Cell-mediated immunity in ferrets delayed dermal hypersensitivity,lymphocyte transformation,and macrophage migration inhibitory factor production

Cell-mediated immune reactions — delayed dermal hypersensitivity, lymphocyte transformation, and macrophage migration inhibitory factor production — were investigated in the ferret. Ferrets immunized with streptokinase in complete Freund's adjuvant had skin test responses characterized by induration with little erythema; skin test biopsies showed mononuclear cell infiltration. In vitro transformation of peripheral blood or splenic lymphocytes was elicited by phytohemagglutinin, concanavalin A, pokeweed mitogen, and streptokinase; macrophage migration inhibitory factor was produced by spleen cells from immunized ferrets. These studies confirm the usefulness of certain tests of cell-mediated immunity in ferrets.     

Lack of effect of azathioprine on phytohemagglutinin-induced lymphocyte transformation and established delayed cutaneous hypersensitivity.

The effect of experimental long-term azathioprine administration on pre-existing in vitro and in vivo manifestations of delayed hypersensitivity was examined. Adult ewes were administered 3 mg/kg/day azathioprine for periods of up to 28 days. Neither phytohemagglutinin (PHA)-induced nor tuberculin-induced lymphocyte transformation was affected. Similarly, pre-existing skin test positivity to tuberculin showed no observable change following drug therapy. These results agree with the majority of those involving clinical situations and indicate that immunosuppression with azathiprine cannot be effectively monitored by either the lymphocyte transformation response to PHA or by changes in the in vivo or in vitro manifestations of pre-existing delayed cutaneous hypersensitivity.     

Comparison of lymphocyte transformation and macrophage migration inhibition tests in the detection of beryllium hypersensitivity.

In seven patients with chronic beryllium disease (Be) the Be lymphocyte transformation test was positive in 100%, independent of steroid therapy, and was reproducible. The Be macrophage migration inhibition test was only positive in four of seven patients (57%) not on steroids, and was not reproducible. In 72 potentially exposed healthy beryllium workers the lymphocyte transformation test was negative in all subjects. The macrophage test was positive in four of 78 and again the results were not reproducible. The workers with positive results showed no differences in age, type or duration of employment from those with negative results and showed no evidence of disease. In addition, the macrophage test was positive in two of 45 non-exposed control subjects. We also confirmed the above advantages of the lymphocyte transformation technique by using tuberculin antigen (PPD). The PPD lymphocyte transformation test gave positive results in approximately 60% of healthy beryllium workers, but the PPD macrophage test was only positive in 7%. We conclude that the Be lymphocyte transformation test is the most sensitive and reproducible and advocate its use in the diagnosis of disease and in monitoring the health of potentially exposed workers.     

In vitro effect of interferon alpha-2b on T lymphocyte transformation and leukocyte migration inhibition in patients with chronic brucellosis

The in vitro effect of IFN-a on lymphocyte transformation and specific immune response against Brucella antigens was studied in 33 patients with chronic brucellosis and 10 normal controls. The following immunologic in vitro tests were applied: PHA activated lymphocyte transformation test using Bromodeoxyuridine and a monoclonal antibody in the presence and absence of 50 and 100 IU IFN Alpha-2b and leukocyte migration inhibition test against Brucella antigens in the presence and absence of 100 and 500 IU of IFN Alpha-2b. Patients were further divided to 2 subgroups according to a positive or negative migration inhibition test. Our results showed that T lymphocyte transformation was similar in patients and controls and that the addition of 50 IU IFN resulted in a significant increase of transforming cells whereas in the concentration of 100 IU IFN only anergic patients and controls responded positively. IFN also resulted in a significant leukocyte migration inhibition only in anergic patients and controls. These findings suggest that the chronic infection is not due to a generalized cellular immunodeficiency state and that IFN Alpha-2b might be a promising therapeutic approach in anergic patients.     

Evaluation of the leucocyte migration test as a measure of delayed hypersensitivity in man: suppression of migration inhibition by puromycin

The response of peripheral blood leucocytes to PPD was studied in vitro by means of the leucocyte migration test, using Mantoux-positive and -negative subjects. The migration of leucocytes from 83% of the positive subjects was significantly inhibited when a concentration of 100 μg PPD/ml was used in the chambers. Leucocytes from the majority of these subjects were also inhibited at a lower concentration of 50 μg PPD/ml and one strongly positive subject showed inhibition at all concentrations tested, down to 1 μg PPD/ml. Stimulation of migration was not observed with leucocytes from any of the positive subjects even when the lowest concentration of PPD was used. Inhibition of migration with PPD was prevented by the addition of puromycin, suggesting that the response is dependent on protein synthesis and may be mediated by a soluble factor produced by sensitized lymphocytes.     

Human transfer factor in vitro. I. Augmentation of lymphocyte transformation to tuberculin PPD

There is recent interest in whether dialysable transfer factor can specifically increase immune responses when added to lymphoid cells in vitro. This report demonstrates that transfer factor preparations (human leucocyte dialysates) `augment' rather than `transfer' lymphocyte transformation responses (DNA synthesis) to tuberculin PPD in vitro and that the magnitude of this augmentation is proportional to the level of DNA synthesis induced by PPD in the absence of added transfer factor. Experiments showed that transfer factor preparations from Mantoux-positive or Mantoux-negative `donors' were equally effective in augmenting `recipient' lymphocyte transformation responses to PPD. Thus the extent of augmentation was related, not to the tuberculin sensitivity of the transfer factor donors, but to that of the recipients. In the absence of tuberculin PPD, transfer factor preparations sometimes stimulated lymphocyte DNA synthesis, but the extent of this was small and inconstant. The results therefore provide evidence for an antigen-dependent, but not an antigen-specific effect of transfer factor in increasing lymphocyte DNA synthesis in vitro. It is suggested that leucocyte dialysates contain an augmenting factor which may facilitate the response of antigen-sensitive cells to PPD in vitro, or may facilitate the recruitment into DNA synthesis of cell populations responding to mitogenic lymphokine produced during lymphocyte transformation.     

