Action of the human lymphokine histamine releasing factor on mouse peritoneal mast cells

Histamine releasing factor (HRF)--a human lymphokine--has been shown previously to release histamine from basophils in vitro. In this paper we show that HRF acted across the species barrier and released histamine from mouse peritoneal mast cells. This response was dose-dependent. Mast cells from both sensitized and non-sensitized mice were equally susceptible to the action of HRF. We observed synergistic action of HRF with specific allergen (ovalbumin) or HRF with anti-IgE antibody in releasing histamine from mast cells. Preincubation of mast cells with calcium ion chelating agent ethylenediaminetetraacetic acid (EDTA) or disodium cromoglycate induces only a small inhibition of histamine release caused by HRF. We conclude that histamine release from mouse peritoneal mast cells can serve as an in vitro test for the assay of human HRF.     

In vitro evaluation of cell-mediated immunity in mice: experiments with soluble and cellular antigens in a spleen-thymus cell leucocyte migration test (LMT)

A modification of the macrophage or leucocyte migration inhibition assay usable in a murine model has been developed. The system operates with mixed spleen–thymus cell populations from mice sensitized to soluble protein or to transplantation antigens. In order to obtain significant inhibition it was found necessary to preincubate the sensitized cells with specific antigen for 24 hr prior to the migration assay. The macrophage inhibitory factor (MIF) has been found to determine the rate of migration inhibition in the mouse system whereas the role of humoral antibody is negligible. The murine MIF is not strain specific and operates in the absence of specific antigen.

Spleen–thymus cells from St/a mice sensitized to a first set C3H skin allograft were inhibited by approximately 30% while cells sensitized to a second set graft showed a 50% inhibition when migrating against C3H cells or MIF produced by confrontation between sensitized St/a lymphoid cells and C3H antigen.

     

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 alpha-L-fucose, but not by beta-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.     

Production of migration inhibitory factor by Listeria-immune mouse T lymphocytes, but not B lymphocytes

Antigens and B cell mitogens have been reported to induce migration inhibition factor (MIF) production by mouse B cells. Immune resistance to the intracellular bacterium, Listeria monocytogenes is thought to involve T cells, but not B cells. Since Listeria-derived components are B cell, but not T cell mitogens, it was important to determine whether these materials could stimulate secretion of the lymphokine, MIF by T cells, B cells, or both. Thus populations of whole, unfractionated spleen cells, obtained from normal and Listeria-immune BDF1 mice, were cultured with or without 100 micrograms/ml of Listeria intracellular product (LIP). The culture supernatants obtained 24 h later were assayed for MIF activity using the in vitro macrophage migration inhibition assay. Data obtained show that immune T lymphocytes release MIF in response to specific Listeria antigens, but that spleen B cells from immune and normal mice, obtained as immune, nylon-wool-adherent cells treated with anti-T-cell serum plus complement, are not capable of releasing MIF. This suggests that release of lymphokines by Listeria-immune or normal B cells stimulated with Listeria-derived antigens and mitogens is unlikely to contribute to resistance against Listeria in vivo.     

Migration inhibition of sensitized mouse spleen cells by cell-associated transplantation antigens: effect of method of antigen presentation.

The migration of specifically sensitized mouse spleen cells following exposure to allogeneic cells (antigen) in vitro was studied. The migration inhibition recorded when sensitized cells were admixed with allogeneic cells in capillary tubes (mix method) was compared to the inhibition observed when allogeneic cells were suspended in the culture chamber media (non-mix method). Specificity as well as a higher degree of migration inhibition were obtained using the mix method, suggesting that this method is superior to the nom-mix method.     

A rapid photoelectric method for reading cell migration from agarose microdroplets

A rapid photoelectric method for reading cell migration from agarose microdroplets is described. Practically instantaneous, the method eliminates drawing and planimetry and makes feasible the use of a wide range of antigen concentrations. The results obtained are similar to those obtained by planimetry, but the photoelectric method is more sensitive. Enhancement of migration as well as inhibition were significantly demonstrated by this method. Migration inhibition of immune mouse spleen cells was found to be bizonal, with 2 peaks, one at very low antigen concentrations (10^?3 μg/ml ovalbumin) and one at 10 μg/ml.     

