Migration Of Peritoneal Exudate Cells Research Articles

Cell‐mediated immunity of the eel, Anguilla japonica (Temminck & Schlegel), as measured by the migration inhibition test

Abstract. Sensitized lymphocytes isolated from the immunized eel, Anguilla japonica, upon interaction with the antigens in vitro, elaborated into the medium a soluble factor which was capable of inhibiting the migration of peritoneal exudate cells harvested from guinea pigs. Production of migration inhibition factor (MIF) was greatest to the specific antigen although there was also some nonspecific release. The elaboration of MIF from the sensitized lymphocytes occurred from the third day after immunization of eels and was detectable until day 106. The inhibitory activity from the blood appeared to persist over a longer period compared to that from the spleen and the anterior kidney. The inhibitory activity, however, was diminished by starvation of the immunized eels.

Cellular immune responses in mice challenged with an amyocarditic variant of coxsackievirus B3

An amyocarditic variant of a temperature-sensitive (ts) mutant derived from the parent myocarditic variant Coxsackievirus B3 (CVB3m) was studied in a murine model of CVB3m-induced myocarditis to assess virus-induced antigens and their possible role in the disease process. Amyocarditic variant ts5R induced a heart tissue antigen(s), extractable by hypertonic KC1, which inhibited migration of peritoneal exudate cells from CVB3-inoculated myocarditic mice in an agarose droplet cell-migration-inhibition assay. The ts5R variant was amyocarditic at inoculum doses of 10(3) to 10(8) plaque-forming units per mouse, but in cyclophosphamide-immunosuppressed mice, ts5R induced myocarditis. Viable ts5R served as a vaccine and protected mice against CVB3m-induced myocarditis. Murine neonatal skin fibroblasts (MNSF) infected with either virus served as in vitro targets and were lysed by splenic cytotoxic T lymphocytes from mice inoculated with either virus variant. ts5R and CVB3m replicated to similar titers in murine neonatal skin fibroblasts (MNSF) at 24 hr postinoculation (pi), but differences in titers were found by 72 hr pi. Levels of natural killer cell activities in spleens of ts5R-inoculated mice were slightly lower than in spleens of CVB3m-inoculated mice at 7 days pi. The data suggest that viral induction of new antigens on target cells and viral induction of specific cytotoxic T lymphocytes that recognize these antigenic changes do not always result in induction of myocarditis.

Amphibian lymphokines: 2 Migration inhibition factor produced by antigenic and mitogenic stimulation of amphibian leucocytes

Migration of Rana temporaria peritoneal exudate cells (PEC) was examined in vitro using both direct and indirect assay systems. After sensitization in vivo followed by in vitro challenge 7–21 days later with the appropriate sensitizing antigen, spleen cell culture supernatants were obtained which inhibited the normal in vitro migration of PEC from non-sensitized animals. Cultures of spleen cells with mitogen also gave rise to supernatants with migration inhibitory properties. Sephadex separation of supernatants showed that maximum inhibitory activity was present in the 27–50,000 MW range and, furthermore, that this inhibition was blocked by α-L-fucose, but not by B-D-galactose. The inhibition did not appear to be species specific. The results indicate that following appropriate stimulation amphibian leucocytes produce a soluble, migration inhibition factor (MIF) with characteristics similar to those described for the mammalian lymphokine MIF.

Induction of tumor-specific cell-mediated immunity by a noninfectious type-C virus.

Hamsters immunized with either noninfectious hamster type-C virus (D9) or irradiated D9 tumor cells were tested for cell-mediated immune reactivity by the macrophage migration inhibition assay. The migration of peritoneal exudate cells from immunized hamsters was significantly inhibited by either D9 virus or D9 tumor extract, but not by extract of an unrelated CELO virus-induced tumor. The virus and tumor extracts had little or no effect on the migration of peritoneal exudate cells from normal hamsters. Noninfectious D9 virus produces a cell-mediated immune response in the hamster and shares antigenicity with D9 lymphoma, which releases the virus.

