Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. IV. Fc receptors on specific peritoneal exudate lymphocytes and their role in delayed-type hypersensitivity reactions.

In mice, delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) is mediated by T cells. Peritoneal exudate T cells (PETLs) from mice optimally sensitized for DTH to SRBC form rosettes when interacted with sensitized sheep red blood cells (EA). The binding of EA to PETLs is mediated by a receptor specific for the Fc portion of the antibody (FcR). Biological activity (mediation of DTH) depends on the unreacted state of PETLs and is lost when the latter are either rosetted with EA or reacted with aggregated IgG. Transfer of EA or aggregated IgG-treated PETLs from mice with DTH to SRBC does not lead to adoptive sensitization of recipients. It is suggested that FcR found on the membrane of T cells mediating DTH play a role in the regulation of the cellular immune response to SRBC.     

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (neurotropin); enhancing effect on delayed type hypersensitivity response through the induction of Lyt-1+2- T cells

To clarify the immunopharmacological action of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin), its effect on delayed type hypersensitivity (DTH) response to sheep red blood cells (SRBC) in mice was examined. Neurotropin enhanced the DTH response in C57BL/6 mice which were low responders to SRBC, but not in either BALB/c or C3H/He mice (high responders) when administered i.p. for 4 consecutive days prior to sensitization. However, Neurotropin did not affect the formation of plaque-forming cells to SRBC in C57BL/6 mice under the condition where it enhanced the DTH response. We further examined the mechanism by which Neurotropin enhanced the DTH response in C57BL/6 mice by means of cell transfer experiments. Spleen cells from mice administered Neurotropin i.p. for 4 days, but not saline, could enhance the DTH response when transferred i.v. into normal syngeneic mice just before sensitization. However, the treatment of the spleen cells with anti-Thy-1.2 + complement (C) or with anti-Lyt-1.2 + C, but not with anti-Lyt-2.2 + C, abrogated its enhancing effect. The depletion of macrophages from the cells had no effect. On the other hand, the spleen cells from mice administered Neurotropin had no enhancing effect in the effector phase of DTH response, and they showed a helper T cell activity in a DTH helper T cell assay system in which cyclophosphamide-treated mice were used as recipients. These results suggest that Neurotropin enhances the DTH response in low responder mice through the induction of Lyt-1+2- DTH helper T cells.     

Cyclosporin A prevents suppression of delayed-type hypersensitivity in mice immunized with high-dose sheep erythrocytes.

When administered intraperitoneally to mice 2 days before immunization with a tolerogenic dose (10(9)) of sheep red blood cells (SRBC), cyclosporin A (CsA; 200 mg/kg) strikingly augmented 4-day delayed-type hypersensitivity (DTH) footpad reactions. These enhanced responses were similar in magnitude to those seen in animals sensitized with an immunogenic, low-dose (10(6)) SRBC. The stimulatory effect of CsA was observed over the dose range of 5-200 mg/kg and was obtained in animals given the drug in one injection, up to 7 days before sensitization. The augmentation of DTH was characterized by footpad swelling, intense mononuclear cell infiltration and increased deposition of 125I-fibrinogen within the challenge site. In addition, increased expression of procoagulant activity by spleen cells in response to antigen was observed. Cell transfer experiments showed that the CsA-enhanced DTH could be adoptively transferred to naive recipients. Additional transfers conducted at the time of antigen challenge suggested that, under the conditions described, CsA inhibited the action of a population of suppressor cells normally effective during DTH reactions.     

Junin virus-induced delayed-type hypersensitivity suppression in adult mice

Junin virus (JV) infection of suckling mice leads to lethal meningoencephalitis consistent with a delayed-type hypersensitivity (DTH)-like immune response. In contrast, there are no central nervous system (CNS) alterations, and high antibody titers are induced in resistant adult mice. As a possible explanation, JV infection in adult mice may provoke DTH depression. Thus in this work we study the alterations induced by JV in the immune response of adult mice by using sheep red blood cells (SRBC) as an unrelated indicator antigen. JV infection was found to abrogate DTH significantly, regardless of SRBC priming time, virus strain attenuation, and viral route of inoculation. The effect proved viral dose-dependent and required live and infectious virus. However, the humoral response to SRBC, as determined by splenic "plaque-forming cell" count was found higher than controls. These results are consistent with adult mouse response to JV infection. In contrast to the guinea pig model, there is no destruction of immunocompetent cells. T-cell percentage in the spleen was high, suggesting involvement with DTH-suppressive action. We suggest that the immune response to SRBC in adult infected mice may be used for understanding the mechanisms involved in resistance.     