Inhibition of tumor growth by peptide specific cytotoxic T lymphocytes in a three-dimensional collagen matrix

Tumor associated, MHC I restricted antigenic peptides have been identified in both human and mouse tumors. Cytotoxic T lymphocytes (CTL) which recognize these tumor associated antigenic peptides are potential anti-cancer effectors. The anti-tumor activity of CTL is usually measured in vitro by the ⁵¹Cr release assay and in mice by tumor growth inhibition which is the most direct assessment of anti-tumor effect. In clinical studies, an in vivo tumor growth inhibition assay is not an option and an in vitro assay which corroborates with in vivo tumor growth is needed to assess the long-term outcome of CTL activity. Here, a three-dimensional (3-D) collagen gel assay was developed to measure in vitro the inhibition of mouse mammary tumor growth by anti-tumor CTL. BALB/c mouse CTL were induced with peptide E474 SFAVATTAL which was expressed by mouse mammary tumor cells D2F2. To measure D2F2 tumor growth inhibition in vitro, a mixture of tumor cells and anti-E474 CTL in a 1 μl cell bolus was embedded in the collagen gel. Complete eradication of tumor growth was observed at E:T ratio of or greater than 1:1. rIL-2 supplementation was necessary to achieve long-term tumor growth inhibition. Even spontaneous D2 tumor explant could be grown in the collagen gel and addition of anti-E474 to this culture reduced tumor growth. This assay system provides a realistic and sensitive alternative to the in vivo tumor growth inhibition assay and allows easy adaptation to test additional therapeutic reagents.     

In vitro assessment of delayed hypersensitivity in the human. Inhibition of cell migration from agarose microdroplets

The in vitro migration inhibition responses of peripheral blood leukocytes from tuberculin skin test positive and negative donors were tested to validate and determine optimal conditions for the agarose droplet method in the human. In vitro migration inhibition was observed in skin test positive donors in the presence of 25 microgram PPD/ml of medium using both unfractionated leukocytes and a mixture of immune lymphocytes with allogeneic polymorphonuclear leukocyte indicators. The supernatants of tuberculin positive lymphocytes cultured with PPD also inhibited the migration of human polymorphonuclear leukocytes and guinea pig peritoneal exudate cells but did not alter that of murine peritoneal exudate cells. These studies establish that the agarose droplet method is an efficient approach to the in vitro assessment of cell-mediated immunity in the human and define suitable indicator cell populations for assay of human lymphokines.     

A simplified in vitro assay of deiayed hypersensitivity in diagnosis of dermatomycoses

A tissue culture technique for detection of cellular hypersensitivity in an animal model of dermatomycoses is presented. It is based on specific inhibition of migration of leukocvtes sensitized to dermatophytic antigens. The application and specificity of this new immunological technique were studied in guinea pigs infected with Trichophyton rubrum, Microsporum vanbreuseghemii, and Epidermophyton floccosum. In all the infected animals, sensitivity to trichophytin was demonstrated to be independent of the clinical disease phase. The in vivre skin-test reactions were common to those of the group of dermatophytes and did not distinguish between the different species. A statistical difference was observed in the leukocyte migration indices of sensitized cells to the homologous and the heterologous antigens of these pathogens (p < 0.05, Student's t test). It is concluded that the leukocyte migration inhibition assay provides a specific expression of cellular hypersensitivity and may be considered suitable for investigation of cellular immunity in vitro in clinical diagnosis or research. This is a simple, rapid, and inexpensive technique that requires small volumes of peripheral blood.     

Random entry of circulating lymphocyte subsets into peripheral lymph nodes and Peyer's patches: no evidence in vivo of a tissue-specific migration of B and T lymphocytes at the level of high endothelial venules.

