L. W. Poulter

Discrimination of Human Macrophages and Dendritic Cells by Means of Monoclonal Antibodies

Two monoclonal antibodies (MoAb), RFD1 and RFD7, have been used to investigate whether human macrophages and dendritic cells represent phenotypically distinct cell types. RFD7 recognizes a 77 kd antigen, and is specific for acid phosphatase positive tissue macrophages, while RFD1 recognizes a unique Class II antigen, which is associated with dendritic cells. With immunohistological/cytological methods it was found that neither of these reagents reacted with granulocytes, monocytes, or lymphocytes, with the exception that a small proportion (<20%) of B cells were stained with RFD1. In tissues, RFD7 reacted with mature macrophages only and did not stain Langerhans cells in the skin or the interdigitating (dendritic) cells of the T‐cell zones of lymphoid tissue and the thymic medulla. Conversely, RFD1 appeared specific for the interdigitating (dendritic) cells and did not react with macrophage populations identified morphologically, geographically, and histochemically in any tissue studied. When peripheral blood monocytes (RFD1−, RFD7−) were matured in vitro, two distinct populations of RFD1+ RFD7− and RFD7+ RFD1− cells emerged. It is concluded that in normal tissues these two reagents identify phenotypic differences between macrophages and dendritic cells that may have functional significance.

Loss of mucosal CD4 lymphocytes is an early feature of HIV infection

T cell subsets in the gut mucosa are distinct populations and their imbalance in HIV has specific implications in infection. Alterations in T cell subsets in duodenal biopsies were investigated in 17 asymptomatic HIV patients, 24 AIDS patients and 10 controls with non‐ulcer dyspepsia. Immunohistochemistry and immunofluoresccnce using MoAbs to CD3, CD4, CDS, CD68, CD45R A, CD45RO and gp 120 were performed on frozen sections. In the lamina propria. there was a significant depletion of CD4+ cells at all stages of HIV, but the density of CDS lamina propria cells was increased. Intraepithelial lymphocytes were decreased in AIDS patients. There was a significant correlation between cellular density and mucosal CD3+ lymphocytes, and between mucosal CD3+ and CDS+ lymphocytes. Although mucosal CD4, CD45RO+‘memory’ cells were decreased, CD8.CD45RO+‘memory’ cells were increased. Mucosal CD4+ lymphocyte depletion occurred early in HIV, and thus their role in mucosal protection against opportunistic infection should be revised. Mucosal CD8+ lymphocytes initially increased, but decreased when CD4 blood counts were depleted, perhaps contributing to loss of host protection against infection. Intraepithelial lymphocyte depletion may also contribute to opportunistic infection.

Characterization of immune inducer and suppressor macrophages from the normal human lung

Monoclonal antibodies (MoAbs) that are able 10 discriminate between dendritic cells (MoAb RFD1+) and mature macrophages (MoAb RFD7+ (in normal tissues were used in combination with density separation techniques to isolate relatively homogeneous subpopulations of macrophages from human bronchoalveolar lavage (BAL). A characterization of surface antigen expression, and functional capacity was then carried out on each isolated alveolar macrophage (AM) subset. One population with the phenotype RFD1+ RFD7‐ obtained from the non‐adherent cell pool showed the characteristics of antigen‐presenting cells having absent or poor expression of Fc and C3b receptors, a low content of lysozomal hydrolase and poor phagocytic capacity. This population strongly stimulated T lymphocytes in allogeneic mixed lymphocyte reactions (MLR). A second AM population, isolated by adherence and density centrifugal ion expressed the phenotype RFD1+ RFD7+. These cells showed the same phenotypic characteristics of mature macrophages with strong expression of C3b and Fc receptors, and marked phagocytic capacity. Such AM were very poor stimulators of allogeneie MLR. Under certain circumstances the RFD1+ RFD7+ cells were shown to actively repress the stimulatory capacity of the RFD1+ RFD7‐ subpopulation. These results suggest that variations within the functional capacity of AM subsets may be capable of influencing the strength of acquired T cell immune responses of the lung.

Immunohistological analysis of lung tissue from patients with cryptogenic fibrosing alveolitis suggesting local expression of immune hypersensitivity.

Immunohistological analysis using monoclonal antibodies in conjunction with histochemical techniques has been applied to lung biopsy material from patients with cryptogenic fibrosing alveolitis. Subsets of lymphoid and non-lymphoid cells have been identified in situ. This analysis showed that the inflammatory cells present were predominantly mononuclear. Most of the lymphoid cells were B lymphocytes, organised into follicles with occasional germinal centre formation. IgM was the major class of immunoglobulin expressed. Both T4+ and T8+ lymphocytes were seen diffusely distributed in the interstitium. The T4+ positive cells were also seen within the B lymphoid follicles. Almost all non-lymphoid cells expressed the phenotype of inflammatory macrophages, but a few also expressed a phenotype characteristic of interdigitating cells. These results suggest that a local B lymphoid immune response is occurring in cryptogenic fibrosing alveolitis. The possibility that a cell mediated immune response is also emerging is discussed.

