Suppression of the cellular interactions of delayed hypersensitivity by corticosteroid

Methylprednisolone sodium succinate was administered to New Zealand white rabbits sensitized with BCG in an attempt to define the effect of corticosteroid upon the interactions of lymphocytes and macrophages in the afferent and efferent limbs of delayed hypersensitivity. Daily intramuscular administration of the corticosteroid, beginning simultaneous with BCG sensitization of rabbits, prevented the inhibition of macrophage migration following tuberculin challenge 14 days after sensitization. If corticosteroid administration was delayed until 10 days following sensitization with BCG, the expected inhibition of macrophage migrations followed tuberculin challenge.

Purified lymphocytes obtained from sensitized rabbits not treated with corticosteroid, when added in vitro to cells isolated from rabbits treated with methylprednisolone from the time of sensitization, re-established the expected inhibition of macrophage migration. In contrast purified lymphocytes obtained from animals treated with corticosteroid from the time of BCG sensitization did not elaborate a factor which inhibited macrophage migration.

Excluding direct effects upon blood vessels, these observations suggest that the afferent rather than the efferent stage of delayed hypersensitivity is suppressed by corticosteroid.

     

Bactericidal activity of rabbit serum containing immunoconglutinin

When lymphocytes from hypersensitive animals are incubated with antigen, biologically active substances are formed which inhibit the migration of mesenchymal cells from normal animals.

These substances were tested by intradermal injection in guinea-pigs and rabbits. The supernatants from incubation of lymphocytes with a high dose of antigen caused immediate pallor which lasted several hours. Later there was a macroscopic inflammation maximal at 24 hours. The histology was characteristic of a delayed hypersensitivity reaction.

The injection of the supernatant from hypersensitive lymphocytes incubated with a small dose of antigen caused little or no pallor and was not followed by a delayed inflammatory reaction. Injection of this supernatant together with the antigen did not potentiate or alter the reaction, in contrast to in vitro experiments where the inhibition of the migration by this supernatant was potentiated by antigen.

Besides this factor a distinct factor occurs in extracts and supernatant fluids of lymphocytes cultivated without antigen and those from control animals, which increases vascular permeability. This substance is probably identical with the lymph node permeability factor (LNPF). The possible role of these biologically active substances in the mechanism of delayed type hypersensitivity is discussed.

     

Pathogenesis of fever in delayed hypersensitivity: role of monocytes.

The present studies were designed to investigate the role of monocytes in the pathogenesis of fever in delayed hypersensitivity. Adherent rabbit blood monocytes (from both normal and sensitized donors) were separated on Ficoll-Hypaque gradients and incubated with antigen (Ag; ovalbumin) and sensitized draining-lymph-node lymphocytes (or their supernatants) from rabbits with delayed hypersensitivity, and release of endogenous pyrogen was assayed. Results indicated that monocytes are activated to produce endogenous pyrogen by Ag and suspensions of draining-lymph-node cells or by an agent (lymphokine) in the supernatants of sensitized lymphocytes preincubated with Ag. The release of lymphokine was Ag specific and was correlated with the skin test reactivity of the donor rabbits to the sensitizing Ag. No evidence was found that Ag-antibody complexes or (in the case of sensitized monocytes) cytophilic antibodies play a role in the activity of this lymphokine which appears to act selectively on monocytes rather than on granulocytes.     

Fraction of spleen cells responsible for suppression of cellular immune response to SRBC in mice treated with outer membrane proteins of Shigella.

Effect of splenocytes isolated from mice immunized with suppressive dose of OMP from Shigella on delayed hypersensitivity, induced in mice with sheep red blood cells was investigated. Only the population of T lymphocytes was found to suppress the delayed hypersensitivity, as measured by the footpad reaction. The results suggest that OMP of Shigella are able to induce in the spleens of animals active T cells which are responsible for the suppression of cellular response induced by SRBC.     

Specificity of a BCG-Induced Pulmonary Granulomatous Response in Rabbits

The specificity of Bacillus Calmette Guérin (BCG)-induced accelerated pulmonary granuloma formation has been evaluated in rabbits by cross sensitization-challenge experiments by using another granulomagenic organism, Corynebacterium granulosum. BCG-sensitized rabbits responded to challenge with homologous but not heterologous antigen, indicating that BCG-induced accelerated granuloma formation displays specificity characteristic of immunological reactions. These differences were also observed in local pulmonary delayed hypersensitivity, as determined by the migration inhibition test. The relationship between local pulmonary delayed hypersensitivity and the accelerated granulomatous response is discussed.     

