The anatomy of peripheral lymphoid organs with emphasis on accessory cells: Light-microscopic immunocytochemical studies of mouse spleen,lymph node,and peyer's patch

Antigen, lymphocytes, and accessory cells interact within peripheral lymphoid organs to generate immunity. Two cell types have been studied for accessory function in culture: mononuclear phagocytes and nonphagocytic Ia-rich dendritic cells. The monoclonal antibodies which have been used to study isolated murine macrophages (MØ) and dendritic cells (DC) include α-macrophage (F4/80, M1/70), α-dendritic cell (33D1), α-Fc receptor (2.4G2), and α-Ia (B21-2) reagents. In this paper, the antibodies have been used to stain accessory cells in cryostat sections of mouse spleen, lymph node, and Peyer's patch. Each organ is known to contain subregions that are rich in either macrophages, B cells, or T cells. We found that the accessory cells in each subregion had a different phenotype. (1) Macrophage-rich regions: Macrophages that lined the site of antigen delivery (marginal zone of spleen, around afferent lymphatics of node, and below the epithelium of Peyer's patch) were stained with M1/70 but not with F4/80. F4/80 was abundant on macrophages in other sites: spleen red pulp, node medulla, and around Peyer's patch efferent lymphatics. (2) B-lymphocyte-rich follicles: Follicular dendritic cells, which retain immune complexes extracellularly, are concentrated on the outer aspect of the germinal center. This region stained strongly with α-Fc receptor antibody 2.4G-2, but not with M1/70, F4/80, or 33D1. (3) T areas: The interdigitating cells of T areas have been linked to isolated dendritic cells. Irregular Ia-rich cells were distributed uniformly in the T areas of each organ. However, staining with 33D1 was not detected and was restricted to foci of nonphagocytic cells at the spleen red/white pulp junction. F4/80, M1/70 or 2.4G2 also did not stain the T area, except for the region close to splenic central arteries. Therefore the principal surface markers and location of the candidate accessory cells in murine lymphoid organs are M1/70⁺ macrophages at the site of antigen entry; F4/80⁺ macrophages around regions of lymphocyte efflux; germinal center dendritic cells, which may be rich in 2.4G2; and Ia-rich interdigiting cells in the T area.     

A discrete population of mononuclear phagocytes detected by monoclonal antibody

Rat monoclonal antibody raised against cultured mouse bone marrow was used to detect an antigenic determinant on a discrete population of mouse mononuclear phagocytes by indirect immunofluorescence. The antigen was expressed on adherent, late-cultured bone marrow macrophages and chronic inflammatory peritoneal macrophages elicited by the injection of thioglycolate broth. Binding of the antibody to resident peritoneal or alveolar macrophages, blood monocytes, or freshly explanted bone marrow cells was not detected. Less than 10% of acute inflammatory mononuclear phagocytes expressed the antigen. The antibody did not bind detectably to lymphocytes, granulocytes, erythrocytes, fibroblasts, or the cells of several murine tumor lines. Results suggesting binding to mast cells were equivocal. The antigen was species, but not strain, specific. It was concluded that maturation, at least, was required for expression of the antigen. Results suggested that additional influences were also involved.     

A novel rat monoclonal antibody reactive with murine tumoricidal Kupffer cells and activated peritoneal macrophages from BCG-infected mice

A rat monoclonal antibody, termed 1A2.10D and raised against mouse BCG-activated peritoneal-macrophages, was used to detect antigenic determinant(s) on mouse tumoricidal activated peritoneal and liver macrophages from BCG-infected mice by indirect immunofluorescence flow cytometry and immunoblot analysis. This antibody was of the rat IgG2b subclass and not cytolytic to BCG-activated macrophages in the presence of rabbit complement. The antigen(s) was expressed on tumoricidal activated peritoneal and liver macrophages from BCG-infected mice and some macrophage cell lines. Its expression could not be detected on resident peritoneal cells, peritoneal exudate cells containing non- or low tumoricidal macrophages, thymocytes, resting bone marrow cells, normal spleen cells, various established mouse tumour cells or one macrophage cell line. Immunoblot analysis showed the antibody to bind to one major protein of 56,000 MW, and to be expressed on the peritoneal and liver macrophages from BCG-infected mice. Using this antibody and a fluorescence-activated cell sorter, selection was made of antibody-positive or -negative macrophages from BCG-infected mice to examine their tumour killing activity. The antibody-positive macrophages clearly showed higher tumour killing activity than the negative macrophages.     