Activated T lymphocytes of the synovial membrane in rheumatoid arthritis and other arthropathies

Immunohistological techniques were used to identify activated T lymphocytes within the synovial membrane of patients with rheumatoid arthritis, using the monoclonal antibody (MoAb) RFT2, which identifies a 40‐k dalton molecule preferentially expressed by T blasts or activated cells. Using this reagent together with a monoclonal ‘cocktail’ that stains all T cells, cell counts on consecutive sections of rheumatoid synovium revealed that up to 50% T lymphocytes were RFT2+ (range 9.3‐50.2%, mean 25.4). Subsequent analysis using combination immunofluorescence demonstrated that over 90% of these activated cells were of the T4+ subset. Furthermore all these cells appeared to be Leu8‐. suggesting that the activated population were exclusively ‘true helpers’ and not suppressor inducers. Studies indicated that 50% of the RFT2+ cells were positive with anti‐Tac MoAb. Comparisons with tissues from other arthropathies demonstrated that this relatively high proportion of RFT2+ cells was a feature restricted to rheumatoid arthritis, although biopsies from patients with psoriatic arthritis and ankylosing spondylitis also contained activated cells. Biopsies of Reiters syndrome, osteo‐arthritis, and pigmented villonodular synovitis contained no activated cells, no were any seen in sections of normal synovium. The presence in rheumatoid synovial membrane of activated T cells which are only of the T4+, Leu8+ subset adds weight to the suggestion that local immunoregulatory dysfunction contributes to the chronic inflammation of rheumatoid arthritis.

Changes in phenotypically distinct mucosal macrophage populations may be a prerequisite for the development of inflammatory bowel disease.

Previous studies have demonslraled the presence of much more marked macrophage heterogeneity in colonic mucosa affected by the idiopathic inflammatory bowel diseases (ulcerative colitis and Crohns disease) than in normal mucosa. This study examines the morphology, distribution and phenotypic expression of mucosal macrophage‐like cells in biopsies from patients with idiopathic inflammatory bowel disease in comparison with disease control samples from patients with colonic infection or ischaemia. Approximately 80% of macrophage‐like cells in histologically normal mucosa co‐express the antigens recognized by the monoclonal antibodies RFDI (an interdigitating cell marker) and RFD7 (a marker for mature tissue macrophages). In idiopathic inflammatory bowel disease, the normal colonic macrophage population is partly replaced by cells staining positively with RFD7 alone, and, to a lesser extent, with RFDI+ dendritic cells. Sections from patients with infections and ischaemia exhibited epithelial HLA‐DR positivity and infiltration of the lamina propria by a more heterogeneous population of macrophages than that seen in histologically normal mucosa. However, the displacement of the normal colonic macrophage phenotype by RFD7+ tissue macrophages occurred to a significantly greater extent in idiopathic inflammatory bowel disease than in disease control mucosa. A pathognomonic feature of the ulcerative colitis and Crohns colitis sections was the clustering of RFD9+ epithelioid cells at the bases of disrupted crypts and adjacent to areas of mucosal damage. It is concluded that a degree of macrophage heterogeneity and macrophage infiltration can occur as a non‐specific response to colonic mucosal damage. The distinctive feature of idiopathic inflammatory bowel disease mucosa is the almost complete replacement of the normal colonic mucosal macrophage population by tissue macrophages and epithelioid cells, and this phenomenon may be important in promoting the development of a chronic inflammatory state

Mononuclear cells in glomeruli and cytokines in urine reflect the severity of experimental proliferative immune complex glomerulonephritis

Immunohistochemical methods were used to investigate the role of macrophages in the progression of proliferative immune complex glomerulonephritis. The mononuclear cell component of glomerular inflammation was analysed in three different stages of chronic serum sickness, each of which was clearly distinguished by criteria of kidney function. Urinary excretion of the macrophage secretory products interleukin‐1 and tumour necrosis factor was also evaluated in relation to the functional severity of kidney disease. T lymphocytes and macrophages began to accumulate in glomeruli at the onset of proteinuria, but not before. Urinary excretion of interleukin‐1 also began with proteinuria. Proteinuria increased in direct correlation with increases in the number of glomerular macrophages. Development of the most severe stage of glomerulonephritis, characterized by cachexia, declining kidney function, and necrotizing glomerular pathology, was accompanied by the disappearance of T cells from glomeruli and the expression of highly abnormal phenotypes by most macrophages. In addition, there was a switch from urinary excretion of interleukin‐1 to excretion of tumour necrosis factor. The progression of proliferative immune complex glomerulonephritis was associated with qualitative as well as quantitative changes in glomerular macrophage populations. Differentiation and/or activation of those glomerular macrophages may have resulted from local T cell‐mediated immunoregulation. Measurements of urinary cytokine excretion provided a reliable means of monitoring disease progression. The local action of tumour necrosis factor probably contributed to declining kidney function in the most severe stage of disease.