Induction of a macrophage migration enhancement factor after desensitization of tuberculin-positive rabbits with purified protein derivative.

The production of a macrophage migration enhancement factor (MEF) has been achieved as a consequence of administering a desensitizing dose of purified protein derivative (PPD) to Mycobacterium bovis BCG-sensitized rabbits. The migration-enhancing effect was first demonstrated when alveolar macrophages (AM) harvested from desensitized rabbits exhibited marked migration stimulation; whereas maximum migration enhancement was observed 8 days after the administration of PPD, migration enhancement of the AM from these rabbits persisted for up to 12 days. Sera from BCG-sensitized, PPD-desensitized animals exhibited a peak of MEF activity 4 days after desensitization. Maximal MEF activity was demonstrated in culture supernatants of nonadherent spleen cells harvested 8 days after the intravenous desensitizing dose of PPD was given. Control spleen cell culture supernatants did not produce detectable MEF. The route of desensitization with PPD was critical. When PPD was administrated intratracheally, MEF activity was not induced. The intravenous administration of BCG after PPD desensitization reversed migration enhancement to strong migration inhibition. Ammonium sulfate fractionation indicated that two fractions contained MEF activity. MEF activity was retained by dialysis membranes with a 15,000-molecular-weight cutoff but passed through dialysis membranes with a 25,000-molecular-weight cutoff. The mixture of migration inhibition factor with MEF-containing supernatants resulted in the mutual cancellation of both activities. These observations suggest that MEF may be a modulator of macrophage effector responses mediated by migration inhibition factor.     

The production of lymphocyte mitogenic factor and migration–inhibition factor by antigen-stimulated lymphocytes of subjects with grass pollen allergy

Peripheral blood lymphocytes of twenty-three subjects with grass pollen allergy were cultured with grass pollen antigen for 3 days. After harvesting, the culture supernatants were added to fresh autologous lymphocytes which were maintained in culture for 6 days. Cellular uptake of [³H]thymidine was measured during the sixth day of culture, and revealed that the lymphocyte culture supernatants stimulated greater thymidine uptake than expected from the lymphocyte transformation response to corresponding amounts of antigen. The supernatant factor which mediated this effect was termed `lymphocyte mitogenic factor' by analogy with a similar response of lymphocytes in clinical and experimental delayed hypersensitivity. Lymphocyte culture supernatants were also tested for migration–inhibition factor by their ability to inhibit the migration of guinea-pig macrophages.

The majority of `allergic' supernatants contained a lymphocyte mitogenic factor active at 1/3 dilution (14/22) and 1/12 dilution (19/21) in contrast to supernatants derived from non-allergic subjects (2/16 and 1/17 respectively). The production of lymphocyte mitogenic factor corresponded to the occurrence of antigen-induced lymphocyte transformation (allergic: 18/22; non-allergic: 1/14); but only a minority of allergic supernatants contained a migration–inhibition factor (6/20). Clinical analysis revealed that migration–inhibition factor was particularly associated with the milder forms of allergy and with a past history of desensitization by depot injection of emulsified pollen antigen. In contrast, lymphocyte transformation and the production of mitogenic factor were uniformly distributed among the various categories of allergic subjects, all of whom had immediate (reaginic) hypersensitivity, but only three of whom had delayed hypersensitivity.

The demonstration of lymphocyte mitogenic factor in a clinical state dominated by immediate hypersensitivity supported the view that antigen-induced transformations are generally mediated by soluble `mitogenic' factors and that such mediators are not necessarily identical to those which can inhibit macrophage migration. It appeared that some features of cellular immunity were associated with grass pollen allergy, in which it is suggested that heterogeneity of lymphocyte-derived mediators may underlie an apparent dissociation of cellular immune functions. On this basis it is proposed that clinical expression of an atopic state may be partly governed by which features of cellular immunity are present.

     

Macrophage migration as an index of immune status.

