Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) or with DT-anti-toxin precipitate, both in Freund's complete adjuvant (FCA). DT-induced inhibition of peritoneal cell migration was equally strong in both groups and increased with time after immunization. Some guinea-pigs immunized with DT-antitoxin precipitate were boosted with DT or DT-antitoxin precipitate, both in FCA. This increased the antibody titre but did not affect 24-hour skin reactivity or migration inhibition. Migration inhibition did not correlate with anti-DT passive haemagglutinins, haemolysins, or cytophilic antibody on peritoneal cells, but did correlate with 24-hour skin reactivity.     

Migration inhibition of peritoneal exudate cells from sensitized and desensitized rats

The migration of peritoneal exudate cells (PEC) from rats with delayed skin reactivity to diphtheria toxoid (DT) and tuberculin (PPD) was inhibited in the presence of antigen. Three days after an intravenous injection of 2.5 mg of DT there was no inhibition with DT. Seven and 28 days later migration was inhibited but less than in sensitized controls. In 6 weeks the difference disappeared. Three days after the DT challenge, the migration inhibition with PPD was weaker than in the sensitized controls. From day 7 on, non-specific reduction of migration inhibition was not detected. The 24-hour skin reactivity to DT and PPD closely paralleled the in vitro phenomena.

The mechanism of desensitization appears to be the lack or a functional defect of the specific cells or a blocking factor unremovable by washing or produced during the migration assay.

     

Sephadex induces eosinophil migration to the rat and mouse peritoneal cavity: involvement of mast cells,LTB4, TNF-α, IL-8 and PAF

OBJECTIVE AND DESIGN: In this study we investigated the chemotactic mediators involved in the Sephadex-induced eosinophil migration into the peritoneal cavities of rats and mice, and which resident peritoneal cells release these mediators. MATERIALS AND METHODS: Sephadex suspension was injected into the peritoneal cavities of rats or mice which were pretreated, or not, with specific drugs that inhibit synthesis or production of the inflammatory mediators and eosinophil chemotactic activities were observed. To investigate the role of resident peritoneal cells as a source of these chemotactic factors, the macrophage population was enhanced or the mast cell population was depleted. The resident cells were also stimulated, in vitro, with Sephadex and the chemotactic activity of the supernatants was determined. RESULTS: Sephadex induced dose and time dependent eosinophil migration in rats and mouse, which were inhibited by dexamethasone and MK 886. BN 52021 only affected the eosinophil migration into the mouse peritoneal cavity. An increase in the macrophage population did not alter the eosinophil migration induced by Sephadex in rat or mouse. However, mast cell population depletion reduced eosinophil migration in rats, but did not alter the migration in mice. Sephadex-stimulated rat mast cells released an eosinophil chemotactic factor whose release was inhibited by dexamethasone and MK 886. Anti-TNF-alpha and anti-IL-8 Abs inhibited the chemotactic activity of the mast cell supernatant. CONCLUSION: Sephadex-induced eosinophil migration into the rat peritoneal cavity is dependent on mast cells, which release LTB4, TNF-alpha and CINC-1. Conversely, Sephadex-induced eosinophil migration into the mouse peritoneal cavity is mediated by PAF and LTB4, which are not released from resident macrophages or mast cells.     

Antibodies against Fc receptors to aggregated IgG of mouse spleen cells: selective blocking of the receptors of splenocytes and peritoneal macrophages, and abolition of the phagocytosis enhancement produced by the opsonizing IgG antibodies.

The Fc receptors to aggregated IgG of mouse spleen cells were solubilized with Nonidet P-40, absorbed by immune precipitate, and the complex obtained was used to raise anti-Fc receptor antibodies in a rabbit. The antibody and its F(ab')2 fragment inhibit binding of heat-aggregated IgG with mouse spleen cells and peritoneal macrophages. When F(ab')2 from the anti-Fc receptor antibody was absorbed exhaustively with mouse peritoneal macrophages, it still partially inhibited the reaction between aggregated IgG and mouse spleen cells devoid of the adherent cells. These data indicate that the Fc receptors to aggregated IgG which are located on the surface of splenocytes and peritoneal macrophages carry both common and private antigenic determinants. It was also demonstrated that the pretreatment of the macrophages with Fab' from anti-Fc receptor antibody abolished completely the phagocytosis enhancement produced by the IgG opsonins.     

Alterations in mouse macrophage migration: a function of assay systems, lymphocyte activation product preparation, and fractionation.