Cell-mediated immunity to mouse adenovirus infection. Blocking of macrophage migration inhibition and T cell-mediated cytolysis of infected cells by anti-S antigen or anti-alloantigen serum.

Migration of peritoneal exudate cells (PEC), which were prepared from mice immunized against mouse adenovirus (M-Ad), was inhibited upon exposure to the antigenic extract of M-Ad-infected cells. This inhibition was shown to be blocked when infected cells or their extracts were pretreated with antiserum against M-Ad-induced cell surface (S) antigen(s) or with antisera against alloantigens of infected cells. Immune spleen cell-mediated cytolysis of M-Ad-infected cells was also blocked in the presence of anti-S, anti-alloantigen or anti-beta 2m serum. Immunofluorescent antibody staining of S antigen(s) was blocked when infected cells were pretreated with anti-alloantigen or anti-beta 2m serum, whereas it was not blocked when they were pretreated with anti-mouse immunoglobulin or anti-Thy-1.2 serum. Conversely, immunofluorescent antibody staining of alloantigens was blocked when infected cells were pretreated with anti-S serum. These findings indicate that S and alloantigens are associated with each other or at least located very close to each other.

Cell-mediated immune responses to chlamydial antigens in guinea pigs injected with inactivated chlamydiae.

Cell-mediated immunity (CMI) to chlamydial antigens was readily induced in guinea pigs by a single injection of Betaprone-inactivated chlamydiae in complete Freund adjuvant. The CMI was measured in vivo by delayed hypersensitivity skin tests, and in vitro by inhibition of migration of peritoneal exudate cells and by proliferation of lymph node lymphocytes. There was an overall correlation between in vivo and in vitro responses. Of the in vitro assays, migration inhibition reflected the state of sensitization, as judged by skin tests, more uniformly than lymphocyte stimulation. Extensive inter- and intra-species cross-reactivity was noted between LB-1, a strain of C. trachomatis, and three strains of C. psittaci, 6BC, GPIC, and 562F. Cross-reactivity between LB-1 and 6BC was one-way only, by all three parameters: LB-1 elicited strong cross-reactions in 6BC-immunized animals but not vice versa. Antichlamydial antibodies could not be demonstrated in any of the animals by microimmunofluorescence.

Assessment of Coxsackievirus B3 ts Mutants for Induction of Myocarditis in a Murine Model

Ten temperature-sensitive (ts) mutants isolated from a myocarditis-inducing wild-type (WT) coxsackievirus B3 parent did not induce myocarditis in adolescent CD-1 mice. An avirulent prototype ts mutant from one of the three complementation groups adsorbed to murine cardiac tissue, as did WT virus. Heart tissues from mice inoculated with WT virus contained 100- to 1,000-fold more virus than heart tissues from mice inoculated with any of the three prototype ts mutants. WT virus exhibited a greater capsid stability and a higher efficiency of replication at 37 degrees C than any of the three prototype ts mutants. All three prototype ts mutants induced less interferon in vivo than WT virus. Cell-mediated immune responses, assessed by the cell migration inhibition assay, were different in mice inoculated with WT virus when compared to ts 5 mutant virus. Peritoneal exudate cells from mice inoculated with WT but not ts 5 virus reacted specifically against antigens in WT virus HeLa cell lysates and antigens extracted with KCl from cardiac tissues of mice inoculated with WT virus. Cardiac tissues of mice inoculated with WT but not ts 5 virus contained KCl-extractable antigens which were able to specifically inhibit the migration of peritoneal exudate cells taken from mice immunized with WT virus. Therefore, ts 5 neither elicited a measurable cell-mediated immune response nor induced antigens in cardiac tissues which were immunoreactive with sensitized-(WT virus)-peritoneal exudate cells. Of 9 revertant viruses isolated from the 10 ts mutants, 5 showed covariance in ability to replicate at 39.5 degrees C and capacity for induction of myocarditis. Some revertants exhibited a reduced capsid thermostability compared to WT virus but yet retained the capacity for induction of myocarditis. The data suggest that induction of myocarditis by coxsackievirus B3 variants depends on a combination of several variables, including capsid stability, capacity for replication at 37 degrees C, and expression of the three identified genes. All three prototype ts mutants served as vaccine viruses in preventing myocarditis in adolescent mice subsequently challenged with WT virus. However, all three prototype ts mutants and their revertant variants retained partial to complete lethality in CD-1 neonates.

