Leucocyte migration inhibition in diphtheria toxoid hypersensitivity

A donor who was highly reactive to diphtheria toxoid (DT) in delayed hypersensitivity and in lymphocyte transformation showed scant evidence of antigen-induced inhibition in the direct leucocyte migration agarose test. Other donors, weak or negative to DT in skin test and transformation, did show evidence of inhibition. Although the migration test is useful in assessing cellular reactivity to tubercular antigen, these results question its suitability for DT.     

In vitro studies of delayed-type hypersensitivity: The time course of the primary immunological reaction in individual rats determined by macrophage spreading inhibition

The aim of this work was to develop a method for the continuous follow-up of the onset and evolution of delayed-type hypersensitivity in individual rats. This hypersensitivity was determined by an in vitro macrophage spreading inhibition test in Wistar rats sensitive to tuberculin and diphtheria toxoid. Circulating antibodies to diphtheria toxoid were evaluated by passive haemagglutination. Samples of peritoneal cells and blood were taken 2 days and a few hours before sensitization, and on days 3, 4, 5, 6, 7, 11, 20, 27 and 40 after sensitization. It was found that: (1) Multiple consecutive washings of the peritoneal cavity, repeated bleedings and sensitization alone produced no change in the percentage of peritoneal macrophages spreading in medium alone. (2) Similarly, the percentage of spread macrophages of non-sensitized rats in the presence of tuberculin remained unaltered during the course of daily peritoneal washings. (3) In sensitized rats, sensitization was followed by a significant inhibition in macrophage spreading in the presence of sensitizing antigens. Thereafter, it was possible to trace individual curves reflecting the onset and evolution of delayed-type hypersensitivity to tuberculin and diphtheria toxoid. (4) The mean macrophage spreading inhibition corresponding to the delayed-type hypersensitivity was found to be maximal for both antigens on day 7 following sensitization, and decreased thereafter. (5) On the other hand, high titres of circulating antibodies to diphtheria toxoid did not appear earlier than 20 days after sensitization and continued to increase towards the end of the observation period.     

Histological and immunopathological studies of delayed hypersensitivity reaction to tuberculin in mice

At 4 to 6 weeks after intravenous infection with 2 X 10(4) CFU of dispersed Mycobacterium bovis bacilli (BCG), C3H/HeNCrIBR and C57BL/6NCrIBR mice exhibited a strong reaction to purified protein derivatives, as evaluated by the increase in footpad swelling at both 24 and 48 h after local antigenic challenge. However, histological studies of the footpad skin demonstrated a prominent perivascular infiltration with polymorphonuclear cells at 6 and 24 h after purified protein derivative challenge, whereas mononuclear cells represented the majority of infiltrating cells only at 48 h. An immunopathological study of the footpad skin showed granular deposits of immunoglobulins and complement in vascular walls and perivascular tissues at 6 and 24 h. These results demonstrate that the footpad swelling observed 24 h after the antigenic challenge is caused by an Arthus-type reaction, whereas that caused by cell-mediated immunity appears at 48 h. Hence, delayed hypersensitivity must be evaluated at 48 and not 24 h after challenge.     

Delayed hypersensitivity to tetanus toxoid in man: in vivo and in vitro studies

The frequency of positive delayed hypersensitivity skin test reactions to tetanus toxoid in 47 healthy volunteers was 80%. The frequency of immediate hypersensitivity was low (8%) and there was less discomfort than with streptokinase or PPD. There was good correlation between leukocyte migration inhibition and delayed hypersensitivity skin testing, and a quantitative relationship was demonstrated between diameter of cutaneous reaction and degree of leukocyte migration inhibition. No relationship was demonstrated between any measures of immune responsiveness and the interval from the last booster immunization. It was concluded that tetanus toxoid is a valuable antigen for assessment of delayed hypersensitivity in man.     

A comparison of the inhibition of leucocyte migration and monocyte spreading as in vitro assays for tuberculin hypersensitivity in man.

The ability of leucocyte migration inhibition and monocyte spreading inhibition test to detect tuberculin hypersensitivity was compared in the same twelve Mantoux-negative and fifteen Mantoux-positive persons. Tuberculin hypersensitivity expressed in vitro as migration or spreading inhibition, induced by 100 mug of PPD/ml, was assessed after 2 and 24, or 4 and 20 hr of incubation. A significant difference was found between negative and positive persons by migration inhibition at the early interval and by spreading inhibition at both intervals. When the two tests were compared on the basis of individual results, monocyte spreading inhibition appeared more discriminating (fewer results in the group of positive persons overlapped with those found among negative persons). Results of the monocyte spreading inhibition test correlated well with cutaneous reactions at both incubation intervals, while with migration inhibition the correlation was not so well expressed at either interval. Furthermore, a given change in skin reactivity of tuberculin-positive persons was reflected better in spreading inhibition than in migration inhibition indices. We conclude that the method of monocyte spreading inhibition compares favourably with the method of leucocyte migration inhibition, and it seems to be a suitable in vitro test for detection of tuberculin hypersensitivity in man.     

