Immunoelectron microscopic demonstration of tissue antigens with monoclonal antibodies

Two methods of demonstrating tissue antigens by ultrastructural enzyme immunohistochemistry were tested. The monoclonal antibodies Ki-M1 and Ki-M4 were chosen for testing the methods because Ki-M1 identifies a relatively stable, and Ki-M4 a very unstable antigen. The two antibodies react selectively with human macrophages and interdigitating reticulum cells or dendritic reticulum cells of lymphoid follicles. The Ki-M1 reaction product is confined to the surface membrane. Ki-M4 reactivity is located on the surface membrane and, less often and to a lesser extent, in the cytoplasm. The technical prerequisites for reliable conservation of the antigens identified by these two antibodies were standardized. The results indicated that prior fixation in 4% paraformaldehyde is preferable for optimum preservation of stable antigens. Application of the primary antibody prior to fixation was found to be the best procedure for demonstrating unstable antigens, although nonspecific reactions were seen more often with this method.     

Ki-M7 monoclonal antibody specific for myelomonocytic cell lineage and macrophages in human

We describe a new monoclonal antibody, termed Ki-M7, which is specific to human myelomonocytic cell lineage and macrophages, as tested by immunohistochemical methods. Ki-M7 recognizes an intracytoplasmic antigen of molecular weight 29,000. Ultrastructurally, the antigen is localized in the lysosome and phagosome compartments and seems to be involved in generation of oxygen radicals during the respiratory burst. Dendritic cells, such as dendritic reticulum cells of lymphoid follicles and interdigitating reticulum cells of lymphoid T-zones, considered as accessory cells of the B- and T-cell immune response, respectively, do not show any reactivity with monoclonal antibody Ki-M7. Ki-M7 seems to be an appropriate reagent to clearly differentiate between the phagocytosing and the immune accessory population of the human monocyte/macrophage system.     

Antigenic profile and functional characterization of human peritoneal macrophages

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.     

Application of liposomes to generation of monoclonal antibody to glycosphingolipid: production of monoclonal antibody to GgOse4Cer

Liposomes were applied to the immunization with GgOse4Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized with GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.     

Characterization of cells containing factor XIII subunita in benign and malignant buccal lesions

Summary In the present study, the distribution pattern and characteristics of cells containing Factor XIII subunita (FXIII A) have been studied in benign and malignant lesions of human buccal mucosa. Tissues from four irritation fibromas and three squamous cell carcinomas were studied by means of double immunofluorescent staining techniques in which the detection of FXIII A was combined with a reaction with CD14 (recognizing a monocyte/macrophage differentiation marker antigen), Mac 387 (reacting with a special subset of macrophages), anti-HLA-DR, Ki-M7 (labelling phagocytosing macrophages) or Ki-67 (visualizing a nuclear antigen associated with cell proliferation) monoclonal antibodies. FXIII A was detected in cells of the connective tissue stroma in both benign and malignant buccal lesions. The number of these FXIII A-reactive cells (FXIII A⁺ cells) increased considerably in the tumour tissues, in particular in those surrounding tumour cell clusters. FXIII A⁺ cells scattered in the fibromatous tissues were spindle-shaped, whereas in the tumour stroma, large stellate cells predominated, and round cells were likewise labelled around blood vessels. FXIII A⁺ cells were labelled with CD14 and Ki-M7 in both fibromatous and tumoural buccal mucosa; however, they failed to show any reaction with Ki-67. FXIII A⁺ cells accumulated in the tumour stroma reacted for HLA-DR as well. These results indicate that in both the benign and malignant buccal lesions FXIII A is contained in a subpopulation of tissue macrophages, which represents a monocyte-derived (CD14⁺) and phagocytosing (KiM7⁺) cell population. The accumulation of the FXIII A⁺ cells in the tumour stroma is believed to be a result of direct migration from the circulating blood. The FXIII A⁺ cells of the tumour stroma may be actively involved in both antigen presentation and matrix remodelling during tumour progression.     

Production and some properties of monoclonal antibodies against human pancreatic secretory trypsin inhibitor.

