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.     

Augmentation of delayed-type hypersensitivity to high dose sheep erythrocytes by cyclosporin A in the mouse: influence of drug dosage and route of administration and analysis of spleen cell populations.

When administered by various routes 48 h before a high systemic dose (10 degrees) of sheep red blood cells (SRBC), Cyclosporin A (CsA) prevented the suppression of delayed-type hypersensitivity (DTH) reactions elicited 4 days later. Augmentation of DTH was observed over a wide range (5-200 mg/kg) and with circulating CsA levels ranging below 45 ng/ml at the time of immunization or antigen challenge. Splenic lymphocytes from vehicle- and CsA-treated mice exhibited good proliferative responses to mitogen in vitro, but only those from CsA-treated animals responded to antigen. Expression of DTH was associated with a progressive, 2-fold increase in the absolute numbers of splenic L3T4+ cells, whereas no significant alteration in the number of Lyt-2+ lymphocytes was recorded. B cell and macrophage numbers in the spleen were unaffected by CsA. In contrast to its potentiating effects on cell-mediated immunity, CsA caused profound (up to 100%) suppression of the concomitant production of splenic anti-SRBC IgM-secreting plasma cells. Circulating anti-SRBC antibody levels were also markedly reduced. These data show that CsA can permit induction of TDTH, whilst suppressing T-dependent humoral immunity and without significant change in absolute numbers of Lyt-2+ cells.     

Influence of vitamin A on immunological response

The influence of vitamin A injections on immunological response in mice was investigated. Two end-points were used: the titre of haemagglutinin at 10 or 15 days after sensitization with sheep red blood cells (SRBC), and the time to reject C57BL/6 male skin grafts by isologous female recipients. Daily vitamin A injections for the 5 days preceding or following sensitization with SRBC led to a large increase in the production of haemagglutinin antibodies. Injection of vitamin A for the 5 days preceding or following grafting, or starting on the 6th day after grafting, significantly reduced the mean rejection time of male skin grafts.     

Enhancement of the Arthus reaction and suppression of delayed type hypersensitivity (DTH) by pluronic F-68, a detergent frequently used to prepare perfluorocarbon emulsions

The effect of a 20% w/v RM101 (perfluorobutyltetrahydrofuran) emulsion containing 5% w/v of the detergent Pluronic F-68 or 5% w/v Pluronic F-68 given alone on the Arthus reaction and on delayed type hypersensitivity (DTH) were evaluated in female A/J mice. The test substances were administered i.v. at 1% body weight at 0,4,7,14 and 28 days prior to the i.p. immunization with 10(7) sheep red blood cells (SRBC). The increase in footpad swelling at 4 h (Arthus reaction) and at 24 h (DTH) after elicitation with the s.c. administration of 10(8) SRBC into the left footpad was used to assess immune competence. Pluronic F-68 given alone enhanced the Arthus reaction only when administered on day 0 of immunization. Pluronic F-68 given alone, as well as the perfluorocarbon emulsion containing Pluronic F-68, suppressed the 24 h DTH for as long as 4 days prior to immunization. Nonemulsified perfluorocarbon, on the other hand, had no effect on either the Arthus reaction or on DTH. The immunostimulatory agent, levamisole, administered (10 mg/kg i.p.) 1.5-2 h prior to immunization with SRBC counteracted both the Arthus reaction and the DTH response produced by Pluronic F-68. The present data clearly demonstrate that the changes in Arthus reaction and the DTH response are due to the Pluronic F-68 used to emulsify the RM101 perfluorocarbon; the changes induced by the detergent in these two immune parameters probably involve separate mechanisms.     

Suppressive and augmentative effects of recombinant human interleukin-2 upon delayed type of hypersensitivity in the mouse

The in vivo action of recombinant human interleukin-2 (IL-2) was investigated against the delayed type of footpad swelling to sheep red blood cells (SRBC) in B₆D₂F₁ mice. A single intravenous injection of 1×10⁵ of SRBC induced transient cell-mediated immunity for delayed type of hypersensitivity (DTH) which peaked four days after the immunization and diminished rapidly thereafter. IL-2 exerted both suppressive and augmentative effects, depending on the timing of its adminstration in relation to the immunization. Daily intraperitoneal injections of as much as 5× 10⁴ Jurkat units of IL-2 from day 0 through day 3 caused marked suppression of DTH elicited on day 4. On the other hand, three or four consecutive injections of IL-2 from day 4 or day 3 through day 6 produced potent DTH on day 7, when DTH was faint, if there was any, without IL-2 administration. Potentiation of DTH was also observed when three injections of IL-2 were given from day 7 through day 9 and the antigen was challenged on day 10. These results suggest that IL-2 acts as an immunomodulator against cell-mediated immunity to SRBC in the mouse.     