Peritoneal macrophages from mice and guinea pigs pretreated with Freund's complete adjuvant (FCA) or any other immunostimulant when packed in a glass capillary and placed in a migration chamber migrated to a larger area than macrophages from normal untreated animals. The extent of migration could be correlated with the dose of FCA and the period of treatment. Under optimum conditions the ratio between the areas of migration of macrophages from FCA-treated animals and of macrophages from untreated animals was above 3.0. A close correlation was observed between macrophage migration and delayed type hypersensitivity (DTH) response in animals sensitized with ovalbumin or sheep red blood cells. The macrophages of immunostimulant-treated animals had relatively higher phagocytic activity. The macrophage migration index (MMI) appears to be a close correlate of macrophage activation and possibly also of the status of cell mediated immune response.     

The immunology of experimental Chagas' disease. II. Delayed hypersensitivity to Trypanosoma cruzi antigens.

Homogenates of suspensions of the trypomastigote and amastigote forms of the Ernestina strain of Trypanosoma cruzi, derived from tissue cultures, yielded two subcellular fractions which elicited strong delayed hypersensitivity reactions in rabbits. The 100,000 g times 90 minute fraction of T. cruzi homogenates gave rise to marked cell-mediated immunity. The 30,000 g times 35 minute fraction of these homogenates was also capable of eliciting a marked cell-mediated immune response. Cell-mediated immunity was assayed by experiments which established passive transfer, inhibition of blood mononuclear cell migration and blast transformation by T. cruzi--sensitized lymphocytes. Sensitized lymphocytes did not have observable effects on trypomastigotes of T. cruzi. The results of the experiments described here strongly suggest that constituents of intracytoplasmic particles of trypomastigotes and amastigotes of T. cruzi are involved in eliciting cell-mediated immunity in rabbits.     

Passive Transfer of Delayed Hypersensitivity to Blastomyces dermatitidis Between Mice

This study further characterized the delayed hypersensitivity state induced in animals by Blastomyces dermatitidis exposure. Passive transfer of delayed hypersensitivity by transfer of cells and inhibition of migration of peritoneal exudate cells were studied, using sensitized mice of two inbred strains. Donor mice were subcutaneously inoculated with viable B. dermatitidis yeast cells. After 15 days, spleen cells or serum from these animals were injected intravenously into normal recipients of the same strain. After 24 h these mice were footpad tested with killed B. dermatitidis yeast cell antigen. Mice receiving spleen cells from sensitized animals had a significant increase in footpad thickness 24 to 48 h after testing. Those receiving only serum remained negative. Migration of peritoneal exudate cells from blastomyces-sensitive donor mice was inhibited by presence of blastomycin but not by mycobacterial antigen. Neither blastomyces-sensitive nor control animals reacted to footpad or migration inhibition testing with mycobacterial antigen.     

New cyclosporin A analogue: synthesis and immunosuppressive activity.

A synthetic analogue of cyclosporine A, in which an unusual amino acid (4R)-N-methyl-4-butenyl-4-methyl-L-threonine (MeBmt) is replaced with L-threonine (Thr), was synthesized by the solid phase method. Its activity in the humoral response to sheep red blood cells in vitro and in vivo in mice was practically the same as that of cyclosporine A used as a standard, whereas the analogue studied exerted a significantly stronger effect in the delayed type hypersensitivity to sheep red blood cells in mice.     

Macrophages and resistance to tumours: I. Inhibition of delayed-type hypersensitivity reactions by tumour cells and by soluble products affecting macrophages

Mice were injected in the footpads with mixtures of an antigen (sheep red blood cells) to which they had developed delayed-type hypersensitivity (DTH) and syngeneic fibrosarcoma cells. DTH reactions were associated with depression of subsequent tumour growth compared with that in control animals. The degree of depression was proportional to the intensity of the reactions. Conversely, the presence of fibrosarcoma cells was associated with depression of the DTH reactions. Serum-free culture supernatants of all tumours tested (mouse, human and rat) depressed DTH reactions in mice, depressive activity being apparent even at dilutions greater than 1:100. Supernatants from cultures of nonmalignant cells had little or no depressive activity. Protein synthesis, but not DNA synthesis, was required for the production of the factor(s) responsible. in vitro, the active supernatants markedly inhibited macrophage migration, either spontaneously or in response to a chemotactic factor, but had much less effect on mitogen-stimulated or unstimulated lymphocyte cultures. The activity was decreased or lost after treatment with proteolytic enzymes, ribonuclease, neuraminidase or hyaluronidase. The supernatants were separated by membrane filtration into fractions of M.W. greater than, or less than 10,000. The fractions differed from each other in their effects in vivo and in vitro. Injection of concentrated supernatants from tumours together with fibrosarcoma cells led to more rapid initial tumour growth.     

