Potentiation of nephrotoxic serum nephritis in Lewis rats by Freund's complete adjuvant--possible role for cellular immune mechanisms

Lewis rats receiving subnephritic doses of nephrotoxic serum (NTS) showed increased albuminuria and glomerular histopathologic alterations during the autologous phase of nephrotoxic nephritis (NTN) when they received simultaneous footpad injections of Freund's complete adjuvant (FCA). Lymph nodal lymphocytes from such experimental rats showed increased in vitro cellular sensitization to the nephrotoxic IgG as measured by [3H]thymidine incorporation. Such a lymphocyte blastogenesis response was not detected in rats receiving the same doses of FCA or NTS alone. Antibody titers to the nephrotoxic rabbit IgG were not different in the two groups of rats as measured by enzyme-linked immunosorbent assay. The transfer of lymph nodal mononuclear cells from rats with NTN potentiated by FCA, was able to induce albuminuria and glomerular histopathologic alterations in recipients treated with NTS. In the above experimental model FCA appears to potentiate the autologous phase of NTN by cellular immune mechanisms.     

The role of serum factors in the suppression of experimental allergic encephalomyelitis: evidence for immunoregulation by antibody to the encephalitogenic peptide.

Lewis rats immunized with myelin basic protein (MBP) in Freund's complete adjuvant (FCA) suffer from a single episode of paralysis from which they recover spontaneously. Subsequent to recovery, further episodes of paralysis cannot normally be induced by reimmunization with MBP in FCA. It is well established that serum, obtained from rats in the refractory state, can suppress the induction of experimental allergic encephalomyelitis (EAE) when given to animals from the time of immunization with MBP in FCA. Here it is shown that treatment with some such sera from Day 7 after immunization also suppressed the disease. However, not all convalescent sera were suppressive, indicating that rats immunized with MBP in FCA could become refractory to EAE without assayable levels of suppressive activity in their sera. In the context of this result it was notable that a correlation was found between the level of antibody specific for the encephalitogenic peptide in sera and the ability to suppress EAE. An inverse relationship was also shown between the amount of anti-encephalitogenic peptide antibody produced after immunization and the severity of EAE induced. Spleen cells from animals treated with Lewis anti-MBP serum after immunization with MBP in FCA could be activated to transfer EAE by in vitro culture with MBP despite the absence of any clinical signs in the donor animals, i.e. the serum inhibited the expansion or differentiation of these cells rather than preventing their priming or bringing about clonal deletion.     

Factors affecting transfer of experimental autoimmune thyroiditis in rats

Actively induced experimental autoimmune thyroiditis in inbred Lewis rats was comparable using standard Freund's complete adjuvant (FCA) and Perrin's modification of FCA. However, adoptively transferred disease using lymph node cells from rats immunized with Perrin's FCA was significantly more severe. With this adjuvant, and pertussis vaccine as co-adjuvant, transfer was uniformly successful when at least 480 × 10⁶ lymph node cells were taken 10 days after immunization and recipients were killed 3 days after transfer. Lymphocytic infiltrates were seen in recipient thyroids as early as 18 hours after transfer. Whole body irradiation of the recipients at 550 r reduced the severity of transferred disease. The frequency and severity of lesions were higher when the lymph node cells were first incubated with low doses of antigen. Thymectomy of the recipients decreased the severity of transferred disease. Under the conditions tested, transfer of disease could not be accomplished by antiserum alone, even using thyroidectomized donors. Administration of an early immune serum with sensitized lymph node cells significantly depressed the severity of transferred disease, while a late antiserum increased it.     

