40C. An isotypematched irrelevant monoclonal antibody was used as a negative control on step sections of tissues (for CCR5 antibodies, UPC-10 (IgG2a); and for RCK108, MOPC21 (IgGl) from Sigma Chemical Co., St. Louis, MO). Subsequently, biotinylated goat anti-mouse IgG and, in most cases, avidin-biotin-alkaline phosphatase complexes (Biogenex, San Ramon, CA) were added in sequence. Fast Red (Biogenex), containing 2% levamisole to block endogenous alkaline phosphatase activity, was used as the chromogen and Mayer's hematoxylin as the counterstain. Brain tissue sections and isolated glial cells were also stained with an immunoperoxidase technique. Tissue preparation and application of primary and secondary antibodies were performed as above. Additional celltype-specific antibodies included anti-CD68 (EBM1 1, Dako) and polyclonal rabbit anti-glial fibrillary acidic protein (anti-GFAP; Dako). After secondary antibody application (biotinylated goat anti-rabbit for anti-GFAP detection; Vector Laboratories, Burlingame, CA), avidinbiotin-immunoperoxidase complexes (ABC standard; Vector) were applied to the sections or cells, and color development was achieved with the chromogen diaminobenzidine (Sigma). To examine specificity of staining, the antibody 2F9 was incubated at 10 p.g/ml for 3 hours at room temperature with 1 08/ml human CCR5 L1 .2 transfectants, induced with butyric acid to express high levels of CCR5. As a control, antibody 2F9 was incubated with nontransfected L1.2 cells or culture media under similar conditions. After incubation, cell suspensions were centrifuged to collect

Cellular Localization of CCR5

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cells at the bottom of the tube, and the supernatant was applied directly to tissues and developed using the immunoperoxidase technique described above. After immunohistochemical staining, quantitative, computer-assisted image analysis was performed on frozen colon sections using a Leica Quantimet 500 Image Analyzer (Leica, Deerfield, IL). Three random sites of lamina propria from each section were surveyed using the 40x objective lens. The number of immunoreactive cells was enumerated on the basis of percentage of immunoreactive area per field as previously described.22

Flow Cytometry Preparation of Human and Macaque Whole Blood Whole blood was collected into EDTA tubes and kept at room temperature until processing. Aliquots of 50 ,ul were distributed to FACS tubes containing 50 ,l of blocking solution (PBS containing 10% normal goat serum and 5% human AB serum) and incubated at 40C for 15 minutes.

Isolation of Macaque Lymph Node, Spleen, and Thymus Mononuclear Cells

Freshly isolated lymph node, spleen, or thymus were placed in a petri dish containing a small amount of RPMI 1640 (Life Technologies, Gaithersburg, MD), minced into 3-mm cubes, and ground through a wire mesh. The cell suspension was layered over Ficoll (Histopaque, Sigma), specific gravity 1.077, and centrifuged at 800 x g for 30 minutes. Cells at the RPMI/Ficoll interface were removed and washed twice with PBS. Fresh mononuclear cells were incubated in blocking solution at a concentration of 1 x 107/ml at 40C for 15 minutes. Culture of Lymph Node Mononuclear Cells

Isolated mononuclear cells were cultured in RPMI 1640 (Life Technologies), 10% fetal calf serum (Life Technologies), and 10 ,ug/ml concanavalin (Con) A (Sigma) at a concentration of 5 x 1 06/ml with 5% C02 at 370C. After 48 hours in culture, the medium was replaced with RPMI 1640/10% fetal calf serum and 1% (v/v) of a culture supernatant derived from rat lymphoblasts stimulated with Con A as a source of interleukin (IL)-2.

Staining for FACS Analysis One hundred microliters of whole blood or cell suspensions was added to tubes containing specific (5C7) or irrelevant (IgG2a, UPC10; Sigma) primary antibodies in blocking solution so the final concentration was 10 ,ug/ml. Cell suspensions were incubated at 40C for 20 minutes, washed twice with PBS, and subsequently incubated with secondary antibody (goat anti-mouse-phycoerythrin; Jackson lmmunoresearch, West Grove, PA) at a final concentration of 10 jug/ml in blocking solution at 40C for 15 minutes. Cell suspensions were washed once with