Hybridomas that secrete monoclonal antibodies against human pancreatic secretory trypsin inhibitor (PSTI) were established by fusion of spleen cells obtained from mice immunized with PSTI with mouse NS-I-Ag 4/1 myeloma cells. One of three resulting monoclonal antibodies (KN-1) was found to recognize the N-terminal moiety of the inhibitor, while the others (KN-2 and KN-3) reacted with other as yet undefined parts of the molecule. Trypsin inhibitory activity of PSTI treated with KN-1 monoclonal antibody was the same as that of PSTI itself, thus indicating no relationship between the N-terminal moiety of the PSTI molecule and its inhibitory activity. We further examined the applicability of one of the monoclonal antibodies (KN-1) for immunohistochemical study of human pancreatic cancer tissue including the normal as a model, and found granular staining of the cytoplasm of the normal acinar and duct cells and also of that of adenocarcinoma cells in formalin-fixed, paraffin-embedded tissue sections.     

A study of lymphocyte subsets in human normal tonsil and lymph node

A series of T and B lymphocyte specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulation in frozen and paraffin tissue sections of human normal tonsil and lymph node by means of immunocytochemical technique. In the paracortical and interfollicular area of tonsil and lymph node, most lymphocytes reacted with Leu 1, Leu 3 a, Leu 4 and OKT4. The numbers of Leu 2 a and OKT8 positive cells were rare in tissue. These cells were not only limited in paracortical area, they also appeared in considerable numbers in medullary cords of lymph nodes. Leu 2 a and OKT 8 positive cells decreased with prominent follicular hyperplasia of tonsils. In addition, substantial leu 3 a and Leu 4 cells were found in the germinal centers. This finding supports the importance of these lymphocyte subsets in regulation of human immune response. In the mantle zone of secondary follicles, the majority of lymphocytes were positive for OKB 2 and BA 1, whereas, the IgM positive cells were predominately observed in the cytoplasma and extracellular substance of B lymphocytes in the germinal centers, but the lymphocytes bearing sIgM were rarely observed. In the mantle zone, the IgM were frequently found on the surface of membrane of small lymphocytes, however, the staining intensity was much than that in the germinal centers.     

Peroxidase-positive mononuclear leukocytes as possible precursors of human dendritic reticulum cells

In this report, we provide evidence that suggests the dendritic reticulum cells (DRC) occurring in germinal centers of lymphatic follicles originate from a distinct endogenous peroxidase-positive mononuclear blood cell subset. The new monoclonal antibody Ki-M4 showed a highly restricted reactivity, tested by immune histochemistry, being confined to DRC, lining cells of lymph node sinuses, and to 0.001% of nonadherent mononuclear blood cells separated at a density of 1.077 g/ml; the latter exhibited endogenous peroxidase activity in cytoplasmic granules. Such granules being specific for myelomonocytic cells implies that DRC may derive from a committed nonadherent subpopulation of this cell line, which in turn originates from bone marrow. The Ki-M4-reactive antigen was found to be destroyed by the majority of fixatives and to resist 4% paraformaldehyde for 10 min and cold acetone for 30 sec. In cell suspensions from human tonsils, Ki-M4 showed strong reactivity with the outer surface of DRC plasma membrane. This observation demonstrates the possibility of using Ki-M4 to separate DRC from cell suspensions in functional tests.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.     

Common immunologic determinant between human immunodeficiency virus type 1 gp41 and astrocytes.

Monoclonal antibodies against a synthetic 12-amino-acid peptide that comprises the immunodominant domain of human immunodeficiency virus type 1 gp41 (amino acids 598 through 609) reacted with astrocytes found in human and rodent central nervous system tissue. The monoclonal antibodies bound to a 43-kDa protein found in central nervous system tissue preparations. These results indicate that human immunodeficiency virus type 1 gp41 contains a common epitope with astrocytes and that an immune response to human immunodeficiency virus type 1 gp41 could generate antibodies that are cross-reactive to astrocytes. Furthermore, anti-astrocyte antibodies, which were directed at a common epitope with the gp41 sequence, were found to be present in cerebrospinal fluid from some AIDS patients with central nervous system complications. Astrocytes regulate the environment for appropriate neuronal function, and astrocyte hyperactivity (astrocytosis) is known to be the common and early pathologic event in brains from patients with central nervous system AIDS. We suggest that antibody-induced effect(s) on astrocytes could lead to the physiologic neuronal dysfunctions observed in AIDS patients.     