Depression by slime-extract from Pseudomonas aeruginosa of delayed type hypersensitivity to sheep erythrocytes

High doses of slime-extract from Pseudomonas aeruginosa was found to suppress the delayed type hypersensitivity response to sheep erythrocytes when administered intraperitoneally 1 to 3 days before or 3 days after intravenous sensitization of mice. Moreover, the spleen cells from sensitized and slime-extract treated mice transferred the depression to normal recipients. Inhibition of DTH response was also seen when recipients were injected with slime-extract 24 h before they were infused with spleen cells from donor mice immunized with sheep erythrocytes. The development of skin DTH was quantitated by footpad increase. The data suggest that slime-extract from P. aeruginosa induces in spleens of mice immunosuppressive cells which affect the immunization with sheep red blood cells.     

A study of the effects of anti-macrophage sera

Anti-guinea-pig and anti-mouse macrophage sera were prepared by immunizing rabbits with peritoneal cells which had been purified by short culture. Such sera caused agglutination and lysis of macrophages in vitro, as well as ⁵¹Cr release, which showed some specificity for macrophages when compared with lymph node lymphocytes. Intravenous administration of anti-macrophage serum to guinea-pigs caused impairment of carbon clearance. Mice given anti-macrophage serum and immunized with sheep erythrocytes showed a normal plaque formation response. In guinea-pigs, local injection of anti-macrophage serum followed by immunization gave rise to a decreased delayed hypersensitivity response, but anti-lymphocyte serum, and to some extent anti-kidney serum, also caused a diminution.     

The effects of oral D-penicillamine treatment on experimental arthritis and the associated immune response in rabbits. II. The effects on cellular parameters.

Prolonged oral treatment (up to 410 days) of rabbits with D-penicillamine at a dose of 15 mg/kg body weight commencing either before or after immunization and the onset of arthritis, diminished and eventually abolished the delayed hypersensitivity response to intradermally administered tuberculin PPD. The 48 h cutaneous hypersensitivity response to the immunizing antigen (ovalbumin) was also significantly reduced, as was the inhibition of leucotye migration by ovalbumin. Cutaneous Arthus reactivity to ovalbumin was unaffected by D-penicillamine treatment. D-penicillamine treatment of normal rabbits was also found to increase the phagocytic index of the reticuloendothelial system as measured by carbon clearance.     

Cell-mediated immunity to Vibrio cholerae with ribonucleic acid-protein fractions of V. cholerae L-form lysates.

Different L-form lysate vaccines of Vibrio cholerae serotypes Ogawa and Inaba and their combination along with ethyl alcohol-precipitated ribonucleic acid (E-RNA) and phenol-extracted RNA (P-RNA) fractions of V. cholerae Ogawa lysates were tested for production of cell-mediated immunity. Both E-RNA and P-RNA fractions induced an increase in leukocyte migration inhibition, macrophage migration inhibition, and macrophage aggregation. They also induced delayed hypersensitivity in rabbits. More consistent results were obtained with the P-RNA fraction.     

Suppression of delayed hypersensitivity by antigen and antibody: Is a common precursor cell responsible for both delayed hypersensitivity and antibody formation?

Delayed-type hypersensitivity and antibody formation to sheep erythrocytes (SRBC) was studied in rats. Two immunologically specific suppressive effects on the primary induction of delayed hypersensitivity were found: one was short-lasting and due to a direct action of intravenous antigen; the other was mediated by anti-SRBC antibody and demonstrable both as a later-occurring and indirect effect of active immunization and as occurring consequent to passive immunization. Either of these two forms of suppression only partially prevented primary induction of delayed hypersensitivity, but used together their effects were synergistic and completely suppressed development of delayed hypersensitivity. The secondary delayed hypersensitivity response was insusceptible to antibody inhibition.