Comparison of Corynebacterium parvum and Bordetella pertussis with Freund's complete adjuvant as immunopotentiators for beta-human chorionic gonadotropin linked to an atoxic fragment of tetanus toxin

Corynebacterium parvum and Bordetella pertussis were compared with Freund's complete adjuvant (FCA) for their abilities to potentiate the immune response to haptenic beta-human chorionic gonadotropin covalently coupled to an atoxic 54,000-molecular-weight fragment of tetanus toxin (beta-hCG-TTII). The ability of each adjuvant to enhance production of antibodies to hCG in rabbits was measured by 125I-hCG radioimmunoassay. At sera dilutions of 1:10,000, analysis of variance for the 8-week postimmunization course showed that the mean 125I-hCG binding capacities of the C. parvum group was significantly greater overall than the B. pertussis group (P = .0002) and that the FCA-treated group had the greatest binding capacity overall (P less than .018). The mean binding capacities at 1:40,000 dilution again showed the FCA-treated group to have significantly higher anti-hCG titers overall (P less than .0015), with C. parvum potentiating a greater overall antibody response than B. pertussis (P = .001). These results indicate that FCA is the most efficacious of the three tested adjuvants in potentiating antibody production to the hapten component of beta-hCG-TTII. C. parvum was also effective at promoting an anti-beta-hCG response, although not to the same degree as FCA. B. pertussis had only minimal potentiating effect compared to FCA or C. parvum.     

Lymphocytes binding basic protein of myelin; cytophilic serum antibody and effect of adjuvant

Splenic lymphocytes from guinea-pigs injected with the basic protein of myelin (BPM) in Freund''s complete adjuvant (FCA) were shown by autoradiography to contain a significantly (P<0.01) greater proportion of lymphocytes binding ¹²⁵I-BPM than splenic lymphocytes from control guinea-pigs injected only with FCA; these in turn contained more than splenic lymphocytes from normal guinea-pigs. Splenic lymphocytes from guinea-pigs injected with a synthetic eleven amino acid peptide (containing the main encephalitogenic determinant, for guinea-pigs, of BPM) in FCA did not contain more lymphocytes binding ¹²⁵I-BPM than control suspensions from guinea-pigs injected only with FCA. Binding ability was confered on splenic lymphocytes of normal guinea-pigs, and especially on splenic lymphocytes of guinea-pigs injected with FCA, when the cells were held with guinea-pig antisera to BPM, the binding capacity of which was present in the IgG₂ subclass. In contrast, splenic lymphocytes from Lewis rats after injection of BPM of rat lacked the ability to bind ¹²⁵I-BPM of rat, as did splenic lymphocytes from normal rats held with potent rat antiserum to BPM of rat. Our results show, for the guinea-pig, that antibody attaches in vitro to the lymphocyte surface as antigen receptor, and, for this species at least, may be an important cause in vivo of the presence of antigen-binding cells after injection of antigen.     

The mode of action of treatment by IgG of haemolytic anaemia induced by an anti-erythrocyte monoclonal antibody

Tolerization of pathogenic antigens is one of the experimental strategies that has been proposed to prevent autoimmune disease. We have investigated here whether neonatal intraperitoneal infection of Lewis rats with Mycobacterium bovis-BCG has any effect on the expression of adjuvant arthritis (AA), an autoimmune disease that is produced by immunization of the rats with dead mycobacteria in mineral oil (i.e. Freund's complete adjuvant (FCA)). We found that neonatal infection with 10⁸ viable BCG bacilli rendered all Lewis rats resistant to the expression of AA after FCA immunization. This BCG-induced protection from reactive arthritis was not seen in Lewis rats infected with smaller inocula (10⁶ BCG bacilli) or if the infection was performed after the neonatal period (e.g. at 3 weeks of age). Neonatal administration of 65-kD mycobacterial heat shock protein (hsp65, a key antigen in the etiopathogenesis of AA) failed to protect Lewis rats from AA; injection of lactoferrin (an autoantigen that may be involved in the physiopathology of autoimmune arthritis) to newborn Lewis rats decreased the severity of AA observed after FCA immunization of the animals. Western blotting revealed that Lewis rats that had acquired resistance to AA also showed changes in their repertoire of antibody specificities; among these alterations was decreased anti-hsp65 reactivity. We conclude that neonatal infection with BCG, but not hsp65 injection, renders Lewis rats resistant to AA and that the phenomenon is associated with change in the repertoire of specificities of circulating antibodies.     

T suppressor lymphocytes regulation of adjuvant arthritis in two inbred strains of rats.