Lymphocytes continuously migrate through the body and thus immune competent cells are constantly delivered to most tissues. They interact with high endothelial venules (HEV) via specific homing receptors and vascular addressins, and these molecules seem to be the reason for a preferential homing of B lymphocytes into Peyer's patches and of T lymphocytes into peripheral lymph nodes. When lymphocytes derived from lymph node cell suspensions were applied in the in vitro lymphocyte/endothelium binding assay, the well-known preference of mouse lymph node B lymphocytes for Peyer's patch HEV compared to peripheral lymph node HEV was confirmed in the rat (2.8 times). When in the same in vitro assay thoracic duct lymphocytes (TDL) were used this preference was far less obvious (1.4 times). However, by injecting rat TDL intravenously and by tracing them directly in HEV, B, T, CD4+ and CD8+ lymphocytes are seen to enter Peyer's patches and peripheral lymph nodes in vivo without preference. Thus, in contrast to lymphocytes from lymph node cell suspensions, no evidence was found of a tissue-specific migration of thoracic duct B, T, CD4+ and CD8+ lymphocytes at the HEV level. This finding demonstrates the importance of considering both experimental conditions and the cell source used when investigating lymphocyte traffic.     

Cell-mediated immunity in the lungs of rabbits using conjugated proteins

A study using direct macrophage migration inhibition of cells from the lower respiratory tract of rabbits immunized with conjugated proteins, incorporated into a killed BCG-oil emulsion, revealed that the reaction was largely carrier specific. The only exception was when bronchoalveolar cells (BAC) from animals immunized with dinitrophenol coupled to ovalbumin (DNP-OA) were significantly immobilized with DNP coupled to bovine gamma-globulin (BGG) in which the ratio of DNP/BGG was high [DNP-BGG(H)]. This result could not be explained by a toxic effect or by a high net negative charge on DNP-BGG(H). In addition, inhibition of BAC from DNP-OA-immunized rabbits was not observed when the ratio of DNP to BGG was reduced approximately 5-fold.     

An in vitro assay for K-papovavirus of mice

Primary cultures of mouse embryu cells were inoculated with K virus, a murine papovavirus, and were examined for cytopathic effect (CPE) or for the development of fluorescent antibody staining specific for K virus V antigen. CPE was not observed. However, numerous cells in infected cultures exhibited positive nuclear fluorescence, and the presence of papovavirus virions was demonstrated by electron microscopy. Extracts from infected cultures produced typical K virus pneumonia in newborn mice. Inoculation of cultures with serial dilutions of virus demonstrated that these cells provide a fluorescent antibody assay for K virus equal in sensitivity to animal inoculation methods. Although specific K virus fluorescence was also detected in cultures of fetal mouse endocardial cells, livers, placentas, and brains, positive cells were much less abundant in these cultures than in cultures of mouse embryo cells. The mouse embryo culture assay described in the present paper represents the first method of measuring K virus infectivity in vitro.     

Activation of pertussis toxin-sensitive CXCL12 (SDF-1) receptors mediates transendothelial migration of T lymphocytes across lymph node high endothelial cells

In this study we examined the role of chemokines in regulating T lymphocyte transmigration across the lining high endothelial cells (HEC) of high endothelial venules (HEV). The roles played by CCL21 (SLC), CCL19 (MIP-3 beta, ELC) and CXCL12 (SDF-1) were assessed using an in vitro transendothelial migration culture system, which constitutively supports high levels of lymphocyte transmigration. We determined that transmigration of T lymphocytes across HEC is inhibitable by treatment of the T lymphocytes with pertussis toxin (PTX) (80% inhibition). This was attributed to blockade of Gi-protein coupled receptors of T lymphocytes, since a non-ADP-ribosylating form of PTX had no significant effect on transendothelial migration. Inhibition of Gi-protein-coupled receptors on the endothelium had no effect on T cell transmigration. Treatment of T lymphocytes with a desensitizing concentration of CXCL12 caused a 60% reduction in T lymphocyte migration across HEC, and the CXCR4 antagonist SDF-1P2G reduced transmigration by 40%. Desensitizing concentrations of CCL21 and CCL19 had no significant effects on T lymphocyte transendothelial migration. Homologous desensitization of T lymphocytes to each chemokine was confirmed in a transwell migration assay. An approximately 3-kb mRNA corresponding to rat SDF-1 beta was constitutively expressed in HEC and cell surface CXCL12 was detectable by enzyme-linked immunosorbent assay. Together, these findings support a pivotal role for HEC-expressed CXCL12 and its receptor on T cells in the regulation of T lymphocyte homing to lymph nodes.     