Studies on delayed-type hypersensitivity (DTH) reactions in the pleural cavity in mice: prolonged DTH reaction and its interruption by cyclophosphamide treatment

DTH reactions to sheep erythrocytes (SRBC) and purified protein derivative (PPD) antigens were produced in the pleural cavity in mice. The profiles of the DTH reactions with respect to time course and cellular exudate reactions differed greatly according to the strains of mice. In particular, HY mice that were established in our laboratory displayed prolonged DTH reactions, characterized by macrophage followed by lymphocyte reactions. HY X C3H F1 mice showed a similar tendency. On the other hand, strains such as C3H, BALB/c, DBA/2 and B6 mice showed short-lived and macrophage-predominant DTH reactions. BALB/c nu/nu mice showed no DTH reactions. Characteristic features of the prolonged DTH reactions in HY mice were transferred with sensitized T cells. However, DTH reactions in HY mice treated with cyclophosphamide (CY) terminated in a short period, and mainly consisted of macrophages and polymorphs, although they were greatly enhanced. Such profiles of the reactions could also be transferred with sensitized T cells from CY-treated and SRBC-sensitized mice. Spleen cells taken from CY-treated and SRBC-sensitized HY mice, when injected intravenously into SRBC-sensitized HY mice just prior to challenge, could not interrupt prolonged DTH reactions. These results thus indicated various phenotypes of the DTH reactions in terms of time course and exudate cellular component involved might be carried by the specific effector T cells in each phenotype of the DTH reactions and could be induced using strains of mice and CY.     

Distinction between suppressors of the delayed-type hypersensitivity and the humoral response to sheep erythrocytes.

Suppressors for both delayed-type hypersensitivity (DTH) and the humoral immune response could be simultaneously induced in the spleens of mice by immunization with a high dose of SRBC. Normal recipient mice of the spleen cells from donors immunized 5 days previously elicited depressed DTH or humoral response when immunized with SRBC. The suppressive activity was found to reside in T not B enriched fraction. Four hundred rad irradiation of the primed spleen cells resulted in complete loss of DTH suppressor activity, but only in some reduction of the suppressor activity for the humoral response. In contrast, hydrocortisone treatment of the donor mice caused no loss of DTH suppressor activity while approximately half of the suppressive activity for anti-SRBC PFC response was lost. Adult thymectomy prevented completely the induction of the DTH suppressor in contrast to little loss of the suppressor activity for the humoral response. DTH suppression was antigen-specific for the induction, but nonspecific for the expression. However the suppression of the humoral response was antigen-specific not only for the induction but also for the expression. In addition, DTH suppressor was capable of suppressing both the induction and expression of DTH while the humoral response was suppressed only in the induction stage by the suppressor.     

Potentiation of immune responses in mice by a new inosine derivative--methyl inosine monophosphate (MIMP).

Inosine 5'-methyl monophosphate (MIMP) is a new immunomodulator designed to improve upon the activity of other thymomimetic purines. In Balb/c mice, MIMP was assessed for toxicity and activity on immune responses. The lethal dose for half the mice (LD50) exceeded 500 mg/kg of body weight by both the parenteral and oral routes. At doses of 1-100 mg/kg, the mice showed no visible untoward effects. The antibody response of splenocytes to sheep erythrocytes (SRBC) was measured by IgM plaque-forming cells (PFC) in soft agar under optimal conditions of immunization and challenge. MIMP (1-100 mg/kg) was given by both the intraperitoneal and oral routes (gavage) at the time of SRBC injection and 4 days thereafter. The PFC response was found to be significantly augmented. The maximum effect (approximately 2x) was observed at 50 and 100 mg/kg, via intraperitoneal (i.p.) and oral routes, respectively. Increases (maximally 1.5x) in the responses of splenic lymphocytes to mitogen stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A) were observed under similar conditions of MIMP treatment. SRBC-induced delayed-hypersensitivity (DTH) was also measured under optimal conditions. By both i.p. and oral routes, enhancement of DTH response was produced by the lower doses of MIMP (0.01-1 mg/kg). Again, a second peak of optimum stimulation of DTH response was produced by 50 mg/kg of MIMP when administered by both routes. The effect was observed mainly on the sensitization rather than on the expression phase. MIMP qualifies as an effective immunopotentiator in normal mice.     

Dissociative effects of malarial infection on humoral and cell-mediated immunity in mice.