Peritoneal macrophages in the immune response to SRBC in vitro

The role of macrophages in the immune response against SRBC in spleen cell suspension cultures obtained from mice was studied.

1. The presence of macrophages increases the number of PFC against SRBC in spleen cell cultures.

2. The ratio peritoneal exudate cells to spleen cells is very critical. Two × 10⁵ peritoneal exudate cells preincubated with sheep red blood cells stimulate 1 × 10⁷ mouse spleen cells to an optimal response. This reaction is suppressed when 2 × 10⁶ peritoneal cells are used.

3. Spleen cell populations which had lost the capability of producing antibodies by elimination of cells adhering to glass yielded a strong immune response upon addition of 2 × 10⁵ peritoneal exudate cells. The significance of macrophages for the induction of the immune response is discussed.

     

In vitro studies of delayed hypersensitivity: inhibition of macrophage spreading in rats sensitive to tuberculin and diphtheria toxoid

Wistar rats were sensitized by footpad injection of BCG in adjuvant, or Mycobacterium butyricum in adjuvant, or diphtheria toxoid in Freund's incomplete adjuvant. It was found that the cell population of the peritoneal washings contained approximately 57 per cent macrophages, 22 per cent lymphocytes, 11 per cent granulocytes and 8 per cent mast cells. The lymphocyte count was significantly reduced and the granulocyte count increased after sensitization. The animals sensitized to M. butyricum exhibited delayed skin reactivity to tuberculin and the spreading of macrophages in vitro was significantly inhibited with the same antigen. On the contrary, the spreading of macrophages obtained from animals sensitized to BCG was not inhibited by tuberculin and there was no cutaneous reactivity. Spreading of macrophages obtained from rats sensitized by diphtheria toxoid was significantly inhibited in the presence of diphtheria toxoid, but not in the presence of tuberculin. These animals displayed delayed cutaneous hypersensitivity to diphtheria toxoid. Spreading of macrophages from normal rats was unaffected by serum antibodies. This was true either when the peritoneal cells were treated with antiserum prior to contact with antigen, or when the antigen—antibody reaction took place in the chamber containing the macrophages ready to spread. These results indicate that the technique of macrophage spreading inhibition is able to detect specifically hypersensitivity of delayed type and offers a convenient method for the in vitro study of delayed hypersensitivity.     

Specific surface antigens expressed on activated mouse peritoneal macrophages and recognized by a novel monoclonal antibody

A hybridoma-secreting monoclonal antibody, designated 2E12D5, was prepared by fusing mouse myelomas with spleen cells from a rat immunized with BCG-elicited mouse peritoneal macrophages. Binding of the antibody to primary mouse cells and cell lines was examined by indirect immunofluorescent flow cytometry. 2E12D5 was cytotoxic and of the rat IgG2a subclass. The antibody reacted to a great extent with the BCG-induced mouse peritoneal macrophages, half the bone marrow cells, macrophage-like cell lines and thioglycollate-induced peritoneal exudate cells to some degree, but not with other cells including BCG-elicited peritoneal lymphocytes, non- or low tumoricidal peritoneal exudate cells, thymomas, myelomas and fibroblasts. Immunoblot analysis showed the antibody to bind to four major proteins, 220,000, 125,000, 105,000 and 92,000 in molecular weight from the BCG-induced peritoneal macrophage. Pretreatment of BCG-induced peritoneal macrophages with 2E12D5 and rabbit complement greatly inhibited macrophage-mediated tumour cell cytotoxicity.     

The monoclonal antibody ER-BMDM1 recognizes a macrophage and dendritic cell differentiation antigen with aminopeptidase activity.