Lymphoid Cell Lines as Indicators of Lymphokine Generation

We investigated the migration characteristics of the cells of four human lymphoid lines, normal peripheral blood leucocytes (PBL), and normal mouse splenocytes (MSC). Two lines (QIMR-WIL and Namalwa) were actively migratory, as were the PBL and MSC. Migration was inhibited at low temperatures and reduced by inhibition of glycolysis, oxidative phosphorylation and RNA synthesis. The migration of QIMR-WIL cells, PBL and MSC but not Namalwa was inhibited by lymphokine-containing supernatants from phytohaemagglutinin-pulsed MSC or MSC stimulated by allogenic cells. The inhibitor of migration in the supernatants had properties similar to those of human leucocyte inhibition factor but distinct from those of macrophage inhibition factor. QIMR-WIL and normal human PBL were compared as indicators in an indirect leucocyte migration assay. QIMR-WIL cells were the more sensitive responder cells and are an abundant, stable, and uniform cell population for lymphokine detection.     

A new assay for rapid measurement of MIF levels by 3H-labelled cells in liquid scintillation counting vials: Statistical implications for the measurement of migration inhibition

A new quantitative assay for migration inhibitory factor (MIF) employs ³H-labelled cultured mouse or human lymphoid cells migrating from capillary tubes. Capillaries filled with labelled cells are placed in liquid scintillation counting vials, along with the MIF- containing sample and are removed at the end of a five-hour incubation period. The residual, labelled cells which have migrated out of the tubes are solubilized and counted in a liquid scintillation counter. While cultured lymphoblast cells are routinely used in the assay, the method was checked against mouse and guinea pig peritoneal exudate cells in both the labelled cell technique and the conventional chamber assay. The assay is technically simple to perform and a useful tool for laboratory research purposes because of the short span of time needed to obtain the results. These advantages indicate a potential for automation and use of this assay in a clinical immunology laboratory. Statistical analysis of data from both assays demonstrated that the relative variation among replicates is lower in the labelled cell assay. The new assay also measured a significant difference between controls and MIF-containing samples when the migration index (MI) was greater than 80%. Criteria for significant inhibition of migration are discussed in regard to the use of analysis variance (ANOVA) and other statistical procedures, and the inadequacy of a single measure, such as the MI, is discussed.     

In vitro correlate of tranplantation immunity: spleen cell migration inhibition in the lizard, Calotes Versicolor.

Sensitization to skin allografts in the lizard, Calotes versicolor, was assessed using the in vitro capillary migration inhibition (MI) assay. In the presence of the respective donor antigen, an appreciable degree of MI of sensitized spleen cells was observed as early as 4 days after grafting. A maximum level was attained on day 7 and this response was maintained as long as one month after grafting with only slight fluctuations in the level of MI. When the clinical manifestations of graft rejection culminated on day 35, the degree of MI was still at the maximum level. MI of allograft-sensitized spleen cells is an antigen specific event, and the same level of inhibition is observed whether the specific antigen is provided in the form of intact spleen cells or spleen extract.     

Migration Inhibition Produced by Sodium Periodate Oxidation of the Macrophage Membrane, and Reversal by Sodium Borohydride

Guinea pig peritoneal exudate cells were harvested 3 to 4 days after the intraperitoneal injection of Marcol oil. The washed cells were exposed to various concentrations of sodium periodate in phosphate-buffered saline (PBS) at pH 7.4 for 10 min at +4 degrees C. The cells were then used in the in vitro migration assay, and migration was consistently inhibited at concentrations from 10(-3) to 10(-5) M. The viability of the macrophages was not affected by this treatment. Sodium borohydride (10(-3) to 10(-5) M) in PBS for 10 min at pH 7.4 reversed the periodate effect. Experiments with purified macrophages showed that sodium periodate has a direct effect on macrophage function rather than an indirect effect via the potentiation of migration inhibition factor. In support of this, the in vitro spreading of macrophages on glass substrate for 1 h has been shown to be inhibited. This spreading inhibition can also be reversed by treatment with sodium borohydride. These results provide a new approach to understanding the biological significance and role of macrophage migration inhibition.     

Immune response to intragraft antigen in draining lymph nodes after corneal transplantation is mediated by interleukin-12.