Cell mediated immunity to herpes simplex in man. IV. A correlation of lymphocyte stimulation and inhibition of leukocyte migration.

The cell mediated immune response to herpes simplex virus was assessed by a modification of the leukocyte migration inhibition test, developed to increase its sensitivity and correlated with the in vitro stimulation of lymphocytes. The introduction of a preincubation step allowed us to demonstrate significant inhibition of leukocyte migration in 15 out of 24 subjects susceptible to recurrent herpes labialis. This was not present in any of the 10 subjects without such infections. However lymphocytes from all 24 subjects demonstrated stimulation in response to herpes virus antigen. The degrees of lymphocyte stimulation and migration inhibition in the susceptible subjects were well correlated and do not support the suggestion that there is a focal defect in this aspect of the cell mediated immune response to herpes simplex virus in those patients.     

Cell-mediated immunity in periodontal disease; cytotoxicity, migration inhibition and lymphocyte transformation studies

Cultures of peripheral blood lymphocytes from patients with gingivitis or mild periodontitis were stimulated with ultrasonicates of Veillonella alcalescens to yield an increase of ¹⁴C-thymidine incorporation and of cytotoxicity against ⁵¹Cr-labelled chicken red cells. The cell-free supernatant from the antigen-stimulated cultures inhibited migration of guinea-pig peritoneal macrophages. In patients with severe periodontitis lymphocytes showed only weak transformation but strong cytotoxic and migration-inhibitory activity. These data suggest that cell-mediated immunity against an oral Gram-negative micro-organism plays some part in the pathogenesis of periodontal disease. The advanced stage of disease, however, revealed a dissociation between lymphocyte transformation and cytotoxicity or macrophage inhibition.     

Cellular hypersensitivity to tuberculin in BCG-revaccinated persons studied by skin reactivity, leucocyte migration inhibition and lymphocyte proliferation.

Some persons seem to lose long-standing peripheral hypersensitivity to tuberculin earlier than others. This was seen in a group of five healthy student nurses who had been BCG-vaccinated as children and were found, in routine skin testing, to be negative to 100 TU of tuberculin purified protein derivative (PPD). They were revaccinated, which resulted in conversion to 10 TU skin test positivity. In agreement with this, their buffy coat cells achieved reactivity to 100 microgram/ml of PPD in a leucocyte migration inhibitory factor (LIF) assay. However, the LIF response, being maximal at 4 weeks, faded away earlier than skin reactivity. Peripheral blood lymphocyte proliferation was studied with several PPD concentrations, 10 microgram/ml always inducing the maximum 3H-thymidine uptake. This was still high at 6 months after vaccination, when the skin reactions tended to be smaller than earlier. The reason why the various parameters of cellular hypersensitivity followed different courses is not known, but it may involve different subpopulations of lymphocytes, activity of suppressor cells or influence by serum factors such as mycobacterial antigen-antibody complexes.     

Parameters of T cell mediated immunity to commensal micro-organisms in patients with chronic purulent rhinosinusitis: a comparison between delayed type hypersensitivity skin test, lymphocyte transformation test and macrophage migration inhibition factor assay.

In 75 patients with unexplained chronic purulent rhinosinusitis T cell mediated immunity to three micro-organisms frequently colonizing the human upper respiratory tract, viz. Haemophilus influenzae, streptococci and Candida albicans, was assessed. Delayed type hypersensitivity (DTH) skin test reactivity was measured in vivo, whereas the blastogenic responsiveness (lymphocyte transformation test; LTT) and lymphokine production (e.g. migration inhibition factor; MIF) of the lymphocytes upon antigen stimulation were measured in vitro. MIF was assayed with a recently developed test system using the human monocytoid cell-line U937 as indicator cells in agarose microdroplets. Two-thirds of the 75 patients tested showed a defective DTH response to one or more of the microbial antigens; this contrasted to the findings in 25 healthy subjects, of whom over 90% showed a positive DTH reaction to any of the three antigens. PHA skin tests were entirely normal in both patients and healthy controls. Microbial antigen-specific LTT responses fluctuated considerably in time from strongly positive to negative and vice versa in healthy individuals as well as in patients. In general however, blastogenic responses in patients were comparable to or even higher than those of healthy persons. In the MIF assay, lymphocytes of all healthy individuals tested showed production of MIF upon stimulation with all three antigens; this again contrasted to two-thirds of the patients, whose lymphocytes showed a defective MIF production. Fluctuations of MIF-production in time could not be established and a very good correlation existed between the data obtained in the MIF assay and those of the DTH skin tests. These results indicate that apart from skin testing, the MIF assay seems to be the most suitable parameter to assess defects in T cell reactivity towards microbial antigens. These defects exist in two-thirds of our patients suffering from chronic purulent rhinosinusitis.