Here we describe the reactivity of monoclonal antibody (mAb) ER-BMDM1, directed against a 160-kDa cell membrane-associated antigen (Ag) with amino-peptidase activity. The aminopeptidase recognized by ER-BMDM1 is present on various mouse macrophage (M phi) and dendritic cell (DC) subpopulations as well as on microvillous epithelia. Analysis of ER-BMDM1 Ag expression in in vitro models of M phi maturation revealed that the Ag is expressed at increasing levels upon maturation of M phi. In vivo, high level expression of the ER-BMDM1 Ag occurs after the monocytic stage of maturation, since bone marrow cells and peripheral blood monocytes are essentially ER-BMDM1 negative. Analysis of isolated-resident and elicited M phi populations showed that ER-BMDM1 recognizes a specific subpopulation of mature M phi: only some resident peritoneal and alveolar M phi are ER-BMDM1 positive, whereas virtually all thioglycollate-elicited peritoneal exudate M phi bind the mAb. In lymphoid organs, a subpopulation of M phi is recognized as well as interdigitating cells (IDC) located in T cell areas. Phenotypic analysis of isolated DC--the in vitro equivalents of IDC--from spleen and lymph nodes confirmed that the majority of this important antigen-presenting cell population expresses the ER-BMDM1 aminopeptidase. The molecular characteristics of the ER-BMDM1 Ag suggest that it may represent the mouse homolog of human CD13.     

Selective chemotaxis of Ia antigen-positive macrophages by macrophage chemotactic lymphokine produced by concanavalin A stimulation.

M1- cells (leukaemic cell line cells established from SL/Am mouse), Mm1 cells (macrophage-like cells subcloned from M1- cells), and undifferentiated B24 cells (subcloned from M1- cells) were similarly Ia-negative and showed little chemotactic response to macrophage chemotactic lymphokine (MCF-LD) produced by Con A stimulation of spleen cells. However, when M1- cells and B24 cells were differentiated by differentiating factor in the fibroblast-conditioned medium, they became positive for surface Ia antigens and exhibited chemotactic responses to MCF-LD. Treatment of the cell-line cells and mouse peritoneal exudate macrophages with anti-Ia antiserum and complement reduced the number of cells responsive to MCF-LD. Moreover, when comparing the chemotactic responses to MCF-LD of Ia-negative and Ia-positive peritoneal exudate macrophages, separated by electric programmable individual cell sorter (EPICS), Ia-positive cells responded more strongly than Ia-negative cells.     

Monoclonal antibody against human macrophages/monocytes and granulocytes

A monoclonal antibody of the IgG2a isotype directed against lymphokine-activated human macrophage cell line (U937) was produced and characterized. By indirect immunofluorescence microscopy or binding assay, this antibody (MaG-1) reacted with human peripheral monocytes, peritoneal macrophages, alveolar macrophages, and peripheral granulocytes but not with lymphocytes, erythrocytes, and platelets. MaG-1 antigen was also expressed by 34% of bone-marrow cells which includes monocytic and granulocytic precursors, whereas lymphoid and erythroid precursor cells were found on MaG-1 negative population. Immunoprecipitation of 125I-labeled U937 cell extract with the Mag-1 antibody under nonreducing and reducing conditions yielded a specific band of 66 kD. Thus, this antibody defines a new human macrophage/monocyte and granulocyte-specific antigen and may be useful for studying differentiation of human macrophages/monocytes.     

Antibody formation by transferred cells in inbred fowls

Specific antibody formation by transferred cells from immunized donors has been demonstrated in adult recipients with cells derived from caecal tonsils, blood and peritoneal exudate as well as from spleen. Cells from all these sources formed both γM and γG antibody, regardless of whether the donors had received one or two injections of antigen. There was no difference in the amount of antibody formed by cells from primary or secondary donors. An autoradiographic method for detecting small amounts of primary 7S fowl antibody (which does not give passive haemagglutination reactions) is described.     

Activity of rabbit monocytes, macrophages, and neutrophils in antibody-dependent cellular cytotoxicity of herpes simplex virus-infected corneal cells.