The Involvement of Dendritic Cells in Chronic Inflammatory Disease

The term dendritic cells has been used collectively to describe a group of non-lymphoid mononuclear cells that appear to act as accessory cells in immune responses and characteristically appear with a dendritic morphology. This group comprises Langerhans cells (LCs), interdigitating cells (IDCs), follieular dendritic cells (FDCs), veiled cells (VCs), and indeterminate cells (ICs). The term dendritic cell has also heen applied to a specific cell type in mice (30], which is thought to be the murine equivalent of the human IDC [11]. In this review the term dendritic cells will be used to refer to those human eells listed above and to distinguish them from the monocyte/ macrophage populations. The need for this distinction particularly when studying immunopathology will be discussed below. The origin and interrelationship of dendritic cells remain ohscure. A current consensus, however, would follow the hypothesis that veiled cells are the circulatory form of dendritic cells, which may become ICs, IDCs, or LCs when they enter the tissues, the different eharacteristics of these tissue-bound cells being influenced by the environment in which they find themselves [1, 19]. The FDCs appear as a separate population restricted to the germinal centres of lymphoid follicles (13]. Since these cells are not directly involved in the development or makeup of chronic inflammatory infiltrates, they will not be discussed further. Using monoclonal antibodies (McAhs) in immunohistologieal studies together with cytochemicai reactions, it has been possible to establish the phenotype of dendritic cells. Phenotype differences can then be used when studying tissue reactions to distinguish specific dendritic cells one from another and from maerophages. These phenotypes are listed in Table I. By using those McAhs that appear specific for one type of dendritic cell, it has been possible to identify discrete cell populations within sections of normal tissues and subsequently within the cellular infiltrates of chronic inflammatory lesions (see helow). The precise identification of these cells was made possible by using combinations of reagents in immunofluoresence or immunocytochetiiical methods [15, 20] or combinations of immunohistology and cytochemistry (20]. Such studies in man were first used to determine the normal distribution of dendritic eells, which seemed rather restricted (Table II). Experimental animal studies have indicated, however, that cells with a similar phenotype may he more widely distributed [10, 32]. Even in man small collections of dendritic cells have been identified in tissues distinct from the classical lymphoid organs [27, 28]. The distribution of these cells may therefore be more extensive than originally thought. The funetional significance of dendritic eells lies in the extreme efficiency of these eells in presenting antigens to lymphocytes. This has been shown true for LCs [3, 29, 31], VCs and IDCs [1, 4, 14] in terms of stimulating T lymphocytes and for FDCs in the induction of B-cell responses [13].

The demonstration of cell surface antigens on T cells, B cells and accessory cells in paraffin-embedded human tissues

This paper describes a method for processing fresh tissue that allows immunohistological analysis on paraffin sections. The method is based on the use of periodate-lysine-paraformaldehyde fixation. The effects of variation in fixation time, concentration of paraformaldehyde, dehydration, clearing, wax embedding and enzyme treatment of cut sections were examined. An optimal processing procedure was established that retains good tissue morphology and allowed 21 out of 27 monoclonal antibodies tested to be used successfully on paraffin sections to identify all major cell subpopulations by their membrane antigenic characteristics. The value of this approach in studying the immunopathology of potentially dangerous infectious diseases and in leukaemia/lymphoma diagnosis is discussed.

Mucosal macrophage subsets of the gut in HIV: decrease in antigen-presenting cell phenotype.

The effect of HIV infection on intestinal lamina propria macrophage subsets was investigated in 41 patients at various stages of HIV infection (asymptomatic HIV infection, n= 17; AIDS, n= 24). Duodenal biopsies taken from HIV patients at endoscopy were snap frozen and cryostat sections cut for immunohistochemical staining. MoAbs CD68 (EBM11. pan‐macrophage marker). RFD1 (antigen‐presenting cells) and RFD7 (mature phagocytic macrophages) were used to identify cell subsets using indirect immunoperoxidase or alkaline phosphatase. Double immunofluoresccncc using MoAbs to HIV proteins (p24, p17 and gp120) and RFD1 were used to identify HIV‐infected antigen‐presenting cells. Double immunofluoresccncc was also used to identify macrophages that expressed both RFD1 and RFD7 (‘suppressor’ macrophages). Intensity of HLA‐DR expression in lamina propria cells was investigated using a MoAb to HLA‐DR directly conjugated to glucose oxidase. The results show that there was no difference in overall density of macrophages, but there was a significant decrease in dendritic cells (RFD1+) in all clinical stages of HIV. There was no difference in the density of RFD7+ macrophages, nor was there a difference in intensity of HLA‐DR expression in lamina propria cells. Only four HIV‐infected cells were positively identified in the 41 patients. This result suggests that the antigen‐presenting arm of mucosal immune defences may be seriously compromised in HIV infection, and represents a further insult to mucosal immunity already impaired as a result of loss of CD4+ T lymphocytes. This may contribute to development of opportunist infection in the gut.