Serological analysis of serogroup icterohaemorrhagiae using monoclonal antibodies

Eighteen serovars (19 strains) of serogroup Icterohaemorrhagiae were serologically analyzed using 18 monoclonal antibodies against serovar copenhageni Shiromizu, M20 and serovar icterohaemorrhagiae RGA strains. The reaction patterns of the serovars against these monoclonal antibodies were different. According to these results, we divided the serovars, except for serovar tonkini, into the following three subgroups: Subgroup 1 reacted to many monoclonal antibodies including serovars icterohaemorrhagiae, copenhageni, hualien, monymusk, mankarso, and budapest. Subgroup 2 fell between subgroups 1 and 3 including serovars dakota, naam, bogvere, birkini, smithi, ndambari, gem, ndahambukuje and mwogolo. Subgroup 3 reacted to only a few monoclonal antibodies: serovars weaveri and sarmin. Serovar tonkini did not react to any of the monoclonal antibodies used. There is a possibility that serovar tonkini does not belong to serogroup Icterohaemorrhagiae. Further studies on the serological reactions of each strain revealed that it was impossible to distinguish the RGA strain from the serovar hualien LT11-31 strain, indicating that they may be identical. It was also observed that serovar copenhageni and monymusk seemed to be closely related. Serovars birkini and smithi, and serovars ndambari and gem were alike in their serological reactivities. Among the 18 monoclonal antibodies, RGAMA-1 was a unique antibody which reacted only to serovar icterohaemorrhagiae and serovar hualien, indicating that it must be the serovar icterohaemorrhagiae specific antibody. On the other hand, SHIRMA-2, 5, 6 reacted to all the serovars except for serovars weaveri, sarmin, and tonkini. These antibodies exhibited a broad reaction spectrum.     

Specific monoclonal antibodies to Opisthorchis viverrini.

A Balb/c mouse was immunized with a crude soluble antigen of Opisthorchis viverrini adult worms (OVAA) over a period of 7 months. Spleen cells from the immune mouse were fused with Sp2/0 myeloma cells. Among the 264 tissue culture wells containing the fused cells, cells of 96 wells (36%) produced antibodies to the immunizing agent. Antibodies produced by cells in several wells reacted with antigens from other species of parasite. Cells of 17 wells produced antibodies specific only to OVAA, thus cells from three representative wells were cloned by limiting dilution. Hybrids obtained produced antibodies which could be classified according to their tissue specificities into three groups. The first group of antibodies reacted strongly to the worm integument and weakly with the muscles while those belonging to the second group reacted only to muscles of the worms. The monoclonal antibodies of the third group gave a positive reaction to both muscles and tegument.     

Mononuclear cell infiltrate, HLA-Dr expression and proliferation in 37 acoustic schwannomas

Frozen sections from 37 schwannomas of the VIII nerve were reacted with a panel of monoclonal antibodies to macrophage, lymphocyte, HLA-Dr invariant chain and nuclear proliferation antigens. A moderate number of macrophages was demonstrated in 96% of tumours. CD8- and CD4-lymphocytes were detected in slightly smaller numbers in up to 87% and 23% of tumours respectively. B-lymphocytes were present in only 2/32 cases and NK-cells were absent from all 16 cases tested. HLA-Dr antigen was expressed by macrophages in most cases and by tumour cells in 13/24 tumours. These findings may represent evidence for a degree of cellular immune response. Occasional cells featuring nuclear proliferation were detected in 15/27 cases.     

Change in tropism upon immune escape by human immunodeficiency virus.

