Relation between Delayed Skin Reactivity and Macrophage Migration Inhibition or Lymphocyte Transformation in Tuberculin-Type Hypersensitivity and Jones-Mote Hypersensitivity

Precise time-course studies on delayed skin reaction, lymphocyte transformation and macrophage migration inhibition were carried out from day 3 to 270 and from day 3 to 120, respectively, in guinea pigs immunized with bovine gamma-globulin (BGG) in complete Freund's adjuvant (CFA) and those immunized with BGG in incomplete Freund's adjuvant (IFA). a) Delayed skin reactions could be elicited for a long period of time after immunization with BGG in CFA in the presence of prominent antibody production and were accompanied by induration. b) Delayed reactions could be elicited transiently after immunization with BGG in IFA and were not accompanied by induration. c) At the peak of hypersensitivity, infiltrating cells at the reaction sites were composed largely of mononuclear cells and basophils, respectively, in the animals immunized with BGG in CFA and those immunized with BGG in IFA. d) Uptake of ³H-thymidine by lymphocytes was increased remarkably in the presence of BGG when cells were obtained at early stages after immunization by both methods. e) Macrophage migration inhibition was strongly positive in animals immunized with BGG in CFA but weakly positive in those immunized with BGG in IFA. Increased lymphocyte transformation preceded the appearance of a positive migration inhibition. f) After immunization with BGG in CFA, Jones-Mote hypersensitivity appeared to precede the development of tuberculin-type hypersensitivity.     

The formation of delayed hypersensitivity to mycobacterial antigens

In experiments on guinea pigs and BALB/c mice delayed hypersensitivity to mycobacterial antigens was induced by the sensitization of the animals with live BCG or killed Mycobacterium bovis or M. avium in incomplete Freund's adjuvant. In the study of the dynamics of the development of skin reactivity to tuberculin some advantages of the sensitization of guinea pigs with live mycobacteria were revealed, while after the revaccination of the animals no development of secondary cell-mediated immune response was observed. The immunization of guinea pigs with atypical mycobacteria prior to their sensitization with BCG was found to lead to the development of higher skin reactivity to allergen prepared from atypical mycobacteria than skin reactivity to tuberculin.     

Immune responses to myelin basic protein in mycobacterial-induced suppression of experimental allergic encephalomyelitis

Pretreatment of guinea pigs with complete Freund's adjuvant (CFA) 21 days prior to injection with myelin basic protein (BP) in CFA resulted in marked attenuation of clinical and pathologic manifestations of experimental allergic encephalomyelitis (EAE). Delayed hypersensitivity skin tests to homologous BP were likewise depressed in protected animals. The protected guinea pigs also demonstrated diminished in vitro reactivity to BP as assessed by BP-induced proliferative response of peritoneal exudate cells (PEC) and BP-induced inhibition of macrophage migration. Broad-based suppression of immunologic reactivity did not occur in these animals, as manifested by larger skin tests to PPD, a greater proliferative response to old tuberculin (OT), by PEC and peripheral blood lymphocytes (PBL), as well as marked PPD-induced inhibition of macrophage migration. Diminution of the degree of cell-mediated reactivity to BP may be one of the mechanisms by which prior treatment with CFA suppresses subsequent development of EAE.     

Leukocyte-migration inhibition in guinea pigs. I. Correlation with skin test reactivity and macrophage-migration inhibition.

Fifteen guinea pigs, immunized with one of three soluble antigens, repeatedly demonstrated inhibition of leukocyte migration and positive skin tests to the immunizing antigens. An additional five animals immunized with ovalbumin demonstrated inhibition of macrophage migration as well as direct and indirect inhibiton of leukocyte migration. Only one of fifteen animals demonstrated inhibition of leukocyte migration and none had a positive skin test with an antigen to which it had not been sensitized, indicating that the assay is antigen specific.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.     

Macrophage migration-inhibition in desensitized guinea pigs

The migration of peritoneal exudate cells obtained from guinea pigs with delayed skin reactivity to egg albumin (EA) and diphtheria toxoid (DT) was inhibited in the presence of antigen. A dose of 2 mg of EA given intravenously 8 days after sensitization specifically abolished the migration inhibition tested 5 weeks later. When the challenge was given into a foot pad 6 weeks after sensitization the migration inhibition was partially suppressed 3 to 28 days later.Repeated skin testing did not affect the migration results of the challenged or unchallenged guinea pigs.The demonstration in vitro of desensitization argues that the mechanism is either a reduced number or a reduced responsiveness of the specific effector cells of delayed hypersensitivity, or an inhibitory effect of cells stimulated by the specific antigen. If a humoral inhibitory factor is involved, it is either tightly bound by the cells or produced during the migration assay.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

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.     

Suppression of the intradermal hypersensitivity reaction of the delayed type by a serum fraction from patients containing leukocyte migration inhibition factor]

The blood serum of patients with active tuberculosis of the lungs and chronic pneumonia inhibited migration of donor's leukocytes and macrophages of the peritonal exudate of guinea pigs when compared with migration of similar cells in the medium with the serum of cattle or donors. After chromatography these sera were fractionated on the columns with Sephadex G-100. Fractions containing the leukocyte migration inhibition factor (LMIF) suppressed, up to complete abolition, the intradermal reaction to tuberculin in man and guinea pigs sensitized with BCG. The LMIF is supposed to act in the regulation of delayed hypersensitivity reaction.     

Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.     