Non-specific MIF-like activity induced by the synthetic immunoadjuvant: N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP)

N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) which represents the active part of mycobacteria for adjuvanticity is able to inhibit the migration of peritoneal exudate cells. This inhibitory action of MDP is not due to cytotoxicity because colchicine which has been shown to inhibit MIF also restores the migratory properties of MDP treated cells, and pretreatment of peritoneal cells with MDP has no influence on their later migration. There could be a relation between structural requirements for adjuvant activity and macrophage migration inhibition because the D-D-stereo-isomer of MDP which is inactive as an adjuvant, has no inhibitory properties.

Studies on carrier protein in contact dermatitis: localization of soluble epidermal carrier proteins by macrophage migration test

The migration of peritoneal exudate cells from picrylchloride-sensitive guinea pigs was inhibited by heteroantigenic components which were isolated by antinormal guinea pig epidermis antibody immunoadsorbent from soluble epidermal proteins of picrylchloride painted guinea pigs. The components were demonstrated by indirect immunofluorescence study to localize in the intercellular spaces of guinea pig epidermis. It is consequently suggested that carrier protein(s) of contact hypersensitivity reside on the cell surface of epidermis.

Development of of macrophage migration inhibition in rabbits infected with virulent Treponema pallidum

Peritoneal exudate cells from rabbits infected with Treponema pallidum Nichols were used as indicators of macrophage migration inhibitory factor activity. Between 3 and 15 weeks after infection, the migration of peritoneal exudate cells was inhibited in the presence of 3 to 25 microgram of T. phagedenis biovar Reiter protein per ml. Before this period, the migration patterns of peritoneal exudate cells from infected animals were uninhibited and similar to those from noninfected control rabbits. These observations were correlated with the development of active cell-mediated immunity during experimental T. pallidum infection.

Cellular and humoral anti-tumor immune responsiveness in chickens bearing tumors induced by avian sarcoma virus.

A systematic comparison was undertaken of the respective abilities of normal chicken embryo fibroblast (CEF) cells, Rous sarcoma virus (RSV)- transformed CEF cells, avian Rous sarcoma (RS) tumor cells and murine RS cells to serve as targets and antigen donors in various assays for the detection of cellular and humoral anti-tumor immunity in chickens bearing tumors induced by Rous sarcoma virus. As measured by a cytotoxicity procedure, avian and murine RS cells were more susceptible to the killing effects of sensitized lymphocytes than were transformed CEF which in turn were more reactive than normal CEF. In contrast, sera from tumor-bearing animals were able to stain by indirect immunofluorescence only the avian RS and transformed CEF cell types. Extracts of both transformed CEF cells and avian RS cells but not normal CEF were equally effective as inhibitors of migration of peritoneal exudate cells derived from tumor-bearing animals. Transformed CEF were found to produce far higher quantities of transforming virus progeny than avian RS cells, although the latter were apparently albe to synthesize defective viral particles. These data indicate the significant variations which may occur under experimental conditions, depending on the types of assays and target cells employed.

Identification and Purification of Immunosuppressive Activity in the Urine of Rats and a Human Patient Treated with Niridazole