Supernatants from mouse spleen cell and peritoneal cell cultures were tested for the presence of lymphocyte activation products. Supernatants from mouse spleen cell and peritoneal cell cultures incubated with brucella antigens contained a macrophage migration inhibition factor(s) and a macrophage spreading factor(s) only if the cells were harvested from Brucella-infected mice. After dialysis and freeze-drying, the supernatants were fractionated by preparative acrylamide-gel electrophoresis. Three fractions with lymphocyte activation product activity were obtained from the fractionated supernatants of mouse spleen cells and peritoneal cells harvested from Brucella-infected mice and cultured with brucella antigen. One fraction inhibited mouse macrophage migration from capillary tubes but not from agarose wells. A second fraction not only inhibited macrophage migration from both agarose wells and capillary tubes, but also contained an activity(s) that stimulated macrophage migration through Nuclepore filters and induced macrophage spreading. A third fraction timulated macrophage migration from agarose wells and also contained an activity(s) that stimulated macrophage migration through Nuclepore filters. Fractionated supernatants of mouse spleen cells and peritoneal cells harvested from uninfected mice incubated with and without brucella antigen, as well as of cells harvested from infected mice and not incubated with antigen, did not contain detectable lymphocyte activation products.     

Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes.

Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration.     

The effect of PHA-activated MN-cell supernatants on polymorphonuclear leucocyte function.

The effect of PHA-activated mononuclear-cell (MN) supernatants on various polymorphonuclear-leucocyte (PMN) functions were assessed. Treatment of PMN with PHA-activated MN-cell supernatants resulted in greater electrophoretic mobility, indicating an increase in the negative surface charge. PMN directional motility was inhibited in the presence of active supernatants but was not affected by a pulse exposure of the PMN to these supernatants. Neither control nor active supernatants were chemotactic for PMN, but treatment of these cells with active supernatants produced an increase in their phagocytic activity, their ability to reduce NBT and in their glucose oxidation through the hexosemonophosphate shunt. Bactericidal capacity of these PMN was unaltered. Specific loss of leucocyte inhibitory factor (LIF) activity from supernatants of PHA-activated MN cells followed their absorption with PMN cells but not with human MN cells or guinea-pig peritoneal exudate cells. Furthermore, acquired inhibition of migration of the absorbing PMN was observed.     

TPA-induced cohort migration of well-differentiated human rectal adenocarcinoma cells: cells move in a RGD-dependent manner on fibronectin produced by cells, and phosphorylation of E-cadherin/catenin complex is induced independently of cell-extracellular matrix interactions

 We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement ”cohort migration”. Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell–cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin–catenin complex, including β-catenin. Cell–extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin–catenin complex induced by TPA, indicating that cell–cell interactions were adjusted to suit cell migration, irrespective of the condition of cell–ECM adhesion, during TPA-induced cohort migration. Received: 31 December 1997 / Accepted: 2 April 1998     

Immunomodulation by corynebacterium parvum in two strains of guinea-pigs and the effect of cyclophosphamide.

An early immunosuppressive phase in the overall stimulatory effect of Corynebacterium parvum on the immune response of two strains of guinea-pigs to ovalbumin (Oa) is described. Boosters given soon after treatment with C. parvum elicited lower secondary responses than those given later on, the peak secondary antibody titre increasing with the interval between primary immunization and the booster injection. Transfer of blood leukocytes (lymphocytes) of immunized donors to virgin syngeneic recipients showed the cellular nature of this effect. Cells transferred from donors boosted on day 40 after primary immunization showed a mean adoptive response that was 3.5 times less than that of a similar number of cells from donors boosted on day 90. The increase in the immunocompetence and/or memory of the transferred cells was related to the moment of injection of the booster antigen (Ag) and not to the interval between priming and transfer, since cells transferred during the primary response failed to show a parallel increase. The early, lesser enhancement of the secondary response by C. parvum would thus appear to be due to a limitation of the number and/or immunocompetence of memory cells developing after the injection of Ag. A marked strain difference was observed in the response of strain 2 and Hartley guinea-pigs to immunization with C. parvum and Oa, delayed-type hypersensitivity (DTH) being relatively inhibited, and antibody (Ab) production relatively increased in the latter, the reverse being true of strain 2 guinea-pigs. The existence of a suppressor or regulatory mechanism sensitive to cyclophosphamide (Cy) during the development of DTH after C. parvum treatment was established. Cy pretreatment, 250-300 mg/kg i.p., of both strains led to the development of a larger number of positive skin tests in animals given C. parvum i.v. and Oa i.d. but not when C. parvum was injected i.d. mixed with Oa. The inhibition of the migration, in the presence of Ag, of peritoneal exudate cells of Cy-pretreated guinea-pigs was also accelerated and enhanced during the first week of the primary response after i.v. C. parvum.     