The effect of malarial infection on immune responses was studied in mice. When sheep red blood cells (SRBC) were injected 2 days before or at the same time as infection with Plasmodium berghei, there was a marked increase in the number of splenic plaque forming cells (PFC) induced by SRBC as compared with uninfected controls. When SRBC were injected 2 days or more after the infection, however, the PFC response was significantly reduced. On the other hand, cell-mediated immunity, as exemplified by delayed-type hypersensitivity (DTH) to a number of antigens, was suppressed whether the infection was introduced before or after antigen stimulation. A similar effect could be produced by injecting the host with the supernatant obtained following incubation in vitro of peripheral blood from heavily infected mice. When this supernatant was injected i.v. into normal mice at the same time as SRBC priming, it enhanced the humoral response to SRBC, but suppressed the DTH to SRBC. The coincident induction of this inverse relationship between humoral and cell-mediated immunities was clearly borne out by a dose response study using different dilutions of supernatant. The active component appeared to be of large molecular weight (greater than 150,000), thermostable and not present in the serum of infected mice.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Regulation of delayed-type hypersensitivity. II. Specific suppressor factor for delayed-type hypersensitivity to sheep erythrocytes in mice.

An antigen-specific suppressor factor for delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is described. Lymph node cells and spleen cells from mice injected intravenously with 1 x 10(9) SRBC 4 days previously were incubated in vitro for 48 h in culture medium. Supernatant obtained from the culture inhibited the induction of DTH to SRBC in normal mice. It also suppressed the expression of DTH in presensitized mice. The suppression is specific as the suppressor factor had no effect on the DTH to noncross-reacting antigen, chicken red blood cells. Treatment of the spleen cells with anti-theta serum and complement prevented the production of the suppressor factor, whereas treatment with anti-Ig serum and complement had no effect. Suppressor factor produced by H-2k mice suppressed the DTH in H-2b mice. The factor thus seems to act across the H-2 barrier. The suppressor factor was not removed by adsorption with goat anti-mouse immunoglobulin immunoadsorbent, but could be adsorbed by SRBC. It was stable at 56 degrees C for 1 h, but was partially inactivated by freezing and thawing. The factor has a molecular weight of less than 35 000 daltons.     

Immunosuppressive effects of<Emphasis Type="Italic"> Ixodes ricinus</Emphasis> tick saliva or salivary gland extracts on innate and acquired immune response of BALB/c mice

Saliva and salivary gland extract (SGE) of Ixodes ricinus ticks have suppressive effects on the innate immune response of BALB/c mice. Tick saliva prevents hemolysis of sheep red blood cells (SRBC) by the human alternative pathway of complement. The adaptive immune response is also modulated by tick antigens (saliva or SGE). When stimulated in vitro with increasing doses of tick antigens, the proliferation and IL-4 production of draining lymph node T cells of mice infested with nymphal ticks increase, peak and then decrease. These results indicate that immunostimulative and immunosuppressive molecules have competing effects in tick saliva or in SGE. I. ricinus saliva inhibits, in a dose-dependent manner, splenic T cell proliferation in response to concanavalin A (ConA). Tick SGE or saliva injected intraperitoneally to BALB/c mice simultaneously with SRBC systemically immunosuppress the anti-SRBC response as shown in vitro by the reduced responsiveness of sensitized splenic T cells to restimulation with SRBC. In brief some components of SGE or tick saliva reduce the responsiveness of draining lymph node T cells and of sensitized splenic T cells in vitro. The responsiveness of naive splenic T cells to ConA stimulation in vitro is also decreased by tick saliva. Modulation of host responses by tick antigens may facilitate tick feeding, transmission and the propagation of pathogens.     

Regulation of delayed-type hypersensitivity. I. T suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

Mice injected with 1 X 10(8) sheep red blood cells (SRBC) into the footpad showed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after the injection. In contrast, mice injected intravenously with 1 X 10(9) SRBC were unresponsive to DTH induction through 1 X 10(8) SRBC injected into the footpad. This suppression of DTH was maintained for at least 6 weeks and was transferable spleen, lymph node and thymus cells to normal syngeneic recipients. Bone marrow cells, on the other hand, did not contain the suppressor cells. The suppression of DTH was antigen-specific in that DTH to chicken red blood cells and contact sensitivity to 2,4-dinitrofluorobenzene was not affected. The suppressor cells were theta-positive and Ig-negative. They appeared in the spleen in optimum number 3-4 days after induction. The suppressor cells affected both the induction and manifestation of DTH. The presence of suppressor and effector cells for DTH inducible by different routes of antigenic presentation reflects the dynamic balance in the regulation of DTH.     

