Cellular localization of simian immunodeficiency virus in lymphoid tissues. II. In situ hybridization

Lymph nodes and spleens were collected at autopsy and by biopsy from 29 rhesus monkeys infected with simian immunodeficiency virus (SIV). Lymph nodes were classified morphologically into stages of follicular hyperplasia, follicular involution, follicular depletion with normal or expanded paracortices, follicular and paracortical depletion, granulomatous lymphadenitis, or normal. The distribution of SIV RNA was determined by in situ hybridization using a nick translated, 35S labeled, SIVmac DNA probe. Numbers of SIV-infected cells were rare during follicular hyperplasia, numerous during follicular and paracortical expansion, and rare during follicular and paracortical depletion. The splenic morphology reflected that of the lymph nodes; however, the numbers of SIV-positive cells were uniformly lower. SIV RNA was frequently restricted to a single nucleus within multinucleate syncytial cells in two cases of granulomatous lymphadenitis. These results, combined with those of a previous study, provide evidence for antigen trapping in SIV-infected hyperplastic lymph nodes and for widespread viral infection of macrophages and lymphocytes during paracortical expansion.     

Cellular Localization of Simian Immunodeficiency Virus in Lymphoid Tissues: I. Immunohistochemistry and Electron Microscopy

Simian immunodeficiency virus (SIV) is a lentivirus with genetic relatedness to the human immunodeficiency viruses (HIV-1 and HIV-2). It induces a fatal syndrome in rhesus monkeys that closely parallels the clinical course of AIDS in humans. The authors used double-labeling immunohistochemical procedures on rhesus lymph node and spleen taken during different time periods after SIV infection to localize the p27 gag protein to specific cellular immunophenotypes. In animals with follicular hyperplasia, viral protein was found associated predominantly with follicular dendritic cells. Many of these cells showed ultrastructural alterations consisting of swollen dendritic processes containing electron-dense material. Lentiviral particles were found associated with this cell type only rarely. In lymphoid tissues with other histopathologic changes, macrophages and multinucleate giant cells were the predominant cell types containing detectable quantities of viral protein; smaller numbers of p27+ lymphocytes were present. Ultrastructurally, viral particles were found within the extracellular space adjacent to tissue macrophages and within membrane-bound vacuoles of giant cells and tissue macrophages. These results show that certain histologic patterns seen during the course of infection correlate with the localization of viral antigen to specific cellular immunophenotypes and that during the disease course, viral protein is preferentially localized in sections of lymph node and spleen to cells of the macrophage and dendritic cell lineages.     

CD163 identifies perivascular macrophages in normal and viral encephalitic brains and potential precursors to perivascular macrophages in blood

Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163(+)CD14(+)CD16(+)) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163(+) blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice.     

Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue.

The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to HIV-1-tat stained solitary cells in the germinal centers and interfollicular zones, and vascular endothelium. Staining by an anti-nef monoclonal antibody was restricted to follicular dendritic cells, whereas anti-rev antibody bound to fibriohistiocytes and high endothelial venules. The antibodies used labeled several cell types in tissues from uninfected individuals. Anti-HIV-1-tat antibodies labeled blood vessels and Hassall's corpuscles in skin and thymus; goblet cells in intestinal tissue and trachea; neural cells in brain and spinal cord; and zymogen-producing cells in pancreas. Anti-rev antibody stained high endothelial venules, Hassall's corpuscles and histiocytes. One anti-nef antibody solely stained follicular dendritic cells in spleen, tonsil, lymph node and Peyer's patches, whereas two other anti-nef antibodies bound to astrocytes, solitary cells in the interfollicular zones of lymph nodes, and skin cells. The current results hamper the immunohistochemical study for pathogenetic and diagnostic use of HIV regulatory protein expression in infected tissue specimens or cells.     

Involution of lymph node histiocytes in AIDS

Lymph nodes of human immunodeficiency virus (HIV)-infected patients were studied histologically and immunohistochemically to elucidate the pattern of involution of various histiocytes in AIDS. Specimens consisted of one node with hyperplasia, five with atrophy, and three with severe atrophy. Antibodies such as L25, ID1, My4, 12, anti-Leu 3a, KiM4, OKT6 and anti-S100 protein were used for identification of the histocytes. Another antibody, VAK5, was used to demonstrate HIV antigen. T-zone histiocytes were mildly decreased in the hyperplastic node, but considerably decreased in the atrophic nodes. My4+ sinus histiocytes were unchanged in number and enlarged in the hyperplastic node, but not decreased in the atrophic nodes. Follicular dendritic cells (FDCs), defined by KiM4, were mostly depleted in the atrophic nodes. The T4 antigen was detected in some of the sinus histiocytes of the atrophic nodes. T6-positive cells were not found in any of the nodes. HIV antigen was detected only in FDCs. It is therefore suggested that various histiocytes respond differently to HIV, and that T-zone histiocytes and sinus histiocytes persist up to the late stage of AIDS.     

