Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

https://science.sciencemag.org/content/sci/249/4975/1431.full.pdf

CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.

Dendritic cells: unique leukocyte populations which control the primary immune response.

Dendritic cells comprise a system of highly efficient antigen-presenting cells which initiate immune responses such as the sensitization of T cells restricted by major histocompatibility complex molecules, the rejection of organ transplants and the formation of T-cell-dependent antibodies. Dendritic cells are found in many non-lymphoid tissues, such as skin (Langerhans cells) and mucosa, and they migrate after antigen capture through the afferent lymph or the bloodstream to lymphoid organs, where they efficiently present antigen to T cells. Dendritic cells are difficult to isolate and, although they originate from bone marrow their site of maturation and the conditions that direct their growth and differentiation are still poorly characterized. Granulocyte macrophage-colony stimulating factor (GM-CSF) favours the outgrowth of dendritic cells from mouse peripheral blood. Here we extend this finding to man and demonstrate that cooperation between GM-CSF and tumour necrosis factor-alpha (TNF-alpha) is crucial for the generation of human dendritic/Langerhans cells from CD34+ haematopoietic progenitors. The availability of large numbers of these cells should now facilitate the understanding of their role in immunological regulation and disorder.

GM-CSF and TNF-α cooperate in the generation of dendritic Langerhans cells

Monocytes are circulating blood leukocytes that play important roles in the inflammatory response, which is essential for the innate response to pathogens. But inflammation and monocytes are also involved in the pathogenesis of inflammatory diseases, including atherosclerosis. In adult mice, monocytes originate in the bone marrow in a Csf-1R (MCSF-R, CD115)-dependent manner from a hematopoietic precursor common for monocytes and several subsets of macrophages and dendritic cells (DCs). Monocyte heterogeneity has long been recognized, but in recent years investigators have identified three functional subsets of human monocytes and two subsets of mouse monocytes that exert specific roles in homeostasis and inflammation in vivo, reminiscent of those of the previously described classically and alternatively activated macrophages. Functional characterization of monocytes is in progress in humans and rodents and will provide a better understanding of the pathophysiology of inflammation.

Blood monocytes: development, heterogeneity, and relationship with dendritic cells.

The immune system generally avoids horror autotoxicus or autoimmunity, an attack against the body's own constituents. This avoidance requires that self-reactive T cells be actively silenced or tolerized. We propose that dendritic cells (DCs) play a critical role in establishing tolerance, especially in the periphery, after functioning T cells have been produced in the thymus. In the steady state, meaning in the absence of acute infection and inflammation, DCs are in an immature state and not fully differentiated to carry out their known roles as inducers of immunity. Nevertheless, immature DCs continuously circulate through tissues and into lymphoid organs, capturing self antigens as well as innocuous environmental proteins. Recent experiments have provided direct evidence that antigen-loaded immature DCs silence T cells either by deleting them or by expanding regulatory T cells. This capacity of DCs to induce peripheral tolerance can work in two opposing ways in the context of infection. In acute infection, a beneficial effect should occur. The immune system would overcome the risk of developing autoimmunity and chronic inflammation if, before infection, tolerance were induced to innocuous environmental proteins as well as self antigens captured from dying infected cells. For chronic or persistent pathogens, a second but dire potential could take place. Continuous presentation of a pathogen by immature DCs, HIV-1 for example, may lead to tolerance and active evasion of protective immunity. The function of DCs in defining immunologic self provides a new focus for the study of autoimmunity and chronic immune-based diseases.

/pdf/avoiding-horror-autotoxicus-the-importance-of-dendritic-21cozhofan.pdf

Avoiding horror autotoxicus: The importance of dendritic cells in peripheral T cell tolerance

Previous studies demonstrated that lymphoid tissues of mice and rats contain small numbers (less than 1 percent of nucleated cells) of dendritic cells (DC) with special cytologic, surface, and functional properties. We show here that similar DC represent 0.1-0.5 percent of human peripheral blood mononuclear cells. DC can be enriched to 20-60 percent purity by a multistep procedure analogous to that used in mice. Adherent peripheral blood mononuclear cells are cultured overnight, and the released cells are depleted of monocytes and B cells by readherence to plastic, rosetting with erythrocytes coated with anti-human IgG, and centrifugation in dense albumin columns. Enriched DC have similar cytologic features to rodent DC by light and electron microscopy. DC express HLA, and HLA-DR and the leukocyte-common antigens. They lack phagocytic capacity, receptors for antibody-coated and neuraminidase-treated erythrocytes, surface and intracellular Ig, esterase, peroxidase, and azurophilic granules. DC do not react with several monoclonal antibodies directed to phagocytes (OKM 1, "mac-1," 63D3, and 61D3) and T cells (OKT 3, 6, 8). Unlike the mouse, human DC express complement receptors. When maintained in culture for 4 d, human DC did not give rise to either B cells or monocytes. Therefore, DC identified by cytologic criteria are distinct from other leukocytes. Enriched populations of DC have been compared to fractions enriched in monocytes, B cells, and T cells in three functional assays: stimulation of the primary allogeneic mixed leukocyte reaction, stimulation of the primary syngeneic MLR, and accessory function for the proliferation of periodate- modified T cells. In each case, the DC fraction was 10-fold or more active than other cell fractions. We conclude that DC circulate in man, and represent the principal cell type required for the initiation of several immune responses.

https://rupress.org/jem/article-pdf/155/4/1172/1393570/1172.pdf

Human dendritic cells. Enrichment and characterization from peripheral blood.