PBS and subsequently with mouse IgG2a at 2 ,tg/ml in blocking solution at 40C for 15 minutes to block exposed valence sites of the secondary antibody. After an additional PBS wash, select tubes were incubated with fluorescein-isothiocyanate- conjugated antibodies against CD3 (Biosource, Camarillo, CA); CD4, CD8, CD14, and CD20 (Becton Dickinson, San Jose, CA); and CD25 and CD45a (Coulter, Miami, FL) in blocking solution at 40C for 15 minutes. Subsequently, 2 ml of FACS lysis solution was added, and tubes were incubated for 10 minutes at room temperature. After two final PBS washes, cells were resuspended in 300 ,l of PBS with 1% fetal calf serum and stored at 40C until analyzed. FACS Analysis Two-color analysis was performed on a Becton Dickinson FACScan flow cytometer. Gating for lymphocytes and granulocytes was based on forward and side scatter characteristics. The monocyte gate was based on identification of CD14+ cells with appropriate forward and side scatter characteristics. For each sample, a minimum of 10,000 cells was counted. For determination of the percentage of CCR5+ cells in two-color experiments, dot-plot quadrants were set based on fluorescence characteristics of cells stained with the isotype-matched irrelevant antibody.

Results Monoclonal Antibodies 2F9, 3A9, and 5C7 Recognize CCR5 on a Similar Population of Peripheral Blood Leukocytes in Both Humans and Macaques The monoclonal antibodies 2F9, 3A9, and 5C7 were first used to stain human and macaque peripheral blood to determine whether the reagents would be useful in species other than humans and also to determine whether differences in CCR5 expression by specific cell types existed between species. Using flow cytometry, all three antibodies recognized macaque lymphocyte CCR5, and antibody 5C7 was chosen for additional flow cytometry studies. As we previously reported,20 CCR5 staining on human lymphocytes was relatively bright with the mean channel fluorescence reaching 1.5 logs above background (Figure 1A). In contrast, CCR5 staining on macaque lymphocytes was approximately one-half log less intense (Figure 1 B). To determine which leukocytes in macaque peripheral blood expressed CCR5, two-color flow cytometric experiments were performed. For these studies, whole blood cells from three rhesus macaques were stained with CD3, CD4, CD8, CD14, CD20, CD25, CD45RA, and CCR5, and results from one animal are demonstrated in Figure 2. Between 5 and 20% of macaque total lymphocytes were CCR5+ (Table 1, blood), and >90% of these cells were CCR5+CD3+ T cells (Figure 2A). CCR5+CD20+ B cells were not detected (data not shown). Additionally, small numbers of

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Rottman et al


A

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13%

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10 10 1 1010 01l:

1010

101 lO

CCR5 Figure 1. Flow cytometric analysis of CCR5 expression on human and macaque peripheral blood lymphocytes as delineated by light scatter characteristics. Fluorescence intensity is greater in the humian sample (A) than the macaque sample (B).

macaque lymphocytes expressed CCR5 and other CD markers including CD4 (2%; Figure 2B), CD8 (4.5%; Figure 2C), CD25 (1%; Figure 2D), and CD45RA (3%; Figure 2E). Thus, CCR5 expression on macaque lymphocyte subpopulations is similar to data we previously reported for hu-

mans.20 The percentage of human monocytes (three individuals) expressing CCR5 varied between individuals from 25 to 40%, but the mean channel fluorescence was only slightly above background (Figure 3A). In contrast, we were unable to detect CCR5 expression on macaque peripheral blood monocytes (Figure 3B). Finally, as we previously demonstrated for humans, CCR5 was not detected on macaque neutrophils or eosinophils (not shown). To determine whether CCR5 expression on macaque lymphocytes can be increased with cellular activation, we performed two-color flow cytometric analysis on macaque lymph node mononuclear cells at the time of isolation and again after 13 days in culture with Con A and IL-2. At baseline, in this single sample, only 5% of lymphocytes were CCR5+CD3+ (Figure 4A), but this percentage was increased to over 70% after 13 days in culture (Figure 4B).