A simple preparation method for mouse eosinophils and their responses to anti-allergic drugs

Objective and design: A simple method for preparing mouse eosinophils was established, and the characteristics of the eosinophils were assessed including their responses to anti-allergic drugs. Materials or subjects: Mouse eosinophils were prepared from peritoneal exudate cells of BALB/c mice primed and boosted with antigen ovalbumin (OVA). Methods: Surface phenotypes, migration activities and leukotriene C₄ (LTC₄) production abilities of these eosinophils were examined. In addition, the effects of anti-allergic drugs, oxatomide and tranilast, on generation of LTC₄ from mouse eosinophils were examined. Results: Eosinophils of mice boosted with OVA were phenotypically and functionally identical with human eosinophils. Around 1 × 10⁷ eosinophils were obtained from mouse peritoneal exudate. It was found that these mouse eosinophils enabled to migrate in response to eotaxin as well as platelet-activating factor (PAF), and generated LTC₄ by IL-5 stimulation. Moreover, it was revealed that clinically used anti-allergic drugs inhibited LTC₄-production dose-dependently. Conclusions: The present study provides a convenient method to obtain fully functional mouse eosinophils that are useful for drug screening and for evaluating implications of eosinophils in allergic responses. Received 20 April 2006; returned for revision 6 July 2006; returned for final revision 29 August 2006; accepted by M. Katori 29 September 2006     

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.     

Cytoplasmic Antigens from Nocardia Eliciting a Specific Delayed Hypersensitivity

Purified cytoplasmic extracts from Nocardia asteroides and N. brasiliensis elicit delayed hypersensitivity in Nocardia-sensitized guinea pigs. The differences in skin reactivity clearly show that it is possible to distinguish between different Nocardia species. When peritoneal exudate cells from the latter animals were obtained and treated with the purified cytoplasmic extracts, their migration was inhibited to a significant degree only by means of the homologous antigen. A mild delayed reactivity was observed when the cytoplasmic antigens were used as skin test materials on animals sensitized with BCG. On the other hand, no inhibition of migration was observed when peritoneal exudate cells from BCG-sensitized guinea pigs were exposed to the cytoplasmic antigens.     

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.     

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.     

Species-restricted effects of human and mouse lymphokines on macrophages

Lymphokines produced by antigenic or mitogenic stimulation of human, guinea pig and mouse lymphocytes were tested for their effects on monocytes or macrophages of the same and heterologous species, to determine whether there is any species restriction in their reactivity. Supernatants from lymphocyte cultures were tested for migration inhibitory activity in an indirect agarose microdroplet assay and for their ability to augument cytolytic activity of macrophages or monocytes in a [3H]thymidine-release assay. Supernatants of human peripheral blood mononuclear cells, stimulated with phytohemagglutinin or purified protein derivative of tuberculin (PPD) were able to strongly inhibit the migration of human monocytes and guinea pig peritoneal exudate cells (PEC), but had no detectable effect on mouse PEC. The human supernatants could also significantly augment the cytolytic activity of human monocytes, but had no effect on cytotoxicity by mouse peritoneal macrophages. Conversely, supernatants of PPD-stimulated spleen cells from mice immune to bacillus Calmette-Guérin (BCG) strongly inhibited the migration of, and significantly augmented cytolysis by, mouse PEC, but had no detectable effects on human monocytes. Moreover, supernatants of concanavalin A-stimulated lymph node cells from guinea pigs inhibited the migration of guinea pig PEC and human monocytes, but had no effects on mouse PEC. The migration inhibitory effects of the human and mouse supernatants did not appear to be mediated by interferon (IF), since partially purified type-1 IF had no detectable effect. In addition, supernatants of human lymphocytes stimulated by Corynebacterium parvum strain 5888, that induced little or no IF, were able to inhibit the migration of, and augmented cytolysis by, human monocytes. These C.parvum supernatants also showed migration inhibitory activity on mouse PEC but did not induce cytolytic activity in these cells.     

Studies on the inhibition of macrophage migration induced by soluble antigen-antibody complexes.

Mixtures of serum of Freund's complete adjuvant (FCA) immunized guinea-pigs and tuberculin PPD consistently inhibited the in vitro migration of peritoneal exudate cells (PEC) of normal guinea-pigs. It is shown that this inhibitory effect is due to a soluble complex between an IgG2 antibody and PPD. By separation of PPD on Sephadex G-200 two peaks were obtained, corresponding respectively to molecular weights of at least 800,000 and 25,000, separated by a plateau. Material derived from both peaks and from the plateau was able to form inhibitory complexes with anti-PPD IgG2. When a mixture of small molecular weight PPD and anti-PPD IgG2 was fractionated on Sephadex G-200, the inhibitory activity was recovered in the void region only. The detection of both IgG2 and PPD in the latter was taken as evidence for the presence of a high molecular weight antigen-antibody complex. When the mechanism of complex-induced inhibition of migration was examined it was found that: (1) complexes act directly on macrophages present in the peritoneal exudate; (2) removal of the Fc fragment of IgG2 by pepsin abolishes its ability to form migration inhibitory complexes; (3) passive sensitization of macrophages with anti-PPD IgG2, followed by exposure to PPD does not result in inhibition of migration; (4) in order to obtain migration inhibition, the complexes must be present during the entire migration period. A 2-hr pulse with complexes does not induce permanent inhibition; (5) the migration inhibitory activity of antigen--antibody complexes can be abolished by certain concentrations of puromycin and aminophylline.     