Levamisole Restored the Compromized State of Immunity after Specific Chemotherapy in Experimental Schistosomiasis Mansoni

Mice infected for 45 days with 120 Schistosoma mansoni cercariae and treated with levamisole (25 mg/kg subcutaneously) have more efficient acquired immunity when challenged with 240 Schistosoma mansoni cercariae the same day of treatment (97.7% # 87.7% in infected challenged controls). In praziquantel-treated mice (500 mg/kg for 2 days orally), the reduction in the percent resistance (45.5%) was accompanied by a significant diminution in the size of granuloma, delayed foot pad swelling and granuloma proportionate T-helper cells number. Levamisole when given two weeks post praziquantel treatment and with the challenge infection increased the percent resistance to 79.2%. The increase in percent resistance recorded in mice receiving both praziquantel and levamisole was accompanied by restoration of granuloma size, delayed foot pad swelling and granuloma proportionate T-helper cells number to infected challenged untreated control values. Results reveal-beside efficacy of levamisole as immunoregulant in schistosome immunity - a possible role for the granuloma as a T-cell mediated response in maintenance of immunity.     

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.     

Immunomodulatory properties of platelet factor 4: prevention of concanavalin A suppressor-induction in vitro and augmentation of an antigen-specific delayed-type hypersensitivity response in vivo.

The immunomodulatory properties of platelet factor 4 (PF4) have been examined in vitro and in vivo. This agent prevented the induction of concanavalin A (Con A)-induced suppressor cells in vitro in a dose-dependent manner but it did not affect the function of established Con A suppressor cells. This effect was not due to an enhanced production of interleukin-2 (IL-2) by lymphocytes exposed to PF4. The delayed-type hypersensitivity (DTH) reaction to sheep erythrocytes (SRBC) was used as a model for the generation of antigen-specific suppression in vivo. PF4 enhanced the magnitude of the swelling following SRBC challenge 10 days after sensitization by the i.p. route or following sensitization by both the s.c. and i.p. routes. These studies show that PF4 has immunomodulatory activities in well-defined models of cell-mediated immunity and suggest that this agent has a potential use in the dissection of events in antigen-specific suppression.     

Styrene-induced immunomodulation in mice

Male mice given different oral doses (0.05, 0.03 or 0.02 x LD50/animal/day) of styrene (LD50 = 1 g/kg) daily for 5 days did not incite any overt toxicity in lymphoid organs or on hematologic parameters. At the tested dose levels styrene produced a mild reduction in the organ weight of adrenal and spleen and slight reduction in the cellular viability of lymph nodes. There was a dose-dependent suppression in the humoral immune response (IgM-producing PFCs of spleen and serum anti-SRBC HA titre) to SRBC. The proliferative response to the B-cell mitogen, LPS however revealed a significant increase in the incorporation of 3HT with middle and lowest doses of styrene. The results of cell-mediated immunity appeared somewhat unexpected and more complex as exposure resulted in a dose-dependent enhancement in the cutaneous DTH reaction to SRBC together with increased blastogenic response of splenic lymphocytes to phytohaemagglutinin (PHA). Additionally, there was significant impairment in the functional activity (NBT reduction, attachment and phagocytic indices) of nonadherent and adherent peritoneal exudate cells. Based on the present data the study identifies the immunotoxic potential of styrene and which acts differently on various arms of the rodent's immune system.     

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.     

The comparative selectivity of adjuvants for humoral and cell-mediated immunity. II. Effect on delayed-type hypersensitivity in the mouse and guinea pig, and cell-mediated immunity to tumour antigens in the mouse of Freund's incomplete and complete adjuvants, alhydrogel, Corynebacterium parvum, Bordetella pertussis, muramyl dipeptide and saponin.