Expression of CCR5 in Human and Macaque Lymph Node CCR5 expression was next evaluated in human and macaque lymph node, and immunohistochemical analysis with monoclonal antibodies 2F9 and 3A9 revealed a similar staining pattern (Figure 5A). Collectively, less than 5% of all lymphocytes within lymph node were immunoreactive for CCR5. Intensely immunoreactive clusters of 10 to 50 lymphocytes were present within many medullary cords and less commonly in the deep paracortex adjacent to the medulla. The majority of immunoreactive lymphocytes within the clusters were moderate in size and had abundant cytoplasm and a small central to eccentric circular nucleus. Some of the CCR5-immunoreactive lymphocytes were very large, with scant cytoplasm and a large central nucleus with a prominent nucleolus. The location and microscopic appearance of the immunoreactive cells within the paracortex and medullary cords were consistent with

mature T lymphocytes and T cell blasts. Only rare, individual immunoreactive lymphocytes were observed within lymphoid follicles. Additionally, the lymph node subcapsular and medullary sinuses of both species contained macrophages (confirmed by staining step sections of both lymph nodes for CD68 expression), which were diffusely, lightly immunoreactive for CCR5. Rare, intensely immunoreactive macrophages occurring individually or in small clusters of up to 10 cells were admixed with the more lightly staining macrophages in both of these locations. In addition, other cell types expressing CCR5 in the lymph node included endothelium, vascular smooth muscle, and scattered capsular fibroblasts (Table 2). To further define the phenotype of the CCR5-immunoreactive cells, macaque lymph node mononuclear cells were isolated from two animals by centrifugation over a discontinuous Ficoll gradient and analyzed by two-color flow cytometric analysis for expression of the CD markers listed above and CCR5. The lymph node contained a lower percentage of CCR5+ lymphocytes than the peripheral blood (Table 1, lymph node), primarily because the lymph node had a greater proportion of CCR5-CD20+ B cells than the blood (data not shown). A

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Figure 2. Flow cytometric analysis of CCR5 expression on macaque peripheral blood lymphocyte subsets as delineated by light scatter characteristics. In this example, 10% of total lymphocytes are CCR5+CD3+ (A), and there are fewer CCR5+CD4+ cells (B) than CCR5+CD8+ cells (C). In addition, CCR5+CD25+ cells are almost undetectable (D) and few CCR5+CD45RA+ cells are present (E).


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Table 1. Mean Percentage of CCR5+ Lymphocytes from Various Macaque Tissues Using Flow Cytometry

Animal

Blood

Lymph node

Spleen

163-95 181-95

15.4 + 1.95 6.75 + 0.92

7.57 + 0.53 5.1 + 1.0

13.35 + 0.78 14.4 ± 0.6

Thymus NA 1.89

+

0.15

Results are expressed as mean percentage ± SEM as a function of the entire lymphocyte pool in each tissue. NA, not available.

However, like the blood, lymph node CCR5 expression by lymphocytes was limited to CD3+ T cells. The pattern of CCR5 expression on lymph node T lymphocyte subsets was similar to the peripheral blood, although proportionately lower on a relative percentage basis (data not shown). Also similar to the peripheral blood, CD14+ monocytes in macaque lymph node did not express detectable quantities of CCR5 (data not shown).

Expression of CCR5 in Human and Macaque Spleen We also studied the pattern of CCR5 expression in both human and macaque spleen. Using immunohistochemistry, less than 15% of lymphocytes in the spleen were immunoreactive for CCR5. Intensely immunoreactive lymphocytes morphologically similar to those described in lymph node were located along the circumference of B cell follicles either as a thin, continuous band of two to five cells in width or as discontinuous clusters of two to five cells. Rare, individual CCR5-immunoreactive cells were located within B cell follicular regions. Within perivascular monocyte/macrophage aggregates (ellipsoids), 50 to 75% of the macrophages were immunoreactive for CCR5. Widely scattered CCR5-immunoreactive cells morphologically consistent with lymphocytes and monocyte/macrophages were observed within the splenic red pulp. Like the lymph node, endothelium was diffusely and lightly immunoreactive for CCR5, and rare, scattered immunoreactive fibroblasts were also observed. To further define the phenotype of the CCR5-immunoreactive cells, macaque splenic mononuclear cells were isolated from two animals by dissociation followed by centrifugation over a discontinuous Ficoll gradient and analyzed by two-color flow cytometric analysis for expression of CCR5 and the CD markers listed above. The spleen contained a greater percentage of total lympho-

cytes that were CCR5+ than the lymph node (Table 1, spleen). Like the blood and lymph node, splenic lymphocyte CCR5 expression was limited to CD3+ T cells, and staining was not observed on CD20+ B cells (data not shown). In general, the pattern of CCR5 expression on other splenic lymphocyte subsets was similar to peripheral blood and lymph node (data not shown). Similar to the peripheral blood and lymph node, we were unable to detect CCR5+CD14+ monocytes in the macaque spleen (data not shown).