Cell-Mediated Immunity in Guinea Pig and Man to a Salmonella typhi Glycoprotein Complex Assessed by Migration Inhibition Methods

A Salmonella typhi glycoprotein preparation in concentrations of 5 to 10 _μg/ml strongly inhibited the migration of peritoneal exudate cells (PEC), obtained from guinea pigs immunized with S. typhi vaccine in Freund's complete adjuvant (FCA) PEC from animals injected with saline in FCA were also inhibited but to a lesser extent, whereas cells from guinea pigs that had received only S. typhi vaccine or normal guinea pigs showed variable and weaker inhibition. PEC from all guinea pigs receiving FCA were strongly inhibited by 5 to 15 _μg PPD/ml. These PPD concentrations had no inhibitory effect on exudate cells from guinea pigs receiving S. typhi vaccine only. The S.typhi glycoprotein concentration (5 and 10 _μg/ml) primarily used in the migration inhibition studies had negligible inhibitory effect on the phytohemagglutinin (PHA)-induced DNA, RNA, and protein syntheses in guinea pig lymph node cells. In the human system only blood leukocytes of donors who had received their third or fourth booster of heat-killed S. typhi vaccine within the latest 2 years were inhibited in their in vitro migration by the S. typhi glycoprotein complex.     

Role of resident macrophages in canatoxin-induced in vivo neutrophil migration

Canatoxin (Cntx), a toxic protein purified fromCanavalia ensiformis seeds, was shown to have lipoxygenase-mediated effects either in vivo or in vitro. Data here show that Cntx induced a dose-dependent migration of neutrophils and mononuclear cells when injected into rat peritoneal cavities. Furthermore, Cntx was able to induce neutrophil migration into pleural cavities and into air pouches. These effects were inhibited by dexamethasone but not by inhibitors of arachidonic acid metabolism (indomethacin, NDGA, and BW-755c) or by a PAF antagonist (BN 52021). In the peritoneal cavity Cntx caused an increase in vascular permeability inhibited by dexamethasone and BW-755c. Neutrophil migration induced by this toxin was dependent on the number of resident macrophages, since the migratory effect was enhanced by increasing the peritoneal macrophage population with thioglycollate pretreatmen; and was diminished when this population was reduced by peritoneal wash. It was also observed that Cntx induced release of a chemotactic factor from macrophage monolayers in vitro. Dexamethasone blocked this release but did not affect in vivo neutrophil recruitment induced by that factor. These data suggest that Cntx-induced neutrophil migration may be mediated by the same macrophage-derived neutrophil chemotactic factor released by other stimuli such as LPS, IL-1, and INF-gamma.     

Blockade of alpha6 integrin inhibits IL-1beta- but not TNF-alpha-induced neutrophil transmigration in vivo

In vitro and in vivo evidence supports a functional role for the integrin alpha6beta1 in neutrophil migration through the perivascular basement membrane, a response that in vivo appears to be associated with platelet/endothelial cell adhesion molecule-1 (PECAM-1)-mediated up-regulation of alpha6beta1 on the cell surface of transmigrating leukocytes. As the involvement of PECAM-1 in leukocyte migration is cytokine-specific, the aim of the present study was to investigate whether alpha6beta1 exhibited a similar profile of stimulus specificity in this context. The cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) were used to elicit neutrophil migration in two murine models of inflammation, migration through cremasteric venules, as observed by intravital microscopy, and migration into the peritoneal cavity. The role of alpha6beta1 was investigated using an alpha6 integrin-blocking monoclonal antibody GoH3. In both models, GoH3 significantly inhibited neutrophil transmigration induced by IL-1beta but not TNF-alpha. This cytokine-specific role of alpha6 integrin was associated with enhanced cell-surface expression of alpha6beta1 on transmigrated neutrophils (as compared with blood cells) in response to IL-1beta but not TNF-alpha. Using lipopolysaccharide as an inflammatory stimulus in the cremaster muscle model, the study also provides evidence for the involvement of alpha6 integrin in leukocyte transmigration as mediated by endogenously generated IL-1beta. Collectively, the findings demonstrate that alpha6beta1 blockade inhibits neutrophil migration induced by exogenous and endogenous IL-1beta but not TNF-alpha, observations that are associated with increased expression of the integrin on transmigrated leukocytes.