In vitro delayed hypersensitivity in normal and hyporeactive patients

The in vitro macrophage migration inhibition test can be used to evaluate human delayed hypersensitivity. Using purified protein derivative of tuberculin (PPD) as the antigen, twenty of twenty-seven in vitro tests in non-anergic persons with negative PPD skin tests were negative and fifteen of sixteen in vitro tests in persons with positive skin tests were positive. In patients with drug or disease-induced cutaneous hyporeactivity, twelve of twenty-eight tests were positive despite negative skin tests. In two anergic patients with mucocutaneous candidiasis positive in vitro tests were obtained with Candida albicans antigen as well. Measurable levels of IgG were seldom detected in the test media.

The results indicate that the macrophage migration inhibition test measures delayed hypersensitivity in man and is sometimes positive in cases of reduced skin reactivity.

     

In vivo macrophage suppression of delayed hypersensitivity in the guinea-pig.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.     

Specificity of the macrophage spreading test with reference to Leishmania antigens and correlation with delayed hypersensitivity.

The inhibition of the macrophage spreading test, claimed to be an in vitro correlate of delayed hypersensitivity, was examined in guinea-pigs immunized with L. enriettii and L. tropica soluble antigens. Cells from peritoneal washings of the guinea-pigs were tested in presence of the homologous and heterologous antigens and also without antigen. Inhibition of macrophage spreading compared to control preparations was noted only in the presence of the homologous antigen when the skin test response of the donor animal was relatively small. The degree of inhibition decreased as the skin test volume increased and when skin test volumes were large there was actual stimulation of macrophage spreading, rather than inhibition. The addition of heterologous antigen to the peritoneal cell preparation always resulted in the augmentation of macrophage spreading above control levels. The possible mechanisms of this in vitro technique and its use as a taxonomic or diagnostic tool are discussed.     

Suppression of delayed hypersensitivity to tuberculin by antigenic competition. A positive immunoregulatory mechanism sensitive to cyclophosphamide.

Effector mechanisms that produce delayed hypersensitivity reactions to tuberculin are subject to positive immunoregulation. Two different immunoregulatory mechanisms can be demonstrated. One is specific and the other, antigenic competition, is non-specific; both are sensitive to cyclophosphamide (CY). Delayed hypersensitivity to purified protein derivative PPD in guinea-pigs can be enhanced by the administration of cyclophosphamide 3 days before but not after immunization. The enhanced response seems to result from the reduced influence on effector cells of CY-sensitive suppressor cells. Passive transfer of delayed hypersensitivity to PPD is facilitated by the use of cells from CY treated animals. The response to both immunization and skin testing with ovalbumin in animals immunized with this antigen in Freud's complete adjuvant (FCA) produces a marked, non-specific reduction in the delayed hypersensitivity response to PPD. CY given 3 days before or 1 day after immunization prevents this suppression of the PPD response by antigenic competition. The data suggests that in the generation of both the specific suppressor cells for tuberculin and the non-specific suppressor cells of antigenic competition, that can influence effector cells for tuberculin, a period of rapid cell proliferation occurs that renders both mechanisms sensitive to cyclophosphamide.     

Transfer of delayed hypersensitivity in rats

Inbred August rats show strong delayed hypersensitivity following immunization with bovine γ-globulin or keyhole limpet haemocyanin in Freund's complete adjuvant as measured by increment of ear thickness at 24 hours. The reactions to PPD and keyhole limpet haemocyanin (but not bovine γ-globulin) are readily transferred by peritoneal exudate cells and to a lesser extent by lymph node cells. In contrast Blackhood rats give weaker active and passive delayed hypersensitivity reactions.     

Relationship between cutaneous delayed hypersensitivity and cell-mediated immunity in vitro responses assessed by diphtheria and tetanus toxoids.