Expression of CCR5 in Human and Macaque Thymus In sections of human thymus, cortical nodules were encircled by a thin, continuous band of CCR5-immunoreactive spindle-shaped to polyhedral cells, one to two cells in thickness (Figure 5B). This thin band of immunoreactive cortical cells was continuous with a focally extensive network of similarly shaped cells within the thymic medulla. By morphology alone, these immunoreactive cells were consistent with thymic cortical and medullary epithelium. To examine the identity of these CCR5+ cells more closely, step sections of thymus were stained for cytokeratin 19, reported to be expressed by thymic epithelium.23 Sections stained for cytokeratin 19 expression revealed a similar but more extensive staining pattern, suggesting that CCR5 was expressed by a subset of the thymic epithelial component. The thymic medulla contained rare, scattered, individual CCR5-immunoreactive mononuclear cells consistent with lymphocytes and macrophages. Other immunoreactive cells within the thymus included vascular endothelium and scattered thymic capsular fibroblasts. To further characterize thymic CCR5 expression, thymocytes were isolated from one macaque by dissociation A

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Figure 4. Flow cytometric analysis of CCR5 expression on macaque lymph node lymphocytes. At the time of isolation, a small number (5%) of freshly isolated lymph node lymphocytes are CCRSCD3+ (A). After a 13-day incubation with Con A and IL-2, the number of CCR5+CD3+ cells is increased to 73%, and the intensity of fluorescence is elevated (B).

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D

E

Figure 5. Immunohistochemical detection of CCR5 on various human tissues. A: Multiple lymphocytes within medullary cords at the corticomedullary junction of a mediastinal lymph node are intensely inmmunoreactive. Magnification, X460. B and C: Cortical and medullary thymic epithelium are also intensely immunoreactive (B; magnification, x 140) as well as cerebral astrocytes (C; magnification, X 180) and neurons (C, inset; magnification, x440). D: In a section of colon from a patient with ulcerative colitis, the lamina propria is massively expanded by CCR5+ mononuclear cells. Magnification, X 120. E: In a blood vessel in the colonic submucosa, the endothelium is immunoreactive for CCR5. Magnification, X600.

followed by centrifugation over a discontinuous Ficoll gradient and analyzed by two-color flow cytometric analysis for expression of CCR5 and the CD markers listed above. Approximately 2% of total thymocytes were positive for CCR5 (Figure 1, thymus), and less than 1% of thymocytes were immunoreactive for both CCR5 and any other CD marker (data not shown).

CCR5 Is Expressed by Neurons and Glia in the Cerebrum and Hippocampus Serial sections of human and macaque brain were examined for CCR5 expression. In addition to endothelium and vascular smooth muscle in the central nervous system, astrocytes (Figure 5C) and neurons (Figure 5C, inset) of

the cerebrum and hippocampus were diffusely immunoreactive for CCR5. In serial sections, cells of identical morphological phenotype were also immunoreactive for GFAP and neuron-specific enolase, respectively. Additionally, numerous small CCR5-immunoreactive polyhedral to spindyloid mononuclear cells were scattered throughout the gray and white matter. Serial brain sections stained for CD68 revealed that these CCR5+ cells were almost uniformly CD68+ and morphologically consistent with microglia. Like other tissues, scattered meningeal and perivascular fibroblasts were also CCR5 immunoreactive. A similar staining pattern was observed with both antibodies 2F9 and 5C7, and CCR5 immunoreactivity to all cells was abolished by preincubating the primary antibody with CCR5+ L1.2 cells.

Cellular Localization of CCR5 1347 AJP November 1997, Vol. 151, No. 5

Table 2. Tissues Used for Immunohistochemical Detection of CCR5

Tissue

Normal Tissue Cerebrum Hippocampus Heart Lung

Number of specimens 1 1 2 3

Kidney Liver

1 3

Skin

1

Foreskin Breast

1 3

Small intestine

1

Colon

1

Rectum Pancreas Skeletal muscle

3 2 2

Thymus

2

Spleen

1

Lymph node

2

Vagina

1

Uterus

1

Chronically inflamed tissue* Kidney (interstitial nephritis)

1

Small intestine (Crohn's disease)

3

Colon (ulcerative colitis)

5

Rectum (radiation injury)