Determination of antibodies and non-specific immunoglobulins produced by single cells

To determine whether one cell simultaneously produces antibodies and non-specific immunoglobulins (nIg), spleen cells from mice immunized twice with SRBC were cultivated on membrane filters, firstly on the layer of native SRBC in agarose and then on the layer of SRBC sensitized with anti-mouse IgG antibodies. A specific immunoadsorbent, eliminating all anti-SRBC antibodies, was placed between the filters with spleen cells and sensitized SRBC. By comparing the location of indirect and reverse plaques (i.e. the location of cells producing antibodies to SRBC and nIg) it was shown that antibody-forming cells do not synthesize nIg.     

Long-lasting enhancement of the delayed-type hypersensitivity response to heterologous erythrocytes in mice after a single injection of cyclophosphamide.

Previous reports have indicated that cyclophosphamide (CY) treatment can enhance delayed-type hypersensitivity (DTH) reactions by abrogating suppressor T cell functions. Such findings have suggested that cells in the suppressor lineage may be particularly sensitive to this alkylating agent. The experiments reported here demonstrate that a single injection of CY before sensitization can induce a long-lasting state of enhanced DTH responsiveness to sheep red blood cells (SRBC) in mice. This enhancement required concurrent antigenic stimulation and appeared to be antigen-specific. Additionally, CY treatment of sensitized mice before the first antigenic challenge for DTH resulted in suppressed responses to that challenge, followed by enhanced DTH to subsequent challenge with the same antigen. The suppressed response was achieved with a lower dose of CY than the subsequent enhancement and also required concurrent antigenic stimulation. These results indicate that the effects of CY on both effector and suppressor mechanisms are critically dependent upon antigenic stimulation, and suggest that apparent suppressor sensitivity to CY may be a function of differential ability to recover from CY treatment in a context of antigenic stimulation.     

Lack of immunosuppression by ketoconazole and itraconazole.

The antifungal drugs ketoconazole and itraconazole were evaluated for their effects in the following test systems: in vitro, phytohaemagglutinin (PHA)-induced proliferation of human peripheral blood mononuclear cells and IL-2-driven proliferation of CTLL-2 cells; in vivo, antibody response to sheep red blood cells (SRBC) and delayed-type hypersensitivity (DTH) reaction to oxazolone. At a concentration of 10 microM, ketoconazole moderately and itraconazole strongly inhibited thymidine (Thd) incorporation in human peripheral blood mononuclear cells cultured in medium supplemented with 5% human serum. Increasing the serum concentration from 5 to 20% almost completely reversed these inhibitory effects. Also, cell viability, found to be less than 15% in cultures containing 10 microM itraconazole was restored by increasing the serum concentrations in the culture medium. Similar observations were made in experiments using IL-2-stimulated CTLL-2 cells: the growth inhibition in the presence of 10 microM ketoconazole or 1 microM itraconazole could be counteracted by increased serum supplementation. In vivo, subchronic intraperitoneal dosing with 40 mg/kg ketoconazole or itraconazole to mice had no effect on the antibody response to SRBC as measured by the number of splenic IgM and IgG plaque-forming cells and did not significantly affect the DTH response to oxazolone. These data indicate that neither ketoconazole nor itraconazole exert immunosuppressive properties in vivo. Their in vitro inhibitory effects on PHA-induced lymphocyte proliferation and IL-2-dependent CTLL-2 growth are reversed by the serum supplementation to the culture medium and these activities should therefore be considered as in vitro artefacts.     

Endothelial cells and renal epithelial cells do not express the Wegener''s autoantigen, proteinase 3.

In vitro and in vivo T cell responses were determined during the course of bronchopulmonary infection with mucoid Pseudomonas aeruginosa. T cell responses were compared in two inbred mouse strains, namely BALB/c mice, which are resistant to the establishment of chronic bronchopulmonary Ps. aeruginosa infection, and C57Bl/6 mice, which have high numbers of bacteria in the lungs through 14 days post-infection. Unseparated lung cells and lung T cells from BALB/c mice exhibited significantly higher in vitro proliferative responses to both heat-killed Ps. aeruginosa and concanavalin A (Con A) than cells from C57Bl/6 mice through 20 days post-intratracheal infection with 10(4) colony-forming units (CFU) Ps. aeruginosa. Proliferation of unseparated lung cells but not lung T cells from BALB/c mice infected 6 days previously with 10(5) CFU Ps. aeruginosa was suppressed in response to Con A; these cells were unresponsive to specific antigen. Suppression of lymphocyte proliferation in the lungs of C57Bl/6 mice infected with 10(4) CFU Ps. aeruginosa and in BALB/c mice infected with 10(5) CFU was found to be mediated by adherent lung cells via the production of nitric oxide and prostaglandins. Determination of in vivo T cell-mediated responses in infected mice demonstrated that resistant BALB/c mice had high DTH and low Pseudomonas-specific antibody responses, while C57Bl/6 mice had low DTH and high antibody levels, in particular, IgG2b and IgM.     