Involution of Lymph Node Histiocytes in AIDS

Lymph nodes of human immunodeficiency virus (HW-infected patients were studied histologically and immuohistochemically to elucidate the pattern of involution of various histiocytes in AIDS. Specimens consisted of one node with hyperplasia, five with atrophy, and three with severe atrophy. Antibodies such as L25, ID1, My4, 12, anti-Leu 3a, KiM4, OKT6 and anti-S100 protein were used for identification of the histiocytes. Another antibody, VAK5, was used to demonstrate HIV antigen. T-zone histiocytes were mildly decreased in the hyperplastic node, but considerably decreased in the atrophic nodes. My4 + sinus histiocytes were unchanged in number and enlarged in the hyperplastic node, but not decreased in the atrophic nodes. Follicular dendritic cells (FDCs), defined by KiM4, were mostly depleted in the atrophic nodes. The T4 antigen was detected in some of the sinus histiocytes of the atrophic nodes. T6-positive cells were not found in any of the nodes. HIV antigen was detected only in FDCs. It is therefore suggested that various histiocytes respond differently to HIV, and that T-zone histiocytes and sinus histiocytes persist up to the late stage of AIDS. Acta Pathol. Jpn. 39: 496∼502, 1989.     

Monocyte/macrophages and their role in HIV neuropathogenesis

Neurological sequelae of human immunodeficiency virus (HIV) infection have been and remain a significant problem. Monocytes and macrophages in humans and monkeys are susceptible to infection by HIV and simian immunodeficiency virus (SIV), and are considered to be a main mechanism by which the central nervous system (CNS) is infected. Within the infected CNS, perivascular macrophages and, in some cases, parenchymal microglia are infected as are multinucleated giant cells when present. While neurons are not themselves directly infected, neuronal damage occurs within the infected CNS. Despite the success of antiretroviral therapy (ART) in limiting virus in plasma to non-detectable levels, neurological deficits persist. This review discusses the continued neurological dysfunctions that persist in the era of ART, focusing on the roles of monocyte and macrophage as targets of continued viral infection and as agents of pathogenesis in what appears to be emergent macrophage-mediated disease resulting from long-term HIV infection of the host. Data discussed include the biology of monocyte/macrophage activation with HIV and SIV infection, traffic of cells into and out of the CNS with infection, macrophage-associated biomarkers of CNS and cardiac disease, the role of antiretroviral therapy on these cells and CNS disease, as well as the need for effective adjunctive therapies targeting monocytes and macrophages.     

The application of the metalophil method for the demonstration of histiocytes

The metalophil method has been performed on 250 sections of a wide variety of inflammatory and neoplastic lesions in which different types of histiocytes might be encountered. In lymph nodes, dendritic histiocytes related to the B-lymphocyte system were consistently metalophil. They appeared as small cells with slender extensions in lymph nodes with follicular hyperplasia and/or sinus histiocytosis and in some cases of Hodgkin's lymphomas or as larger pleomorphic cells in primary or secondary malignancies of lymph nodes. Small, rounded cells were seen in some cases of marked paracortical reaction, in dermatopathic lymphadenitis and in some cases of mycosis fungoides. These cells most probably represented Langerhans cells and interdigitating reticulum cells, which are related to the T-lymphocyte system. Interdigitating cells become metalophil when they are activated or proliferating. Epithelioid cells in different benign and malignant lesions were metalophil like the sinus histiocytes of the lymph nodes, the Kupffer cells of the liver and the alveolar histiocytes in the lung. Foreign-body giant cells in lymph nodes after lymphography were also metalophil. The sinus lining cells lymph nodes were also well-delineated. Histiocytes of malignant histiocytic proliferations were sometimes metalophil as were the so-called histiocytes in malignant fibrous histiocytomas. Epithelial cells, particularly the basal cells of squamous epithelium often take up the silver. Carcinoma cells were sometimes metalophil and the method appeared not to be of value in the differentiation between metastatic carcinomas and lymphomas. The most promising application seems to be the study of the distribution of dendritic histiocytes in malignant proliferations of B-lymphocytes.     

Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys.

Simian immunodeficiency virus (SIV), like the human immunodeficiency virus (HIV), is a lentivirus that is both immunosuppressive and neurovirulent. Rhesus macaques (Macaca mulatta) inoculated with SIV often develop a giant cell encephalitis similar to that seen in humans infected with HIV. The authors examined SIV expression by immunohistochemistry and RNA in situ hybridization in the cerebrum, cerebellum, choroid plexus, and spinal cord from five macaques with and two macaques without giant cell encephalitis. Selected portions of the central nervous system (CNS) also were examined by electron microscopy. Simian immunodeficiency virus was detected in the CNS of all seven monkeys whether or not they had giant cell encephalitis. Both SIV antigen and RNA were present in all levels of the CNS examined. Macrophage/giant cell lesions always contained viral RNA and antigen and were the only sites where viral particles were detected by electron microscopy. However, SIV antigen and RNA also were commonly associated with small vessels, the choroid plexus, and meninges; these were the only locations where virus was detected in animals without giant cell encephalitis. Immunophenotyping showed that the cellular infiltrates consisted primarily of monocyte/macrophages and occasional CD8-positive T cells. Macrophages and T cells also were present in the stroma of the choroid plexus and were intimately associated with vessels in the CNS of SIV-infected but not uninfected macaques. Simian immunodeficiency virus infection of the macaque CNS provides an excellent model for studying the pathogenesis, treatment, and prevention of HIV-1-encephalitis.     

Lymphadenopathy in macaques experimentally infected with the simian immunodeficiency virus (SIV).

A T-cell tropic lentivirus of macaques the simian immunodeficiency virus (SIV), has morphologic, growth, and antigenic properties that indicate that it is related to the human immunodeficiency virus (HIV), the etiologic agent of the acquired immune deficiency syndrome (AIDS) in humans. Six juvenile macaques developed persistent lymphadenopathy (greater than 3 months in duration) after inoculation with SIV. The histologic appearance of the lymph nodes was characterized by marked follicular hyperplasia with abundant proliferative B cells infiltrating into the paracortex. The number of T8-positive lymphocytes equaled or exceeded the number of T4-positive lymphocytes in the paracortex. These findings, in association with immunologic abnormalities and a previously observed fatal immunodeficiency syndrome in SIV-infected macaques, provide further evidence of the importance of SIV-induced disease in macaques as a model for the study of AIDS.     

Simian immunodeficiency virus infection of macaque bone marrow macrophages correlates with disease progression in vivo.

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 SIV-infected rhesus monkeys were examined by immunohistochemistry and in situ hybridization. By immunohistochemistry, only four monkeys were positive for SIV core 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 SIV were mainly mononuclear cells that appeared to be of myelomonocytic lineage. In contrast, monkeys with severe lymphoid depletion had SIV RNA localized to larger mononuclear cells with abundant cytoplasm often located in small lucent areas of the stroma. These SIV RNA-positive mononuclear cells were positive for the macrophage determinant CD68 as demonstrated by immunohistochemistry. Furthermore the stage of simian acquired immune deficiency syndrome, as indicated by lymphoid morphology, and SIV localization in the bone marrow were correlated with the incidence of anemia, bone marrow hyperplasia, 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 SIV infections in the bone marrow.     

The Lymph Node in HIV Pathogenesis

Lymph nodes play a central role in the development of adaptive immunity against pathogens and particularly the generation of antigen-specific B cell responses in specialized areas called germinal centers (GCs). Lymph node (LN) pathology was recognized as an important consequence of human immunodeficiency virus (HIV) infection since the beginning of the HIV epidemic. Investigation into the structural and functional alterations induced by HIV and Simian immunodeficiency virus (SIV) has further cemented the central role that lymphoid tissue plays in HIV/SIV pathogenesis. The coexistence of constant local inflammation, altered tissue architecture, and relative exclusion of virus-specific CD8 T cells from the GCs creates a unique environment for the virus evolution and establishment of viral reservoir in specific GC cells, namely T follicular helper CD4 T cells (Tfh). A better understanding of the biology of immune cells in HIV-infected lymph nodes is a prerequisite to attaining the ultimate goal of complete viral eradication.     

Early appearance of simian immunodeficiency virus (SIV) antigen and antibodies as variables in evaluating antiviral drugs in macaques.