1

Vagina (vaginitis)

4

*Characterized

Cells immunoreactive Neurons, astrocytes, microglia, endothelium, vascular smooth muscle Neurons, astrocytes, microglia, endothelium, vascular smooth muscle Endothelium, scattered mononuclear cells, fibroblasts Alveolar macrophages, vascular smooth muscle, endothelium, rare interstitial mononuclear cells Rare interstitial mononuclear cells Hepatocytes, ductal epithelium, rare periportal mononuclear cells, endothelium Endothelium, vascular smooth muscle, scattered fibroblasts, mononuclear cells Endothelium, vascular smooth muscle Ductal epithelium, rare interstitial mononuclear cells, endothelium, scattered fibroblasts Rare mononuclear cells in lamina propria/submucosa, endothelium, vascular smooth muscle, scattered fibroblasts, myenteric plexuses 25-50% of mononuclear cells in lamina propria/submucosa, endothelium, vascular smooth muscle, scattered fibroblasts, myenteric plexuses Similar to colon Acinar cells, rare interstitial mononuclear cells Endothelium, vascular smooth muscle, scattered fibroblasts, mononuclear cells Cortical and medullary epithelium, endothelium, vascular smooth muscle, rare medullary mononuclear cells, rare capsular fibroblasts Lymphocytes, monocyte/macrophages, endothelium, vascular smooth muscle, scattered fibroblasts Lymphoblasts, macrophages, endothelium, vascular smooth muscle, scattered fibroblasts Vaginal epithelium, endothelium, vascular smooth muscle, fibroblasts, rare interstitial mononuclear cells Rare mononuclear cells, vascular smooth muscle, and endothelium

Rare interstitial mononuclear cells, arteriole endothelium, and vascular smooth muscle 50-75% of mononuclear cells in lamina propria/submucosa, endothelium, vascular smooth muscle, scattered fibroblasts, myenteric plexuses 50-90% of mononuclear cells in lamina propria/submucosa, endothelium, vascular smooth muscle, scattered fibroblasts, myenteric plexuses >75% of mononuclear cells in lamina propria/submucosa, endothelium, vascular smooth muscle, scattered fibroblasts, myenteric plexuses 20% of perivascular mononuclear cells, >75% of intraepithelial lymphocytes, endothelium, vascular smooth muscle, scattered fibroblasts

as chronic by the predominance of mononuclear cells within lesions.

To further define the phenotype of the immunoreactive glial cells, we evaluated CCR5 expression on cytospin preparations of freshly isolated macaque glial cells. The preparations, consisting of a mixture of microglia, astrocytes, and oligodendrocytes, were stained for CCR5, CD68, GFAP, and neuron-specific enolase. Cells morphologically consistent with microglia were immunoreactive for CCR5 and CD68, and astrocytes were immunoreactive for CCR5 and GFAP. Interestingly, neurons of the peripheral nervous system also express CCR5. In sections of normal and inflamed small intestine, colon, and rectum, neurons within the myenteric and Auerbach plexuses were intensely immunoreactive for CCR5.

CCR5 Expression in Multiple Normal Human Tissues We also surveyed other normal human tissues to determine the pattern of CCR5 expression (Table 2). In most every tissue reviewed, a small percentage of intravascular and perivascular mononuclear cells consistent with lymphocytes and monocyte/macrophages were immunoreactive for CCR5. In parenchymal tissues such as liver, pancreas, and kidney, rare CCR5-immunoreactive cells were most often located in the perivascular connective tissue. In the lung, heart, uterus, and skeletal muscle, immunoreactive mononuclear cells were associated with supporting connective tissue. In the small intestine and

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Association of Inflammation Score With % CCR5 Positive Area

tive staining did not correlate with any particular type of endothelium. In addition to endothelial expression, CCR5 expression was also observed on vascular smooth muscle in multiple tissues. In general, vessels with positively staining smooth muscle also were lined by immunoreactive endothelium. Additionally, CCR5 was detected on epithelium in multiple tissues (Table 1). Similar epithelial staining patterns were observed using CCR5 antibodies that recognize different CCR5 epitopes (2F9 and 3A9). Finally, CCR5 expression was also detected on scattered fibroblasts in almost every tissue examined.