To examine the molecular basis of immunity generated to intragraft antigens and determine whether it differs between acceptor and rejector hosts, we used a novel in vitro system to assay the T cell response to a specific antigen, ovalbumin (OVA), in the graft. OVA-containing corneas were orthotopically grafted into syngeneic or allogeneic hosts. Draining cervical lymph nodes (cLN) were assayed for OVA-specific T cell proliferation and cytokine production. In addition, cytokine production was assayed in cultures of antigen-presented cells (APC) isolated from cLN cultured with OVA-specific DO11.10 T cells and OVA. OVA-specific immunity was detected only in the draining cLN of mice following allogeneic, but not syngeneic, grafting, and this immunity was evident well before any demonstrable alloresponse in the graft. In addition, cLN cells from mice that accepted their corneal allografts produced significantly less interferon-gamma (IFN-gamma) when stimulated in culture than cells harvested from cLN of rejector hosts. Moreover, APC isolated from cLN of acceptor hosts produced significantly lower levels of IL-12. These data suggest that the induction of immunity to corneal antigens in the draining cLN occurs via an interleukin-12 (IL-12) and IFN-gamma-dependent mechanism. Targeting this process may serve as an effective immunomodulatory strategy in corneal transplantation.     

Therapeutic effects of cysteine protease inhibition in allergic lung inflammation: Inhibition of allergen-specific T lymphocyte migration

OBJECTIVE AND DESIGN: We have evaluated the effects of the broad-spectrum cysteine protease inhibitor E64 on allergic lung inflammation in the mouse ovalbumin model of human asthma. We have also characterised membrane-associated cathepsin enzyme activity on a range of cell types. MATERIALS: Balb/C mice, E64 and CA074, various cell lines. TREATMENT: E64 was administered by subcutaneous minipump into ovalbumin-sensitised mice prior to intranasal ovalbumin challenge. The effect of E64 on ovalbumin-induced inflammation in vivo and ovalbumin-specific T cell proliferation in vitro and ex vivo was examined. Membrane-associated cathepsin activity on various cell types was measured. RESULTS: E64 treatment (0.36-0.48 mg/day) led to a significant reduction in eosinophil numbers and lung weights in the mouse model. Histological examination of lungs confirmed the anti-inflammatory effect. E64 greatly reduced ovalbumin-specific T cell numbers in the lymph nodes draining the lung following intranasal challenge whilst an accumulation of these T cells was found in the 'priming' lymph nodes. An analysis of various cells involved in lymphocyte priming and migration revealed that monocytes, dendritic cells and endothelial cells express high levels of membrane-associated cathepsin B activity. CONCLUSIONS: Since E64 is not cell permeable and does not inhibit antigen-induced T cell proliferation in vitro or in vivo, the data indicate that membrane-associated cysteine proteases, possibly cathepsin B, may regulate T lymphocyte migration in vivo.     

The initial characterization of a thymus factor chemotactic to bone marrow cells

Thymus supernatants were produced by cluturing minced newborn CBA/J mouse thymuses in serum-free media for 48 h. Supernatants thus obtained were chemotactic to a subset of bone marrow cells as assessed in blind well chambers, and enriched for immature lymphoid cells in the migrating cell population. The enriched population of cells was shown to be capable of homing to the thymus of an irradiated mouse in vivo in a significantly higher percentage than nonmigrated bone marrow cells. In this report, initial characterization of the factor(s) responsible for this in vitro migration is presented. Several well studied thymic factors were compared with the thymus supernatants for their ability to induce migration of bone marrow cells in vitro. Thymulin (FTS-Zn), FTS, and TP-5 (the pentapeptide fragment of thymopoietin) were used. None of these factors demonstrated chemotactic properties in the migration assay using concentration ranges in which other in vitro activities have been observed. The chemoattractive activity of the supernatant was unaltered by ultracentrifugation. The effects of temperature on the chemotactic properties of thymus supernatant were examined, and a fifty percent decrease in observed migration occurred with thymus supernatant heated to 100 degrees C for 1 h. In addition, incubation of the supernatant for 1 h at 37 degrees C with chymotrypsin, but not with trypsin, inhibited migration, presumably by inactivation of the active factor. Using Amicon microconcentrators, the supernatant was separated into several fractions based on molecular weight. Initial data suggest that the active fraction is in the less than 10,000 mw range.