Corynebacterium parvum was the only adjuvant of those tested which consistently potentiated delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) in the mouse, although this required the antigen-adjuvant mixture to be injected subcutaneously in the footpad rather than the flank. For induction of DTH to ovalbumin (OVA) in the guinea pig, C. parvum and MDP could be substituted for the mycobacteria in FCA. C. parvum was effective in aqueous solution provided that the OVA was absorbed onto alhydrogel. Saponin also potentiated DTH when injected with OVA in aqueous solution. C. parvum was the sole adjuvant of those tested to promote protective cell-mediated immunity (CMI) to M4 fibrosarcoma cells. It is concluded that C. parvum is a promising candidate adjuvant for promoting CMI induction without recourse to a water-in-oil emulsion.     

Preferential induction of cell-mediated immunity by chemically modified sheep erythrocytes

Sheep red blood cells (SRBC) almost exclusively induce humoral antibodies when injected in saline into adult rats. Chemical modification of SRBC by either periodate oxidation or acetoacetylation resulted in preparations which provoked lower antibody responses than did normal SRBC, but induced much higher levels of delayed-type hypersensitivity. Furthermore, SRBC which had been both periodate oxidized and acetoacetylated induced even higher delayed responses, but were unable to stimulate detectable antibody formation. Thus, by two simple chemical treatments, SRBC have been converted from an antigen which predominantly induces humoral antibodies tc an antigen which exclusively provokes cell-mediated immunity. The chemically modified SRBC had some notable immunological properties. (I) Antigenically, they were still strongly agglutinated by anti-SRBC antiserum. (II) They very effectively induced delayed-type hypersensitivity when injected either in saline or Freund's complete adjuvant. (III) They lost their immunogenicity when lysed just prior to injection. (IV) They induced delayed-type hypersensitivity which cross-reacted with goose, rabbit and horse red blood cells, a result consistent with the notion that antigens cross-react more broadly at the cell-mediated immune level than at the antibody level. (V) Comparatively high levels of both humoral and cell-mediated immunity were achieved when a mixture of periodate oxidized SRBC and acetoacetylated SRBC was injected. The theoretical and practical implications of these findings are discussed.     

Effects of Antiarthritic Compounds on Sheep Erythrocyte-Induced Delayed-Type Hypersensitivity

Delayed hypersensitivity (DTH) to sheep erythrocytes (SRBC) in mice is a simple model of cellular immunity mediated primarily by Ly 1⁺ T-cells. Because little is known about the pharmacology of this model, the therapeutic effects of a variety of antiarthritic agents were tested. Swelling was determined using mercury plethysmography and used to determine drug activity. Two immunosuppressive agents (cyclophosphamide and azathioprine) and two steroids (dexamethasone and hydro-cortisone) significantly reduced swelling. Only one of six nonsteroidal antinflammatory drugs (NSAIDs), aspirin, reduced swelliny. Indomethacin, phenylbutazone, and benoxaprofen augmented swelling and ibuprofen and isoxicam were inactive. Four disease-modifying antirheunatic drugs (DMARDs), D-penicillamine, levamisole, chloroquine, and aurothioglucose, were tested and none inhibited swelling. Aurothioglucose significantly augmented swelling. In conclusion, only steroids and immunosuppressive agents had consistent effects on DTH to SRBC. In general, NSAIDs and DMARDs were either inactive or augmented swelling. These results suggest that this model may be of use as a routine screen for immunomodulatory agents.     

Effects of Antithymocyte Sera and Antimacrophage Sera on Cell-Mediated Immune Reactions in Listeria-Infected Mice