0

Inflamed Mucosal Tissues Contain Increased Numbers of Mononuclear Cells Immunoreactive for CCR5

% Area of Laniina Propria Occupied by CCR5+ Cells Figure 6. Association of inflammation score with percentage of CCR5-posiarea in ulcerative colitis. To determine whether the number of CCR5+ mononuclear cells present in the lamina propria of colon biopsies correlated with the severity of the lesion, colon biopsies were examined microscopically and assigned an inflammation score (Table 3). Serial sections were stained for CCR5 expression, and morphometric analysis was used to determine the percentage of area within the lamina propria occupied by CCR5+ cells. The percentage area of the lamina propria occupied by CCR5-immunoreactive cells was positively correlated with the inflammation score (r2 = 0.872). tive

colon, rare, scattered CCR5-immunoreactive mononuclear cells were present in the lamina propria and, to a lesser extent, submucosa. Rare individual lymphocytes within Peyer's patches were also immunoreactive for CCR5. Additionally, in the lung, alveolar macrophages expressed CCR5, and the intensity of staining was highly variable such that 25% of the macrophages were lightly stained, 25% were intensely stained, and the remainder were intermediate in staining intensity. In the majority of tissues reviewed, CCR5 was also detected on vascular and lymphatic endothelium. However, the level of endothelial expression was highly variable within each tissue examined. Although staining was observed in arterioles, capillaries, venules (Figure 5E), and lymphatics, in each tissue section, some vessels were lined by intensely staining endothelium, some vessels were unstained, and others were lined by endothelium that stained with intermediate intensity. Thus, posi-

To determine whether CCR5+ cells are preferentially recruited to sites of inflammation, we examined CCR5 expression in a variety of inflamed tissues including kidney (interstitial nephritis), vagina (vaginitis), small intestine (Crohns disease), colon (ulcerative colitis), and rectum (radiation injury) (Table 2). In general, all inflamed tissues contained a greater number of CCR5-immunoreactive cells than the corresponding normal tissue. The kidney biopsy was characterized by focal glomerulosclerosis, multifocal dilated renal tubules, interstitial fibrosis, and multifocal aggregates of mononuclear cells that caused expansion of the renal interstitium. Within these mononuclear aggregates, less than 5% of cells were immunoreactive for CCR5. In the samples of inflamed vagina, the submucosa contained variably sized perivascular aggregates of mononuclear cells consisting primarily of lymphocytes and fewer numbers of macrophages. Approximately 20% of perivascular mononuclear cells as well as individual submucosal mononuclear cells were immunoreactive for CCR5. In contrast to the inflamed kidney and vaginal specimens, which contained relatively few CCR5+ cells, up to 90% of mononuclear cells in the lamina propria and submucosa of inflamed gut biopsies were immunoreactive for CCR5 (Table 2). To determine whether the number of CCR5+ mononuclear cells present in the infiltrate was associated with the severity of the lesion, five colon biopsies from patients with ulcerative colitis of varying severity were examined microscopically and assigned an inflammation score (Table 3). Briefly, patients with mild colitis (score of 1) had multifocal aggregates of lymphocytes and macrophages within the lamina propria and rare crypt abscesses. As lesions increased in severity (score of 2), the mononuclear infiltrate and edema caused diffuse expansion of the

Table 3. Colonic Biopsy Scoring Criteria

Score 0 1 2 3

Epithelium Intact Intact Intact Ulcerated

Lamina propria

Scattered WBCs, no expansion Focal expansion Diffuse WBC infiltrate, diffuse expansion Diffuse WBC infiltrate, diffuse expansion

Mucosal glands Normal Rare crypt abscesses >1 crypt abscess Extensive effacement

Submucosa No WBC infiltration No WBC infiltration Minimal WBC infiltration Diffuse WBC infiltration


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lamina propria, and crypt abscesses were more abundant. The most severe case (score of 3) was characterized by massive distension of the lamina propria with mononuclear cells, multifocal crypt dropout, and/or crypt abscesses, extensive epithelial ulceration, and extension of the mononuclear infiltrate into the submucosa and lamina muscularis. After scoring, serial sections were examined for CCR5 expression using immunohistochemistry (Figure 5D), and morphometric analysis was used to determine the percentage of area within the lamina propria occupied by CCR5-immunoreactive mononuclear cells. The percentage of area of the lamina propria occupied by CCR5-immunoreactive mononuclear cells was positively associated with the inflammation score (Figure 6; r2 = 0.872). Serial sections of these tissues stained for CD3, CD4, CD8, CD68, and PCA-1 (plasma cells) revealed that the immunoreactive mononuclear cells consisted of approximately equal numbers of CD68+ monocyte/macrophages and CD3+ lymphocytes. CCR5+CD8+ lymphocytes were more abundant than CCR5+CD4+ lymphocytes.