Rabbit phagocytes were examined for their ability to kill target cells infected with herpes simplex virus by antibody-dependent cellular cytotoxicity. Two sources of rabbit corneal cells were used as targets: Staaten Seruminstitut Rabbit Cornea (a continuous cell line) and stromal keratocytes from the middle layer of corneas excised from New Zealand white rabbits. Peritoneal exudate and alveolar macrophages were found to be the most active effector cells, followed by blood neutrophils and monocytes. Peritoneal exudate and alveolar macrophages killed target cells with dilutions of antibody as high as 1:10,000. Monocyte antibody-dependent cellular cytotoxicity activity was absent in over one-third of the rabbits tested and was only weakly active in positive rabbits. In vitro aging of monocytes did not enhance activity. Antibody reactive with peritoneal exudate macrophages, alveolar macrophages, and blood neutrophil effector cells appeared 7 days after intracorneal injection of infectious herpes simplex virus. Results of these studies show that in vitro assays with a complete rabbit system (effectors cells, antibody, and target cells) can be developed to monitor herpetic disease and suggest an active role for rabbit phagocytes in cytotoxicity of herpes simplex virus-infected cells.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

Cytotoxicity of immune guinea-pig cells: I. Investigation of a correlation with delayed hypersensitivity and a comparison of cytotoxicity of spleen, lymph node and peritoneal exudate cells

The work was intended to show whether any correlation could be established between delayed hypersensitivity and the appearance of specifically cytotoxic cells in the spleen, lymph nodes and peritoneal exudate.

Using chicken erythrocytes as target cells, the cytotoxicity of cells from immunized guinea-pigs was assessed by the ⁵¹Cr release technique. After immunization with chicken erythrocytes and complete adjuvant, spleen, lymph node and peritoneal exudate cells were active, the overall time course of cytotoxicity corresponding broadly with intensity of delayed hypersensitivity reactions. Following intravenous immunization with erythrocytes, there was no delayed hypersensitivity response, and lymph node cells were not cytotoxic; spleen cells and peritoneal exudate cells showed cytotoxicity. A further partial correlation with delayed hypersensitivity was shown in immune-deviated guinea-pigs. Lymph node cell cytotoxicity and delayed hypersensitivity were markedly depressed while spleen cytotoxicity was unimpaired and peritoneal exudate cytotoxicity only slightly lowered.

Assessed by effector: target cell ratio, peritoneal exudate cells were found to produce greater cytotoxicity than spleen or lymph node cells. Partial cell separation by adherence showed that cytotoxicity was associated with peritoneal macrophages. Cytotoxicity directed against chicken erythrocytes was found to be target-cell specific.

     

In vivo depletion of macrophages by desulfated iota-carrageenan in mice

Almost 90% of the sulfate groups of iota-carrageenan (CGN) was removed with acid-methanol in an attempt to obtain a product which would selectively eliminate macrophages in mice. Desulfated CGN(DS-CGN) failed to induce in vivo polyclonal antibody production in DBA/2 mice. However, the number of phagocytes in the peritoneal cavity, spleen, thymus and lymph node of DBA/2 mice was reduced stringently by DS-CGN treatment. The number of Mac-1 positive cells(macrophages) in DS-CGN-treated mice gradually decreased for at least 7 days after the last injection of DS-CGN. In contrast, the relative proportion of T and B lymphocytes in the lymphoid organs was unaffected by DS-CGN treatment. DS-CGN suppressed antibody responses to SRBC, a T cell and macrophage-dependent antigen, but no such suppressive effect was observed in the polyclonal antibody responses to LPS, a T cell and macrophage-independent B cell activator. Furthermore, the impaired SRBC antibody responses in DS-CGN-treated mice were restored following transfer of adherent cells but not T cells. These experimental results indicate that DS-CGN selectively eliminates macrophages without influencing lymphocyte function in vivo.     

Evaluation of accessory cell heterogeneity. II. Failure of dendritic cells to activate antigen-specific T helper cells to soluble antigens

The activation of antigen-specific T cells requires Ia⁺, antigen-presenting accessory cells (AC). Dendritic cells (DC) and macrophages (M?) isolated from spleen and peritoneal exudate were tested as AC for the activation of T helper cells and the induction of T cell proliferation. The cell separations to obtain DC and splenic M? were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes. DC as well as the M? were able to induce antigen-specific T cell proliferation, but only the M? and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens. Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity. The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway. Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells. Based on these results and on previous data using Ia⁺ tumor cell line as AC, we confirm the existence of functional AC heterogeneity.     