The V3 loop of human immunodeficiency virus type 1 is both a determinant of viral cell tropism and a target for neutralizing antibodies. This relationship was investigated. Selection of a dual-tropic (T cells and macrophages) virus to replicate in CD4+ brain cells results in loss of macrophage tropism and of neutralization by an anti-V3 loop monoclonal antibody. Moreover, selection of the brain-selected variant to escape from V3 loop-specific neutralizing monoclonal antibodies results in the reduction or loss of brain cell tropism and the reacquisition of macrophage tropism. These data may indicate that the antigenic diversification of human immunodeficiency virus type 1 apparent after seroconversion can be selected either by immune responses or by colonization of new cell types.     

Modulation of surface antigens of a human monocyte cell line, U937, during incubation with T lymphocyte-conditioned medium: detection of T4 antigen and its presence on normal blood monocytes

The human monocyte line, U937, derived from an individual with histiocytic lymphoma, undergoes morphological and functional changes when incubated with medium conditioned by lectin-stimulated cloned human T lymphocytes. Using monoclonal antibodies and flow cytometry, we therefore analyzed alterations in surface components that might accompany these morphological changes, in comparison with components present on normal blood monocytes. The U937 cells possess three surface antigens in common with blood monocytes, detected with OKM1, 4F2, and anti-monocyte.2 (the last monocyte specific). DR antigen was not detectable on U937 cells with three anti-DR framework antibodies but was detected on blood monocytes. Unexpectedly, OKT4, a monoclonal antibody to T4 antigen previously believed to be restricted to helper T lymphocytes, also reacted with U937 cells. Six monoclonal antibodies to other epitopes on T4 also reacted with U937 cells. None of these could be inhibited by blocking of Fc receptors. T4 with its various epitopes were also expressed on normal human blood monocytes. Other lymphocyte surface markers (T3, T8, T6) and fibronectin were not detectable on U937 cells or monocytes. An individual, whose lymphocytes lacked the epitope detected with OKT4 but had epitopes detected with OKT4 A, B, C, and D, had monocytes with identical reactivity, evidence that the T4 on monocytes and lymphocytes are products of the same structural gene. Stimulation of U937 cells for 24 hours with supernatants from Con A-stimulated T lymphocyte clones caused an increase in expression of OKM1 and Fc receptor activity and a decrease in expression of T4, consistent with a more mature phenotype of blood monocytes. Although the function of the T4 molecule is unknown, it is notable that it is displayed by two cells of distinct lineage which interact in the response to soluble antigens.     

Quantitative immunocytochemical characterization of mononuclear phagocytes. I. Monoblasts, promonocytes, monocytes, and peritoneal and alveolar macrophages

The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.     

Monoclonal antibodies directed against mouse macrophages in different stages of activation for tumor cytotoxicity

Three rat monoclonal antibodies against mouse peritoneal macrophages in different stages of activation were produced and characterized. One of these (AcM.1) bound to activated macrophages induced by pyran and Corynebacterium parvum, but not to resident and thioglycollate medium- (TGC) or proteose peptone- (PP) elicited macrophages. On the contrary, the antigen identified by MM9 monoclonal antibody was expressed only on resident and TGC- or PP-elicited macrophages. WE15 monoclonal antibody, on the other hand, reacted with all of the macrophages described above. In the assay for function, AcM.1 and WE15 monoclonal antibodies in the presence of complement (C) abolished the capacity of activated macrophages induced by pyran or C. parvum but not the capacity of killer T cells and natural killer (NK) cells to kill tumor target cells. On the other hand, MM9 and anti-Thy-1.2 monoclonal antibodies in the presence of C, as expected, did not affect the cytotoxicity of activated macrophages. However, none of the four monoclonal antibodies in the absence of C had any blocking effect on macrophage-mediated cytotoxicity. AcM.1 antibody reacted with two polypeptides with m.w. of 70,000 and 45,000 on pyran-activated macrophages; however, the antigens recognized by WE15 and MM9 have not been determined yet. These results indicate that the three rat monoclonal antibodies define different antigens present on macrophages at different stages of activation for tumor cytotoxicity, and that these antibodies should prove to be useful probes for analyzing the mechanism of activation of macrophages for tumor cytotoxicity.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.