Discussion Using a panel of monoclonal antibodies specific for human CCR5, we have shown that CCR5 is expressed by a small subpopulation of lymphocytes and monocyte/macrophages in human and macaque blood, primary and secondary lymphoid organs, and noninflamed tissues. Interestingly, neurons, astrocytes, and microglia also express CCR5. Other cells expressing CCR5 include epithelium, endothelium, vascular smooth muscle, and fibroblasts. By morphometric analysis, there is an increased number of CCR5-immunoreactive cells in a setting of chronic inflammation, and the number of immunoreactive cells is positively associated with a histopathological correlate of inflammatory severity. For many years, it was known that HIV-1 required a co-receptor in addition to CD4 for efficient infection of cellular targets.2425 In addition to their significant role in leukocyte chemotaxis and emigration, chemokine receptors have emerged as important co-receptors for HIV-1 infection.9-13 Of the chemokine receptors, CCR5 may play a particularly important role in the early stages of sexually transmitted HIV-1 infection, because it is used as a co-receptor by macrophage-tropic strains of HIV-1,9-12 which are most likely to be transmitted by this route.14 Interestingly, macrophage-tropic strains of SIV also use CCR5 as a co-receptor for entry into cellular targets.17 In this paper, we show that CCR5 is expressed by a spectrum of human and macaque cells (T lymphocytes, monocytes/macrophages, and microglia) known to co-express CD4 and serve as cellular targets for macrophage-tropic strains of HIV-1 and SIV, respectively.26-28 The lymph node is an important viral reservoir for both HIV-129 and SIV30 infection. Furthermore, HIV-1 replication in vivo occurs at continuously high rates,31 and the average life span of a productively infected lymphocyte has been estimated to be 2.2 days.32 Viral survival in vivo, therefore, is

dependent on replenishment of the target cell population. In this study, we demonstrate that cells putatively susceptible to infection with macrophage-tropic strains of SIV and HIV-1 (CD4+CCR5+ phenotype) constitute a relatively small percentage of the total cellular population within lymph node, blood, and other lymphoid tissues. However, additional work is required to understand the cellular dynamics of CCR5+ cells during retroviral infection and how any putative changes to numbers and/or distribution affect viral load, viral distribution, and pathogenicity. In addition to leukocytes in the peripheral blood and lymphoid organs, we also show that CCR5 is expressed on neurons, astrocytes, and microglia of the cerebrum and hippocampus. The observation that neurons and astrocytes express CCR5 is intriguing because low-level infection or rare infection of these cell types with HIV-1 has previously been described.3334 However, as neurons and astrocytes are not known to express CD4, if they are infection competent, HIV-1 may use alternative receptors, such as galactosyl ceramide as one example.35 Interestingly, other investigators have failed to document infection of neurons in AIDS dementia patients.36 Expression of other chemokine receptors have been described in the central nervous system, including the IL-8 receptor, CXCR2, identified on neurons in specific regions of the brain,37 and the Duffy antigen on central nervous system endothelium and cerebellar Purkinje cells.38 Another group demonstrated expression of a putative seven-transmembrane-spanning chemokine receptor in the mouse brain by Northern analysis. Interestingly, this murine receptor also binds human and mouse MIP-la, MIP-1,B, and RANTES with high affinity.39 Chemokine expression on malignant astrocytes has also been detected.40 Thus, it is likely that chemokines and chemokine receptors play roles in central nervous system development, function, and potentially HIV-1 dissemination in brain, although their roles in these functions have yet to be defined.37 Additionally, we demonstrate that CCR5 is expressed on endothelium, smooth muscle, and fibroblasts in a variety of tissues. Chemokine receptor expression by mesenchymal cells in general and endothelial cells specifically has been described. For example, the Duffy antigen has been detected on vascular endothelium.38 Moreover, IL-8 promotes41 and IP-10 inhibits42 angiogenesis, suggesting that these cells express functional chemokine receptors. Also, in some circumstances with select HIV-1 isolates, endothelial cells can support low-level HIV-1 replication. For example, viral gene expression has been detected in the central nervous system34 and other tissues including lymphoid organs.43 HIV-1 will infect and replicate in freshly isolated human brain capillary endothelial cells in vitro44 as well as proliferating umbilical vein endothelium,45 and the infection of both types of endothelium appears to occur independent of CD4 expression. As CCR5 is a co-receptor for CD4, it is unclear whether CCR5 plays a significant role in HIV-1 infection of these types of endothelial cells under these conditions. However, because HIV-1 infection of brain has profound pro-inflammatory and degenerative effects on parenchymal cells, it is possible that the expression of CCR5 and other receptors for HIV-1 may change during the course of infection resulting in increased or decreased efficiency of HIV-1 transmission to parenchymal cells. Further