The cell-mediated immune responses of allograft rejection, delayed hypersensitivity, and resistance to Listeria monocytogenes were suppressed by injections of antithymocyte serum (ATS), but the immune responses were not significantly altered by antimacrophage serum (AMS) or normal rabbit serum (NRS). Antisera were prepared in rabbits against purified mouse thymocytes and purified peritoneal macrophages. When mice were injected with ATS near the time of skin grafting, allografts survived significantly longer. Similar administration of AMS or NRS failed to alter the course of graft rejection. Decreased footpad swelling indicated the suppression of delayed hypersensitivity in mice injected with ATS 6 days after a sublethal inoculation of Listeria cells. None of the serum treatments affected the ultimate survival of mice infected with a small number of bacteria. Either ATS or NRS was injected into immunized mice 1 day before and 2 days after a challenge inoculation of Listeria cells. Pronounced suppression of delayed hypersensitivity was found in the ATS-treated groups, along with extensive mortalities that reached 100% in the group receiving the largest dose of ATS. All control animals survived and demonstrated strong delayed hypersensitivity reactions. Antimacrophage serum had no significant effect on the three mechanisms of cell-mediated immunity that were tested. The lymphoid cells which mediate delayed hypersensitivity, antimicrobial cellular immunity, and allograft rejection possess antigenic determinants in common with thymocytes.     

Adjuvant effect of Bordetella pertussis vaccine to sheep erythrocytes in mice: enhancement of cell-mediated immunity by subcutaneous administration of adjuvant and antigen.

The subcutaneous route (s.c.) was used to study the adjuvant effect of Bordetella pertussis vaccine (PV) on cell-mediated immunity to sheep erythorcytes (SRBC). The immune response was measured by a sensitive assay procedure in which the antigen is injected intracutaneously into the mouse ear and the inflammatory swelling is measured with calipers. PV significantly enhanced cell-mediated immunity to SRBC, and the enhancement persisted for at least 3 weeks. PV administered up to 6 days before SRBC also significantly enhanced the response; PV injected 1 or more days after SRBC was not effective. In addition, it was found that PV per se released into the suspension medium a cell-free component(s) (pertussis supernatant) that contributed significantly to adjuvanticity. The adjuvanticity of both PV and PS was completely eliminated by heat.     

Simultaneous stimulation and suppression of two different indicators of the cell-mediated immune response by the immunoregulator dextran sulphate.

The effect of high mol. wt dextran sulphate (DS) on two different indicators of the cell-mediated immune response was studied simultaneously in the same animal. The two indicators of the cell-meidated immune response chosen for this study were the footpad swelling response to sheep red blood cells (SRBC) and skin allograft rejection. Mice receiving DS and SRBC showed a significant increase in the delayed-type footpad swelling response over the controls at 24 and 48 h after challenge, while in addition, the same mice showed a significant suppression of the delayed-type response to allograft antigens as indicated by a significant delay in rejection time of allografts. The results presented here show that DS is capable of simultaneously stimulating and suppressing two indicators of the cell-mediated immune response in the same animal.     

Influence of tumour carriage on the production of lymphokines affecting macrophage behaviour.

Normal and cyclophosphamide (Cy)-enhanced delayed-type hypersensitivity (DTH) reactions to sheep red blood cells (SRBC) were severely impaired in CD1 mice injected with 10(6) viable Landschütz ascites carcinoma (LAC) cells at the time of immunization. The tumour-induced suppression of DTH was accompanied by inhibition of splenic T-lymphocyte responses to antigen and mitogen. This was assessed by measuring lymphocyte transformation and the production of two lymphokines affecting macrophage behavior, i.e. lymphocyte-derived chemotactic factor (LDCF) and macrophage procoagulant-inducing factor (MPIF). Our results imply that impaired production/function of lymphokines affecting macrophage behaviour may play a key role in tumour-induced suppression of cell-mediated immunity and that this inhibition is independent of Cy-sensitive suppressor cells.     

Altered immune response (humoral and delayed-type hypersensitivity reactions) to sheep red blood cells in the course of experimental filarial infections (Litomosoides carinii,Brugia malayi,Acanthocheilonema viteae) ofMastomys natalensis