A new monoclonal antibody, TRPM-3, binds specifically to certain rat macrophage populations: immunohistochemical and immunoelectron microscopic analysis

An anti-macrophage monoclonal antibody designated TRPM-3 was produced using thioglycollate-elicited rat peritoneal macrophages as immunogen. By the immunoperoxidase method, TRPM-3 was found to be specific for certain macrophage populations, such as marginal zone macrophages and marginal metallophils of the spleen, sinus macrophages of the lymph nodes, and omentum macrophages. No epithelial cells, peripheral blood monocytes, granulocytes, or lymphocytes had reacted with TRPM-3. Immunoelectron microscopically, reaction to TRPM-3 was clearly demonstrated on the plasma membrane of splenic marginal zone macrophages, lymphatic sinus macrophages, omentum macrophages, and peritoneal macrophages. Accordingly, TRPM-3 was considered to have recognized the particular membrane antigen expressed by restricted macrophage populations.     

Recognition of Rat Dendritic Cells by a Monoclonal Antibody

A mouse monoclonal IgM antibody reactive with dendritic cells (DC) from the Brown Norway (BN) rat was prepared. This antibody (1F119) binds to a membrane-bound antigen present on DC from thoracic duct lymph, spleen, thymus, and lymph node. The antigen is present only in low density on 5% of splenic macrophages (M phi) and absent from peritoneal M luminal diameter. In situ, the antibody exhibits a strong reactivity towards DC in the thymic medulla, whereas no reaction is observed with cortical cells. Furthermore, cells positive for 1F119 can be identified in T-cell areas of spleen, lymph node, and Peyers' patches. 1F119 was genetically restricted in that a strong reactivity was found with DC from rats of the RT1n and RT1u haplotypes, an intermediate reactivity with the RT1c haplotype, only a weak reactivity with the RT1l and RT1b haplotypes, and no reactivity with the RT1a and RT1k haplotypes. The relatively weak reactivity of 1F119 with respect to the RT1l haplotype also appeared from a weak binding of 1F119 to DC from Lewis rats, as was assessed by FACS analysis. This result was comparable to the binding of OX3 (RT1.Bl and RT1.Bu) to DC from BN rats. Studies performed on thymus sections of recombinant rat strains indicate that 1F119, despite its apparent specificity for DC, reacts with a polymorphic RT1.B product.     

分娩后小鼠腹腔巨噬细胞的吞噬功能

本文证明分娩后4天小鼠腹腔巨噬细胞和注射葡聚糖后的小鼠腹腔巨噬细胞,以及正常未妊娠小鼠腹腔巨噬细胞吞噬鸡红细胞和酵母菌的功力明显不同。其中。分娩后4天和注射葡聚糖腹腔巨噬细胞,其吞噬率和吞噬指数都明显高于对照组。该结果表明分娩后小鼠体内单核——吞噬细胞系统活性增强,吞噬活性增高。     

New monoclonal antibody that specifically recognizes murine interdigitating and Langerhans cells

A new monoclonal antibody, M1-8, that recognizes murine interdigitating cells (IDC) and Langerhans cells was obtained from a hybridoma prepared by fusion of SP2/0 mouse myeloma cells with splenic cells of rats immunized with IDC-rich cell suspension obtained from lymph nodes of athymic nude mice (BALB/c nu/nu). The specificity was assessed immunohistochemically on frozen sections of lymph nodes and epidermal sheets from both nude and normal mice. M1-8 reacted with paracortical IDC, veiled cells of the marginal sinus, and epidermal Langerhans cells in both normal and nude mice. In simultaneous staining by M1-8 and nonspecific esterase or anti-Ia or anti-Thy-1,2 antibody, the same epidermal dendritic cells were positive for all these antigens except Thy-1,2. Immunoelectron microscopy of the lymph node suspension using gold colloid particles revealed the attachment of gold particles to the cell membrane of IDC. Analysis by flow cytometry of the lymph node cell suspension showed 14 or 6% of M1-8-positive cells in nude or normal mouse, respectively. Immunohistochemical analysis showed that M1-8 also reacted with dendritic cells in the thymus and spleen and had a different distribution from F4/80. M1-8 also reacted with monocytes in bone marrow and peripheral blood, alveolar macrophages, and thioglycollate-stimulated peritoneal exudate macrophages. The antibody belongs to the immunoglobulin M class, reacts immunochemically with a glycoprotein in the cell membrane, and has a molecular mass of approximately 15 kDa.