SIV infection in macaques has become an important animal model for HIV-1 infection in humans. An antibody assay was therefore developed and compared to a commercially available antigen assay with respect to their usefulness to monitor the course of simian immunodeficiency virus (SIV) infection in cynomolgus monkeys. A peptide, JB6T, consisting of 21 amino acids with the sequence NSWGCAFRQVCHTTVPWVNDS corresponding to a segment in the env protein of human immunodeficiency virus (HIV) type 2 was used as antigen in an enzyme-linked immunosorbent assay (ELISA). JB6T was found to detect IgG and IgM antibodies to viral antigens with high specificity. The earliest anti-SIV IgM antibodies were detected at days 13-16, with a maximum at day 20 and subsequently the levels fell. Specific IgG antibody levels increased at day 16-20 after SIV infection and reached a plateau at day 60. The commercially available HIV-1 p24/26 antigen test could, due to cross-reactivity, be employed to detect SIV antigen delay, peak and duration.     

Pathogenicity of HIV in lymphatic organs of patients with AIDS

HIV antigens were searched for in the thymus, lymph nodes, bone marrow, and spleen of AIDS patients, by means of immunofluorescence technique. Human IgG against HIV and monoclonal antibodies against viral gag P24 protein yielded strong cytoplasmic fluorescence of cells in sections of the thymus, lymph nodes and spleen. Some cells containing HIV antigens were morphologically multinucleated giant cells. They reacted with monoclonal antibodies against helper/inducer T-cells (OKT4+), and were complexed with antibody or with complement as demonstrated by double-staining immunofluorescence technique. A large number of inflammatory cells infiltrated the thymus in areas containing cells expressing HIV antigens. These studies demonstrated an association of HIV virus with cytopathic and immunopathogenic reactions in lymphatic organs of AIDS patients, and are consistent with previous results, as well as indicative of a primary aetiologic role for the virus.     

Comparative histopathological studies in the early stages of acute pathogenic and nonpathogenic SHIV-infected lymphoid organs

To clarify the early pathological events in simian and human immunodeficiency chimeric virus (SHIV)-infected lymphoid organs, we examined rhesus macaques infected with an acute pathogenic SHIV (SHIV89.6P) or a nonpathogenic SHIV (NM-3rN) by sequential biopsies and serial necropsies. In the SHIV89.6P-infected monkeys, acute thymic involution as shown by increased cortical tingible-body macrophages and by neutrophilic infiltrates without follicular aggregation in the medulla began within 14 days postinoculation (dpi). Cells that were strongly positive for the virus were identified in the thymic medulla. SHIV89.6P-infected lymph nodes showed severe paracortical lymphadenitis with scattered virus-positive cells at 14 dpi and they developed paracortical depletion without the obvious follicular involution. In contrast, NM-3rN-infected monkeys showed no signs of thymic dysinvolution and the lymph nodes exhibited only follicular hyperplasia. NM-3rN-infected monkeys showed much fewer virus-positive cells in these lymphoid tissues than did SHIV89.6P-infected monkeys during the same period. These differences clearly reflect the difference in the virulence of these SHIVs.     

Induction of MHC class I antigen expression following infection of a human esthesioneuroblastoma cell line with cytomegalovirus and human immunodeficiency virus.

Productive infections with cytomegalovirus (CMV) and human immunodeficiency virus (HIV) were established in the Tp41ON cell line derived from a human esthesioneuroblastoma. HIV antigen expression was highest in cultures coinfected with CMV and HIV. Viral infection caused increased MHC class I antigen expression while class II and CD4 antigens remained undetectable using immunofluorescence methods. Uninfected cultures showed 10% and coinfected cultures 80% class I antigen positive cells. In coinfected cultures, CMV and HIV antigens were detected in 4% and 8% of the cells, respectively. The detection of CMV antigens in some multinucleated cells suggests coinfection with both viruses in these cells, as multinucleated cells were not found in cultures infected with CMV only. The study shows that a cell line showing neuronal differentiation in vitro can be infected with CMV and HIV and that this infection increases MHC class I antigen expression.     

Trafficking of inflammatory macrophages from the kidney to draining lymph nodes during experimental glomerulonephritis.