Administration of the antischistosomal compound niridazole to mice, guinea pigs, and humans results in the suppression of several manifestations of cell-mediated immunity. Sera from animals treated with niridazole blocked the in vitro production of migration inhibitory factor (MIF) while niridazole itself was inactive, suggesting that these effects are caused by water soluble mediators. We now report that crude extracts prepared from the urine of rats and a patient receiving nirdazole, but not from pretreatment control urine, similarly suppress antigen-induced inhibition of migration of peritoneal exudate cells from sensitized guinea pigs. With immunosuppressive activity monitored by the direct MIF assay, combined solvent extraction and chromatographic techniques were used to fractionate immunosuppressive activity from the urine of niridazole-treated rats and the patient; the most active fractions, purified about 100-to 1000-fold as compared to methanol-water extracts of dried voided urine, inhibited MIF production at 0.1 to 0.01 ng/ml of assay mixture. These purified fractions also showed immunosuppressive activity by an in vivo assay wherein doses as low as 1 mug/kg injected intravenously (i.v.) into mice suppressed cell-mediated granuloma formation around Schistosoma manisoni eggs. Identically purified fractions prepared from urine of rats and the patient before they received niridazole showed no immunosuppressive activity either in the MIF or in the granuloma assay systems.

Isolation of Dinitrophenyl-Skin Protein Conjugates by Immunoadsorbent Chromatography

Immunoadsorbent columns charged with rabbit anti-dinitrophenyl (DNP) antibody were used to isolate and concentrate DNP-protein conjugates from the 105,000 g supernate of extracts prepared from guinea pig epidermis previously painted with dinitrofluorobenzene (DNFB). Conjugates isolated in this manner elicited positive delayed hypersensitivity skin tests when injected intradermally into DNFB-sensitized animals. Dinitrophenylated epidermal protein conjugates prepared in vitro were capable of specifically inhibiting the migration of peritoneal exudate cells obtained from DNFB-sensitized guinea pigs. These results demonstrate the application of immunoadsorbent chromatography and the migration inhibition assay to the further definition of the epitopes involved in contact sensitivity.

Studies on carrier protein in contact dermatitis: macrophage migration inhibition by soluble epidermal substances as carrier proteins

The migration of peritoneal exudate cells from picrylchloride-sensitive guinea pigs can be inhibited by the water-soluble proteins present in the picrylchloride-treated guinea pig epidermis. Further examination of these proteins revealed that antigens were present in fractions with the molecular weights ranging between 10 000 and 200 000.

Experimental autoimmune encephalomyelitis in mice: genetic control of susceptibility.

Experimental autoimmune encephalomyelitis (EAE) was induced in inbred and congenic strains of mice by injection of mouse spinal cord homogenate (MSCH) in Freund's complete adjuvant (FCA) with pertussis vaccine. Genetic analyses showed that susceptibility to EAE in mice was inherited as a dominant trait and was in part controlled by genes located in the centromeric half of the H-2 complex. Mice with EAE developed cell-mediated immune responsiveness to basic protein of myelin (BPM), as judged by the macrophage migration inhibition assay, using peritonealyexudate cells; this was not observed with mice of resistant strains. However, the migration of peritoneal exudate cells of both susceptible and resistant strains was significantly inhibited in the presence of purified protein derivative (PPD) of M. tuberculosis. Thus, the genes involved in the control of susceptibility to EAE also influence T cell responsiveness to BPM. Antibody to BPM, as judged by radioimmunoassay, was detected in susceptible and resistant strains but there was no correlation between the presence or levels of antibody and susceptibility or resistance to EAE. It is suggested that resistance to EAE is associated with failure of T cells to recognize and/or respond to the encephalitogenic determinant of the BPM molecule.

Agarose Droplet Method of Macrophage Migration-Inhibition Test for Newcastle Disease Virus in Chickens

The migration of peritoneal exudate cells collected from chickens immunized against Newcastle disease virus was inhibited by the inactivated virus antigen. The agarose droplet method for macrophage migration-inhibition test in chickens is described.