Modulation of the immune responses against SRBC after oestriol treatment in mice.

Delayed-type hypersensitivity (DTH), primary direct and indirect PFC, memory antibody response and suppressor cell induction against sheep red blood cells (SRBC) have been examined in oestriol (E3) pretreated mice. The results showed that DTH and primary direct and indirect PFC responses were suppressed by E3 treatment. These suppressive effects could, however, be overcome when oestrogenized mice were given supra-optimal doses of SRBC for each response. On the other hand, the memory antibody response was markedly enhanced when E3 was given prior to the primary antigen stimulation. Induction of the suppressor cells for the antibody response seemed not to be affected by E3 treatment, but the characterization of the suppressor cells revealed that those obtained from E3 treated mice were surface immunoglobulin positive (sIg+) cells whereas those from control mice were not. These results were discussed in terms of the altered antigen distribution due to the activated phagocytic activity of the reticuloendothelial system (RES) after E3 treatment.     

Modification of Immune Response by Cinnarizine

Effects of cinnarizine on immune response in mice were investigated. Mice were orally administered with cinnarizine and were immunized with sheep red blood cells (SRBC) intravenously. Numbers of plaque forming cells (PFC) to SRBC in spleen of these mice were assayed and delayed-type hypersensitivity (DTH) response to SRBC was measured. 1) PFC response in immunization with 5 × 10⁶ cells/mouse of SRBC was enhanced by administration of 25 mg/kg of cinnarizine, while the response in immunization with 5 × 10⁸ cells/mouse was suppressed by 25 to 200 mg/kg of cinnarizine. 2) From study on timing of administration, suppression of PFC response by 6.25 to 200 mg/kg of cinnarizine was observed at 24 hr. after the immunization. 3) 12.5 to 200 mg/kg of cinnarizine suppressed polyclonal B cell activation induced by lipopolysaccharide (LPS). 4) Colchicine induced suppressor T cell inactivation was prevented by administration of 50 mg/kg of cinnarizine and it was suggested that cinnarizine may induce suppressor T cells from the study of adoptive cell transfer system. 5) 50 mg/kg of cinnarizine showed the suppression of DTH response in expression phase, but not in induction phase. It was concluded that immune responses in mice were modified by cinnarizine.     

Induction of suppressor T cells for lymph node cell proliferation after contact sensitization of mice with a poison oak urushiol component.

Contact sensitivity with properties of delayed-type hypersensitivity (DTH) can be induced in mice by 3-heptadecylcatechol (HDC, a component of poison oak urushiol oil). Sensitization is effected by painting on abdominal skin and is assessed by measuring ear swelling produced after ear challenge. Further studies on the nature of this sensitization were made by monitoring the induction of lymph node cell (LNC) proliferation (as indicated by increased in vitro uptake of [14C]-thymidine into DNA) after cutaneous treatment with HDC. Draining inguinal LNC proliferation peaked 5-6 days after abdominal application of HDC. LNC taken from sensitized mice at times later than this peak suppressed HDC-induced proliferation when transferred into recipient mice. Such suppressor cells were T lymphocytes as implied by their sensitivity to anti-Thy-1.2 antibody and complement. The suppressive effect appeared to have both specific and non-specific components. LNC containing these T suppressor cells could not suppress the optimal proliferation in vitro of previously sensitized cells, nor was suppressive activity observed against the induction of contact sensitization itself. Thus, although the suppressor cells appeared to act on the afferent phase of sensitization, they may not be directed against the effector cells of DTH.     

Effect of antiserum against isologous aggregated immunoglobulins on delayed-type hypersensitivity reaction in mice immunized with sheep red blood cells

Injection of mouse antiserum against isologous aggregated immunoglobulins (MAAS) into mice previously receiving 10⁵ sheep red blood cells (SRBC) did not affect the intensity of the delayed-type hypersensitivity (HDT) reaction when tested at the peak of sensitization (4th day), but led to a marked increase in the intensity of the reaction in the later stages (6th day). MAAS completely abolished suppression of HDT observed after sensitization with 5·10⁷ SRBC. Transfer experiments showed that under the influence of MAAS the number of suppressor cells of HDT in the spleen of the sensitized mice was reduced. MAAS had no effect on proliferation of antibody-forming cells or on the intensity of hemagglutinin production, but reduced by 70% the number of rosette-forming cells (RFC) detectable in the spleen at the peak of the primary immune response. The results may be evidence that RFC take part in the suppression of HDT.Laboratory of Immunochemistry, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 11, pp. 578–580, November, 1979.