Macrophage accumulation within the glomerulus and renal interstitium is a prominent feature of most forms of glomerulonephritis, but the fate of these inflammatory cells is unknown. Macrophage trafficking to the draining kidney lymph nodes (KLN) was assessed in a detailed kinetic analysis of accelerated antiglomerular basement membrane (GBM) disease in the rat. Leucocytes draining to KLN via lymphatic vessels were identified within the marginal sinus by MoAb labelling of tissue sections. In anti-GBM disease, there was a significant increase in the weight of the KLN due to both lymphoproliferation within the nodes and increased lymphatic drainage from the inflamed kidney, as evidenced by prominent dilation of the marginal sinus and increased numbers of cells within the sinus. In non-inflamed lymph nodes, few ED1+ macrophages were present within the marginal sinus (3.0 +/- 0.6/100 nucleated cells). However, in anti-GBM disease, macrophages became the major cell type within the dilated marginal sinus of the KLN, as shown by labelling with ED1, ED2 and ED3 MoAbs, peaking at 74 +/- 2.6 ED1+ cells/100 nucleated cells at day 14. These changes were not simply due to systemic antigen administration, since in the axillary lymph node (ALN) there was no obvious dilation of the marginal sinus and macrophages accounted for a maximum of only 15 +/- 4.6 ED1+ cells/100 nucleated cells. In conclusion, this study provides indirect evidence that there is significant trafficking of the renal macrophage infiltrate to the KLN during experimental glomerulonephritis. This may be a mechanism whereby nephritogenic antigens, released as a consequence of the local inflammatory response, may be presented to T and B lymphocytes within lymph nodes, resulting in the amplification of the immune response in glomerulonephritis.     

Immature sinus histiocytosis. Light- and electron-microscopic features, immunologic phenotype, and relationship with marginal zone lymphocytes.

The light-microscopic, ultrastructural, and immunohistochemical features of immature sinus histiocytosis were studied in 10 lymph nodes with the histologic picture of toxoplasmic lymphadenitis and compared with the features of lymphoid cells present in the marginal zone of the splenic white pulp. Areas of immature sinus histiocytosis consisted largely of medium-sized lymphoid cells with markedly irregular nuclei and abundant pale cytoplasm. Using a panel of monoclonal antibodies, the predominating lymphoid cells were found to carry the B-cell phenotype B1+Ba1-sIgM+sIgD-OKIa1+. Admixed were variable numbers of larger, blastic lymphoid cells, small lymphocytes, histiocytic elements, and polymorphonuclear granulocytes. The marginal zone of the splenic white pulp was composed of a similar mixture of cells, and marginal-zone lymphocytes demonstrated an analogous immunohistochemical phenotype. Our results indicate that immature sinus histiocytes are B-lymphoid cells that are closely related to marginal zone lymphocytes. As such, immature sinus histiocytes may have a role similar to that of marginal-zone lymphocytes, which have been claimed to transport antigens or immune complexes toward the follicular center or to serve as precursors of plasma cells. We suggest that immature sinus histiocytosis represents an abnormal expansion of the marginal zone, normally present at the sinusoidal pole of lymphoid follicles. The reason for this marginal-zone hyperplasia, recognized as immature sinus histiocytosis in a variety of reactive lymph node conditions, may be a maturation arrest in the normal development of immature sinus histiocytes into small, sIgM+ sIgD+ lymphocytes.     

A rational basis for mucosal vaccination against HIV infection

Summary: The lack of success in the development of an effective conventional vaccine against HIV has focused attention on mucosal immunity. This is a rational move, since HIV is transmitted mostly by the mucosal route. The mucosal strategy is based on the concept that: a) HIV/SIV has to cross the mucosal-regional lymph node-blood barriers, each of which can prevent viral transmission or decrease the viral load, b) Immunization has to target directly the mucosal tissues or indirectly the regional lymph nodes, in order to prevent or control viral replication. This strategy is consistent with antigen localization and effective entry into the lymph nodes, driving the immune response, c) A dual immune mechanism may be necessary for effective mucosal protection, mediated by specific CD4 and CD8 T-cell and antibody responses to the immunizing antigens, and innate antiviral factors and [i-chemokines which downmodulate CCR5 co-receptors. Targeted iliac lymph node immunization with SIVgp120 and p27 in alum prevents SIV infection or significantly decreases the viral load when challenged by the rectal route. Indeed, in addition to specific immunity, including significant sIgA antibody-forming cells in the iliac lymph nodes, CD8-suppressor factor and the three β-chemokines (RANTES, macrophage inflammatory protein (MIP)- lα and MlP- 1β) are significantly associated with protection against rectal mucosal SIV infection.