The data concerning delayed hypersensitivity to SRBC in the rat were in some ways analogous to previous findings concerning induction of haemolytic plaque-forming cells to SRBC in the same animal. The interpretation that delayed hypersensitivity and humoral antibody formation could represent alternative responses of potential antibody-forming cells to immunological induction was considered.

     

Relationships among differentiated T-cell subpopulations: II. Cytotoxicity and other functions of T cells specific for nucleated chicken erythrocytes

Relationships among T-cell mediated cytotoxicity, tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper T cells were studied in AKR mice by means of target cell destruction (⁵¹Cr-release), footpad reaction, migration inhibition test and antibody production against the trinitrophenyl group. (1) Immunization with chicken red blood cells (CRBC) in saline, Freund's incomplete (FIA) or complete adjuvant (FCA) and fixed-CRBC (FRBC) in FIA or FCA induced delayed hypersensitivity as demonstrated by footpad swelling. (2) Migration inhibition was positive in the group immunized with CRBC in saline or FCA, or FRBC in FCA, but negative in those immunized with CRBC or FRBC in FIA. This may suggest that the former has to be assigned to tuberculin type and the latter to Jones-Mote type. (3) T-cell mediated cytotoxicity by immune spleen cells was detected only in mice immunized with CRBC in saline. (4) Pre-treatment with cyclophosphamide augmented delayed footpad reaction in mice immunized with CRBC in saline, but suppressed cytotoxic activity. (5) FRBC in saline scarcely induced delayed footpad reaction and cytotoxic activity, whereas they activated helper function efficiently. Thus, four types of immunological phenomena, attributable to the functions of T cells, may depend upon distinct subpopulations of differentiated T cells which are raised by different methods of immunization.     

Inhibition of polymorphonuclear leucocyte locomotion by surface-bound antigen-antibody complexes

Delayed-type hypersensitivity responses to sheep erythrocytes were studied in inbred C57BL/6 and outbred NMRI mice fed either protein-deficient diets containing 8% and 4% casein or a normal diet with 27% casein. Following sensitization with optimal doses of antigen, the magnitude of the response was similar in mice fed the 8% protein and the normal diet. Large numbers of sheep red blood cells which suppressed the delayed hypersensitivity response in normal mice, failed to inhibit this response in animals fed the 8% casein diet. However, the titres of serum haemagglutinins were similar in mice of either dietary group immunized with high doses of antigen. Sensitized spleen cells from deficient mice kept on the 8% casein diet, had lower suppressor capacity than those from normal mice upon transfer into syngeneic hosts. Delayed-type hypersensitivity was significantly depressed in mice fed the 4% protein diet whereas the titres of serum antibodies to sheep erythrocytes were not diminished.     

Effect of metabolic inhibitors on the formation of antibody to sheep erythrocytes, on development of delayed hypersensitivity, and on the immune response to infection with Mycobacterium tuberculosis in mice.

The effect of a number of metabolic inhibitors was determined on: (i) the production of cellular immunity to infection with Mycobacterium tuberculosis in mice by vaccination with mycobacterial ribonucleic acid (RNA), (Ii) the production of cellular immunity to infection with M. tuberculosis in mice with viable H37Ra cells, (iii) the induction of antibody formation to sheep erythrocytes, and (iv) the induction of delayed hypersensitivity in mice to purified protein derivative. The pattern of inhibition produced by metabolic inhibitors on cellular immunity to infection with M. tuberculosis produced by mycobacterial RNA was entirely different from the pattern of inhibition produced by the same metabolic inhibitors on antibody formation to sheep erythrocytes. The effect of the metabolic inhibitors on the induction of delayed hypersensitivity to purified protein derivative did not correlate with the pattern of inhibition produced by the same compounds on antibody formation or on the development of immunity produced by mycobacterial RNA. Cellular immunity to infection produced in mice by viable H37Ra cells was not reduced by any of the metabolic inhibitors except actinomycin D. The possible reasons for the lack of activity of the metabolic inhibitors on the immune response to viable H37Ra cells and the lack of correlation with the pattern of inhibition found in mice vaccinated with mycobacterial RNA is discussed.