Cutaneous delayed hypersensitivity (CDH) testing with microbial antigens in man is thought to reflect the status of cell-mediated immunity (CMI). We have evaluated diphtheria-tetanus (DT) and tetanus (T) toxoids by comparing the CDH response with in vitro parameters of CMI: lymphocyte deoxyribonucleic acid (DNA) synthesis, leukocyte inhibition factor (LIF) in 5 immunized adults, and lymphotoxin release in 2 adults. Cord blood lymphocytes were used as controls for each assay. A dose response with both toxoids was used to compare the CDH reaction with each in vitro assay, establishing the maximum response and threshold dose which gave a positive response. All subjects had a positive CDH response to both antigens (≥5 mm induration at 48 hr), positive DNA synthesis (stimulation index ≥3), LIF release (migration ≤80%), and lymphotoxin production, while cord blood lymphocytes were usually negative to all in vitro assays. No consistent quantitative relationships between CDH reactions and in vitro CMI responses were seen. Threshold antigen dose data revealed that DNA synthesis was approximately ten times as sensitive an assay as CDH, and 10⁵ times as sensitive as the LIF technique. No difference in sensitivity was noted between DT and T toxoids. Three subjects re-evaluated 16 mo after the initial study showed positive CDH and CMI responses to tetanus toxoid, although the antigen dosage required varied considerably. We conclude that the CDH response with either toxoid in the concentrations used is a good indicator of CMI in the immunized individual. Although recommended starting antigen dose is given for each assay, a dose response for each assay must be performed to adequately evaluate the CMI responsiveness to a test antigen.     

Absence of association between delayed type hypersensitivity to tuberculin and atopy in children in The Gambia

BACKGROUND: An inverse association between delayed type hypersensitivity to tuberculin and atopy has been observed in children, suggesting that exposure to mycobacteria may influence the immune response to allergens. OBJECTIVE: To investigate the relationship between tuberculin responses and atopy in children living in three different environments in The Gambia. METHODS: In this cross-sectional study a total of 507 school-aged children were recruited from rural, urban poor or urban affluent communities. They were assessed for skin responses to five common allergens and tuberculin, presence of bacille Calmette-Guérin (BCG) scar, presence of intestinal parasites, and total serum IgE. Atopy was defined as the presence of a skin prick test response > or = 3 x 3 mm to at least one allergen. RESULTS: The overall prevalence of atopy was 33% but there was a significant variation among the three study groups. The prevalence of atopy was 22% in urban poor, 36% in urban affluent, and 43% in rural children. Controlling for potential confounding factors, children in the rural community had a significantly higher odds ratio, 3.3 (95% confidence interval 1.8-6.0) of being atopic than children from the urban poor community. No association between atopy and tuberculin response or BCG scar was observed in any of the three groups. Serum IgE levels were higher among children of the urban poor group but were not associated with tuberculin response or BCG scar in any of the groups. CONCLUSION: Environmental factors have an important influence on the development of atopy in children in The Gambia but delayed type hypersensitivity to tuberculin is not a protective factor.     

Immunosuppressive factors released by transforming lymphocytes in the delayed hypersensitivity skin response to tuberculin

Experiments were carried out in BCG-sensitized cattle to see if factors produced by transforming blood lymphocytes could modify the tuberculin skin reaction or the uptake of [³H]thymidine by sensitized bovine lymphocytes. It was found that the subcutaneous injection on one side of the neck of tuberculin, tuberculin-stimulated autologous blood lymphocytes or culture supernates from tuberculin-stimulated lymphocytes depressed the response to intradermal injection of tuberculin on the other side. The suppression of the response appeared to be antigenically specific since the delayed skin reaction to the intradermal injection of brucallergen was not suppressed by the injection of the same lymphocyte culture supernates.

In vitro studies of [³H]thymidine uptake by autologous lymphocytes showed that the culture supernates from tuberculin-stimulated lymphocytes had a mitogenic activity, but at higher concentrations of supernate, this mitogenic activity was depressed. The higher concentration of supernate also non-specifically suppressed [³H]thymidine uptake by autologous lymphocytes stimulated with phytohaemagglutinin or brucallergen.

Since an immunosuppressive α-globulin fraction had been demonstrated in bovine serum by other workers, it was thought that this may have been the factor released in vitro by the transforming lymphocytes. The α-globulin fraction was therefore isolated from sera of four tuberculous cattle, obtained 10 days after tuberculin skin testing, and from four uninfected and untested control cows. This serum α-globulin fraction from both groups of cattle, suppressed [³H]thymidine uptake by homologous lymphocytes stimulated with tuberculin or PHA but levels of this factor in sera of tuberculous cattle were not raised above those in control cattle.

It was concluded that the antigenically-specific suppression of tuberculin skin-reactions was most likely mediated by antigen—antibody complexes. On the other hand results with cultured lymphocytes may have been due to a non-specific immunosuppressant released from lymphocytes and which had properties in common with a serum α-globulin fraction.