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examination of tissue from patients at various stages of infection will be needed to address this issue. To our knowledge, epithelial expression of a chemokine receptor has not previously been described. In this report, CCR5 was localized to a wide variety of epithelial cells. This pattern of staining was demonstrated using three different antibodies that recognize different CCR5 epitopes. Furthermore, incubation of the primary antibody with CCR5+ transfectant cells abrogated recognition of CCR5 in tissues. Epithelial cells, however, have been documented to express chemokine protein, including eotaxin,46 MOP-1, GRO-a, -,3, and -_,47 and the CCR5 ligand RANTES.48 It is interesting to speculate that epithelial expression of both chemokines and chemokine receptors may, under certain circumstances, involve autocrine feedback regulatory functions in growth and differentiation. In addition, in patients with AIDS and symptoms of bowel inflammation, HIV-1 infection of bowel epithelium has been described,49 and HIV-1 also infects established colorectal epithelial cell lines.50 Similar to endothelial expression of CCR5, however, examination of the putative role of CCR5 in HIV-1 infection of these cells will be required before it can be implicated in HIV-1 infection and cellular dysfunction. Follicular and interdigitating dendritic cells in lymph node have been implicated in retroviral replication and persistence.51-53 However, we were unable to demonstrate CCR5 expression by follicular dendritic cells in either human or macaque lymph nodes. Although it is accepted that follicular dendritic cells entrap HIV-1-associated antigen and germinal centers contain abundant HIV-1 RNA by in situ hybridization, the mechanism by which this localization occurs remains unclear. Interestingly, other phenotypically related dendritic cells, such as those from the thymus or tonsil, express low levels of CD454 and can bind and internalize HIV-1 rapidly, which is followed by viral integration and replication.55 In addition, HlV-1-infected Langerhans cells can infect activated T cells, suggesting a role in viral dissemination.55 However, only certain strains of HIV-1 are apparently able to infect dendritic cells. For example, thymus and tonsillar dendritic cells become infected by macrophage-tropic but not lymphocyte-tropic strains of HIV-1.54 Although we were unable to detect CCR5 expression within the follicular dendritic cell labyrinth of lymph node germinal centers or within cells of a dendritic morphology within the paracortex, it is possible that these dendritic cells express CCR5 at a level that is below the detection limits of our assay. Alternatively, these cells may not express CCR5. Therefore, a more thorough analysis ofCCR5 expression on dendritic cells in general will be necessary to determine whether this receptor is important for HIV-1 infection of phenotypically distinct members of the dendritic cell family. Lastly, we also demonstrate an increased number of CCR5-expressing mononuclear cells at sites of chronic inflammation. There exists significant epidemiological evidence that HIV-1 is more efficiently transmitted from an infected individual to a recipient when the mucosal surface of the recipient is inflamed. Specifically, genital ulcerative disease is epidemiologically associated with more efficient transmission of HIV-1 during heterosexual intercourse.56 Other non-ulcerative sexually transmitted

diseases that may cause inflammation of the urogenital tract have also been identified as risk factors for HIV-1,5 and rectal gonorrhea has been identified as a risk factor for HIV-1 transmission between homosexual men.58 In this study, we demonstrate that inflamed colonic mucosa contains increased numbers of target cells expressing CCR5. In this regard, HIV-1 has been detected within mononuclear cells of the colonic and rectal lamina propria of AIDS patients with inflammatory bowel disease by in situ hybridization.49 Collectively, these results suggest that CCR5+ cells are recruited to inflammatory sites and, as such, may facilitate viral transmission. Additional work will be required to understand the role of CCR5 in inflammation, the importance of CCR5+ cells in the inflammation cascade, and whether the presence of these cells at inflamed mucosal sites predispose individuals to HIV-1 infection.

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