A comparison of peritoneal exudate cells and peripheral blood leukocytes in direct and indirect migration inhibition tests as in vitro assays for tuberculin hypersensitivity in guinea pigs

Peritoneal exudate cells (PEC) and peripheral blood leukocytes (PBL) are most frequently used in the migration inhibition test. The aim o this work was to compare the ability of these two types of cells to reflect tuberculin hypersensitivity in the migration inhibition test. We sensitized 36 guinea pigs with complete Freund's adjuvant and 20 controls were injected with incomplete Freund's adjuvant. Migration of PEC in medium containing 5, 15, or 75 μg of PPD/ml was assessed after 30 min, and 1, 2, 4, 18, 24, and 48 hr of incubation. The migration of PEC from sensitized animals was inhibited, the inhibition being dose dependent and, with lower concentrations of the antigen, becoming significant only after 4 hr or later. With both PEC and PBL from the same sensitized animal we observed virtually identical migration inhibition in the presence of 75 μg of PPD/ml. A correlation was found between the migration inhibition indices of PEC and PBL. In the indirect test, active supernatants containing lymphokines caused nearly identical migration inhibition of PEC and PBL from normal animals. It follows that in the guinea pig PEC and PBL behave alike both in the direct and in the indirect migration inhibition tests. Thus, PEC and PBL appear to be equally valuable sources of cells for migration inhibition tests.

Rejection of tumor cells in vitro: a T-cell-mediated reaction.

Cytoxic lymphoblasts (LBC) were produced by the co-cultivation of guinea-pig lymphoid cells from the thymus, lymph nodes, spleen, bone marrow and blood with syngeneic methylcholanthrene-induced sarcoma (MC-D) cells. The cytotoxic reaction was inhibited by pretreatment of the lymphoid cell populations with a rabbit antiserum specific for guinea-pig thymus-derived (T) lymphocytes. Supernatants of LBC inhibited the migration of peritoneal exudate cells, but did not contain lymphotoxin or a skin reactive factor. It is concluded that the cytotoxic cells are of T-cell origin.

Macrophage migration inhibitory activity of mycobacterial growth inhibitory factor and the effect of a number of factors on mycobacterial growth inhibitory factor activity.

Mycobacterial growth inhibitory factor (MycoIF), which inhibits the intracellular multiplication of virulent tubercle bacilli within normal peritoneal macrophages in vitro, was tested for its ability to inhibit the migration of normal peritoneal exudate cells. The migration of peritoneal exudate cells was not inhibited by MycoIF. It was also shown that normal peritoneal macrophages infected with virulent Mycobacterium tuberculosis, strain H37Rv, required 72 h of incubation with spleen cell culture supernatant fluids containing MycoIF in order to inhibit intracellular bacillary multiplication. Treatment of infected macrophages with trypsin before their exposure to MycoIF abolished the ability of MycoIF to inhibit intracellular mutiplication of tubercle bacilli. Incubation of infected macrophages with goat anti-mouse globulin before their exposure to MycoIF also blocked the action of MycoIF.

Studies on sperm antigenicity. 2. in vivo and in vitro cellular reactivity in guinea pigs sensitized to homologous and heterologous spermatozoal autoantigens

In vitro delayed type hypersensitivity was demonstrated with peritoneal exudate cells from guinea pigs of the Rockefeller and Hartley strains, immunized with different preparations of the guinea pig male reproductive tract (RMT) emulsified in complete Freund's adjuvant-H37Ra. The RMT preparations were purified guinea pig spermatozoal autoantigens S, P, and T; whole guinea pig spermatozoa, and extract from epididymal tissue. The cellular sensitivity in vivo was demonstrated by injecting the proper antigen into the skin of the tested animals and in vitro by the macrophage inhibition technique. Peritoneal exudate cells from guinea pigs sensitized with whole guinea pig spermatozoa cells were inhibited in vitro by the specific antigen, epididymal extract, and autoantigen-T. Autoantigen-S was found to be a weak immunogen. However, the migration of peritoneal exudate cells from guinea pigs sensitized with large amounts of antigen-S was inhibited by whole spermatozoa in vitro. This cross-reactivity revealed the possibility that the immunogenicity of purified autoantigen-S might be connected to its molecular size. According to the immunizing dose of the antigens, testicular lesions of either the aspermatogenic or orchitis type were found in the testes of sensitized guinea pigs. Lesions in the testes of the guinea pigs were not detectable by cross-immunization with heterologous human or rat spermatozoa, although some degree of in vitro cross-reactivity was detected by skin test studies.