Monocyte-specific monoclonal antibodies (7) were used to compare the efficacy of monocytes and dendritic cells as accessory or stimulator cells for human T cell replication. Both unfractionated and plastic-adherent mononuclear cells were first treated with a cytolytic antimonocyte antibody that kills greater than 95% of monocytes but not dendritic cells. When tested as stimulators of the mixed leukocyte reaction (MLR) and of oxidative mitogenesis (the proliferation of T cells modified with sodium periodate), the monocyte-depleted cells had normal or enhanced stimulatory capacity. Monocyte-depleted mononuclear cells also proliferated normally to soluble antigens (Candida albicans, tetanus toxoid), even under limiting conditions of cell dose, antigen dose, and culture time. Adherent blood mononuclear cells were next separated into monocyte-enriched and -depleted components using fluoresceinated antimonocyte antibody and the cell sorter. The depleted fraction (less than 2% monocytes by esterase staining and by cytology) contained the dendritic cells and exhibited at least 75% of the accessory activity. The monocyte-rich fraction (approximately 97% esterase positive) stimulated the MLR and oxidative mitogenesis weakly, and was comparable in potency to nonadherent cells. Cell-specific antibodies and complement were also used to prepare dendritic cells that were thoroughly depleted of monocytes and lymphocytes. The dendritic cells (70-80% pure) were potent stimulators of the allogeneic MLR, syngeneic MLR, and tetanus toxoid response, being active at stimulator to responder ratios of 1:100 or less. Taken together with previous studies (1, 2), these experiments indicate that the dendritic cell is the major stimulator of T cell replication in man. The contribution of class II products of the major histocompatibility complex (7) was then evaluated with a new monoclonal, 9.3F10. Accessory function was dramatically inhibited if cells bearing class II antigens were killed with 9.3F10 and complement, or if class II molecules were blocked by the addition of 9.3F10 Fab to the culture medium. The expression of 9.3F10 class II products was therefore studied on purified monocytes and dendritic cells. Most if not all cells in both populations reacted with 9.3F10, and each population exhibited approximately 150,000 125I-Fab 9.3F10 binding sites per cell. Since Ia+ dendritic cells are active accessory cells, but Ia+ monocytes are not, class II products are necessary but not sufficient for the stimulation of T cell proliferation in man.

https://rupress.org/jem/article-pdf/158/1/174/1093524/174.pdf

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication.

We report on the characteristics of cells in the cutaneous lesions and blood of 21 patients with lepromatous, tuberculoid, and intermediate forms of leprosy. A large proportion of the infiltrates in lepromatous lesions consist of macrophages heavily parasitized with Mycobacterium leprae. The T cells in the lesions are devoid of OKT4/Leu 3a-positive ("helper") cells and consist almost exclusively of OKT8/Leu 2a-positive ("suppressor") populations. In contrast, the tuberculoid infiltrates contain well-organized epithelioid and giant-cell granulomas and only remnants of bacilli, and the predominant T cell is from the OKT4/Leu 3a-positive subset. In both tuberculoid and lepromatous infiltrates, T cells and macrophages expressed HLA-DR antigen. No marked alteration in the distribution of blood T-cell phenotypes was noted. We conclude that there is a marked difference between T-cell subsets in lepromatous and tuberculoid infiltrates, which may influence the microbicidal activity of macrophages in the l...

The cutaneous infiltrates of leprosy: cellular characteristics and the predominant T-cell phenotypes.

3C10 and 1D9 are two related monoclonal antibodies that specifically identify human mononuclear phagocytes in a large number of sites, including blood monocytes, alveolar macrophages, and macrophages in tissue sections of spleen, lymph node, and skin. The antigen persists on monocytes cultured for greater than 4 wk, but it is not found on giant cells. The 3C10-1D9 determinant is carried by a 55 kD polypeptide, is expressed at approximately 40,000 copies per monocyte, and is protease sensitive. The antigen is clearly different from HLA-class II or Ia-like antigens that have been studied with a new monoclonal 9.3F10. The 9.3F10 antigen is found on B cells, dendritic cells and monocytes; is protease resistant, and occurs on a 33-29 kD doublet typical of class II products. The 3C10 monoclonal provides a clear distinction between human mononuclear phagocytes and dendritic cells. First, monocytes and lymphocytes can be eliminated from plastic-adherent mononuclear cells using 3C10, complement, and two previously described cytotoxic antibodies, BA-1 (anti-B cell) and Leu-1 (anti-T cell). As a result, the trace dendritic cell component of blood can be enriched to considerable purity (65-75%) and yield. Second, immunocytochemical staining of tissue sections reveals that 3C10+ macrophages are anatomically segregated from dendritic cells. Large numbers of 3C10+ cells are found in red pulp of spleen and in regions surrounding lymphatic channels of lymph node. However, 3C10+ macrophages are scarce in white pulp of spleen and the lymphocyte-rich cortex of node that are the sites where dendritic cells are localized. 3C10+ cells in skin are found in the dermis, particularly in leprosy infiltrates, but the Langerhans' cells of epidermis are 3C10-. The distinctive localization of macrophages and dendritic cells is consistent with their respective functions as effector and accessory cells in the immune response.

https://rupress.org/jem/article-pdf/158/1/126/1093518/126.pdf

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.

L S Hair

Papers

Human dendritic cells. Enrichment and characterization from peripheral blood.

Relative efficacy of human monocytes and dendritic cells as accessory cells for T cell replication.

The cutaneous infiltrates of leprosy: cellular characteristics and the predominant T-cell phenotypes.

Specific antimononuclear phagocyte monoclonal antibodies. Application to the purification of dendritic cells and the tissue localization of macrophages.