Coccidioidin sensitivity in control,immunized and infected guinea pigs

Summary Delayed hypersensitivity to potent coccidioidin developed in guinea pigs immunized with deadC. immitis arthrospores and guinea pigs infected with an aerosol ofC. immitis arthrospores at weeks 1 and 2. Delayed hypersensitivity in control animals sensitized by repeated intradermal testing developed at weeks 3 and 4. The delayed hypersensitivity responses were characterized grossly by indurations larger than 25 mm² and could be seen at 6 and 24 hours after testing. Retesting reduced the size of the 24 hour indurations when compared to virginal reactions. The retest delayed reactions in infected animals had indurations at 24 and 48 hours that were larger than those in the other groups.In those animals that were skin test positive but not challenged no tube precipitins, agar gel precipitins, CF antibodies, anaphylaxis or immediate hypersensitivity were detected. Because of the inability to detect precipitins the early phase of the hypersensitivity seen at 6 hours was not considered an Arthus reaction.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

Non-specific antimicrobial cellular resistance in sensitized guinea pigs.

Specific delayed-type hypersensitivity was induced in guinea pigs with bovine albumin + complete Freund adjuvant, bovine gamma globulin + complete Freund adjuvant and BCG vaccine. The animals were subsequently tested for nonspecific antimicrobial resistance. Sensitized and control groups were challenged intraperitoneally with Listeria monocytogenes 2 hr after reinjection with the sensitizing antigen. The listeria content of the spleens was determined 1 or 5 days after the infection. The number of organisms recovered from the spleen one day after infection was significantly less in guinea pigs sensitized with bovine gamma globulin and BCG than in the control group; after 5 days no such difference was recorded. There was no difference between the bovine albumin sensitized and the control group 1 day after infection, while on the 5th postinfection day listeria counts were higher in the sensitized than in the control animals.     

Guinea pig macrophage agglutination factor is antigenically distinct from migration inhibition factor and immunoglobulin.

Lymph node cells from guinea pigs with specific delayed hypersensitivity release macrophage agglutination (MAggF) and migration inhibition factors (MIF) upon exposure to antigen or concanavalin A in serum-free medium. MAggF in culture supernatants was absorbed neither by immunoabsorbents made with a rabbit anti-guinea pig lymphokine serum that removed MIF, nor by immunoabsorbents made with rabbit anti-guinea pig Ig. These results suggest that MAggF is antigenically distinct from MIF and Ig.     

RNA extracts of lymphoid cells sensitized to DNP-oligolysines convert nonresponder lymphoid cells to responder cells which release migration inhibition factor

Strain 13 nonresponder peritoneal exudate cells were converted to responder status to α or ?,DNP-oligolysines after incubation of the cells with RNA extracts prepared from responder guinea pigs skin test sensitive to these synthetic antigens. The conversion of nonresponder strain 13 cells was assessed by the direct cell migration inhibition correlate of delayed hypersensitivity. Nonresponder cells were not converted by RNA extracts prepared from unimmunized responder guinea pigs or from non-responder strain 13 guinea pigs previously injected with DNP-oligolysines. Thus, it seems possible to correct immunological unresponsiveness in vitro in spite of a specific genetically determined deficiency of the immune response related to the Ir gene.     

Heterologous antigenic stimulation in induction of delayed hypersensitivity.

An influence of a delayed hypersensitive reaction to a primary antigen on the induction of delayed hypersensitivity to a second unrelated antigen was observed in guinea pigs immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABAT), and injected intradermally 3 weeks later with a mixture of ABAT and secondary antigen. Animals so treated developed delayed hypersensitivity to sheep red blood cells (SRBC) or Type II pneumococcal polysaccharide as secondary antigens, as measured by skin test reactivity and inhibition of macrophage migration, whereas ABAT unsensitized control groups did not. However, attempts to induce delayed reactivity to proteins as secondary antigens were unsuccessful. The injection of secondary antigen into a mineral oil-induced inflammatory lesion did not induce delayed hypersensitivity, suggesting that specific reactivity to ABAT is a prerequisite for heterologous induction. Possible mechanisms for the observed phenomenon, including a role for macrophages, are discussed.     

Delayed Hypersensitivity in Relation to Suppression of Growth of Listeria monocytogenes by Guinea Pig Macrophages

Prototypes of delayed hypersensitivity (tuberculin allergy, graft rejection immunity, and contact dermatitis) were established in guinea pigs. The macrophages from peritoneal exudates of such animals were examined for their capacities to suppress the growth of Listeria monocytogenes in vitro. Only the macrophages from animals sensitized to BCG clearly exhibited this property.     

Lymphokine production in the cell-mediated immune response (author's transl)

In response to tuberculin, lymphocytes derived from BCG-sensitized guinea pigs elaborated soluble materials capable of inducing some reactions in vitro which correlated with the cell-mediated immune response in vivo. The lymphokine activity of these soluble materials was demonstrated by the following biological effects: macrophage migration inhibition, macrophage aggregation, cytotoxicity to HeLa cells and skin reaction.     

The effect of hydrocortisone and cyclosporin A on bacillus Calmette-Guérin epitheloid cell granulomas

The injection of bacillus Calmette-Guérin (BCG) intradermally into the ear of guinea pigs leads to the formation in the draining lymph node of granulomas containing epithelioid cells with rough endoplasmic reticulum (RER) and an absence of phagocytosed material. BCG granulomas in hydrocortisone- or cyclosporin A (CsA)-treated animals contain mononuclear phagocytes with no RER. In CsA-treated animals, these cells contain fragments of phagocytosed organisms. CsA was given at two doses, 25 mg/kg orally and 50 mg/kg ip. The higher dose completely suppressed the delayed hypersensitivity (DH) response to purified protein derivative (PPD) but the lower dose did not affect the level of the DH response, but had a profound effect on epithelioid cell formation. The role of lymphokines in the maturation of the monocyte/macrophage to epithelioid cells is discussed.