Litomosoides carinii-, Acanthocheilonema viteae- orBrugia malayi-infectedMastomys natalensis were sensitised against sheep red blood cells (SRBC) on various occasions after infection to determine the effect of filarial infections on the immune response to a non-filarial antigen. The phagocytic activity of the reticuloendothelial system (RES) was controlled in vivo by the elimination of⁵¹Cr-labelled SRBC. Antibody titres against SRBC (agglutinating and lytic antibodies) were similar to those of uninfected controls inL. carinii-orB. malayi-infectedMastomys sensitised during prepatency or early patency up to 90 days post infection (p.i.) but were reduced in animals sensitised during patency. A significant inverse correlation existed between anti-SRBC antibody titres and microfilaraemia levels. In contrast,A. viteae-infectedMastomys showed reduced humoral anti-SRBC responses at the end of prepatency, whereas the response tended towards normal with increasing parasitaemia. Delayed-type hypersensitivity (DTH) against SRBC was measured as footpad swelling after sensitisation by the s.c. or i.v. route and intraplantar challenge. DTH reactions were reduced during prepatency in all infections after s.c. sensitisation. During patency, 24-h reactions were similar to those of age-matched controls but the swelling persisted 24 or 48 h longer than in the latter. InA. viteae infections, even enhanced 24-h reactions were found during patency. Histological investigations did not reveal differences in the type of cell infiltrations between infected and control animals. After i.v. sensitisation with SRBC,L. carinii- andA. viteae-infected animals showed weaker DTH reactions than the controls, independent of the period after infection. In the case ofB. malayi infections, DTH reactions were similar to those of controls during early prepatency, whereas reduced DTH responses were observed later than 50 days p.i. As shown inL. carinii-infected animals, depressed DTH reactions after i.v. sensitisation did not depend on an altered expression phase but rather on an altered regulation during the inductive phase of the response: increases in the sensitising SRBC doses that caused decreasing DTH reactions in uninfected animals led to enhanced reactions in infected animals. Phagocytosis of i.v. injected⁵¹Cr-labelled SRBC was enhanced during prepatency inL. carinii infection and during patency in all infections.Dedicated to Prof. Dr. W. Peters on the occasion of his sixtieth birthdaySupported by a DAAD grant     

Lack of correlation between delayed-type hypersensitivity and host resistance to Plasmodium chabaudi infection.

C57B1/6J mice immunized with Plasmodium chabaudi antigen plus saponin exhibited strong delayed-type hypersensitivity (DTH) reactions to footpad injections of P. chabaudi antigen. However, when immunized mice were challenged with P. chabaudi parasites, DTH was significantly depressed after 3 days of infection. This DTH depression coincided with a steep rise in the titre of malarial antibody in these immunized challenged mice when compared to unchallenged immunized mice. Serum from mice recovering from P. chabaudi infection depressed DTH levels of immunized mice significantly, but spleen cells from convalescent mice only slightly reduced the DTH levels. These results are discussed with respect to the role of cell-mediated immunity in malaria infections.     

Modulation of delayed-type hypersensitivity during the time course of immune response to a protein antigen

Delayed-type hypersensitivity reactions elicited in the footpad of ovalbumin-sensitized mice after challenge with aggregated ovalbumin on day 4 or 8 of immunization are distinct. The former was characterized by a dense mononuclear infiltrate and, macroscopically, the reaction peaked at 48 hr after antigen challenge; the latter was preceded by immediate-type reactions, reached the maximum at 24 hr and faded drastically later. Histologically, oedema and a mixed granulocytic-lymphocytic infiltrate was found at this time-point. Immunoglobulin G1 (IgG1), IgG2a and IgE antibodies were detected only in plasma obtained after 8 days of immunization. Regarding the cytokines produced by draining lymph node cells after in vitro restimulation, interleukin-4 (IL-4) and IL-10 were predominant after 4 days and interferon-gamma and IL-2 after 8 days of immunization. These two types of delayed-type hypersensitivity (DTH) were used to study the influence of antibody-mediated responses on the inductive and effector phases of cell-mediated immunity. The effector phase of DTH was not affected by immediate-type reactions, as abrogation of these reactions by mediators' antagonists on day 8 or induction of passive reactions by transfer of immune serum on day 4 did not change the extent or kinetics of either type of DTH. Only transfer, before immunization, of whole or T-cell-enriched spleen cells, but not sera, from hyperimmunized donors (high antibody producers) abolished the induction of pure DTH in 4-day immunized recipient mice and changed their cytokine profile to a T helper 2 type. These results indicate that in a non-polarized immune response to a protein antigen there is initially a bias towards cell-mediated immunity, which is gradually dampened by the development of antibody-mediated immunity.