Adjuvant arthritis can be induced by a single injection of Freund's complete adjuvant (FCA) in the highly susceptible Lewis (LEW) rat strain, but not the resistant Wistar A.G. (WAG) strain. This strain-dependent susceptibility to the disease is correlated with differences in T suppressor cells regulation. In WAG rats, indeed, the in vitro response in LEW alloantigens was highly inhibited 11 days after FCA injection, while LEW rats in vitro response to WAG alloantigens was slightly increased. Furthermore, spleen cells from WAG rats given FCA 4 days before exhibited T cell-mediated active suppression of WAG in vitro response to LEW alloantigens when they were co-cultured with WAG normal spleen cells. This suppression was abolished by removal of T cells on nylon wool column. A previous irradiation of these T cells also inhibited their suppressive effect, suggesting that FCA-induced suppression might be due to soluble suppressor factor(s). On the other hand T cells from FCA treated LEW rats did not produce any modification of LEW in vitro response to WAG alloantigens. This suggests that the severe arthritis induced in LEW rats could be correlated with a defect of their suppressor cells functions, while in WAG rats FCA activated suppressor T cells could control the disease.     

Precipitation of experimental autoallergic uveoretinitis by cyclosporin A withdrawal: an experimental model of uveitis relapse.

This study set out to determine whether withdrawal of cyclosporin A (CyA) in Lewis rats sensitized to retinal S antigen would precipitate experimental autoallergic uveoretinitis (EAU), and whether challenge of such animals with S antigen or an unrelated stimulus would accelerate EAU onset after drug withdrawal. Rats were sensitized with 50 micrograms S antigen in Freund's complete adjuvant (FCA) and EAU onset was suppressed by 18 days of treatment with CyA at doses ranging from 3 to 10 mg/kg daily. Without challenge, seven out of 11 animals developed EAU with a median onset of 78 days. This was reduced to 68 days in rats challenged on day 32 with FCA alone, to 48 days with 10 micrograms S antigen in FCA, and to 41 days with 50 micrograms S antigen in FCA. The incidence, onset and severity of anterior uveitis and extent of photoreceptor destruction were related to both CyA dose and nature of challenge. The extent of photoreceptor destruction ran parallel with severity of anterior uveitis; and delayed-type hypersensitivity reactivity on day 43 was related to both severity of anterior uveitis (P less than 0.001) and photoreceptor damage (P less than 0.002). At the highest dose, CyA also delayed the appearance of antibody to S antigen; however, subsequent antibody levels were unrelated to EAU severity or to nature of challenge. The results indicate that CyA-induced suppression of the immunological response to S antigen can recover spontaneously after drug withdrawal, that challenge with either S antigen or FCA alone can accelerate the subsequent onset of EAU, and that these phenomena may provide a basis for investigating mechanisms underlying relapse of human uveoretinitis.     

Comparison of Corynebacterium Parvum With Freund's Complete Adjuvant as a Potentiator of the Immune Response To β-Human Chorionic Gonadotropin Linked to Tetanus Toxoid

ABSTRACT: Corynebacterium parvum was compared with Freund's complete adjuvant (FCA) for potentiation of the rabbit immune response to β-human chorionic gonadotropin linked to tetanus toxoid (β-hCG-TT). With each adjuvant, antibodies to hCG were detected using passive hemagglutination. Higher antibody titers were produced by FCA-treated animals. The ability of antisera to β-hCG-TT to neutralize the biological action of native hCG was determined by the rat uterine weight assay. Anti-β-hCG-TT sera from C. parvum-treated rabbits were not significantly different (p > 0.10) from FCA potentiated anti-β-hCG-TT sera in neutralizing hCG-induced uterine weight gain. C. parvum has potential for future application in active immunization studies of fertility regulation.     

Humoral and cellular immune response of the rat to immunization with bee venom.

Lewis rats were immunized with bee venom allergen in Freund's complete adjuvant (FCA) or with FCA only. Animals immunized with bee venom developed specific IgG antibodies but no specific IgE antibodies were detected. Lymphocytes from lymph nodes when cultured with antigen in vitro showed an increased stimulation index from day 17 onwards. A concomitant augmentation of T suppressor cells was observed; the T helper/T suppressor cell ratio declined from 4.5:1 before immunization to 1:1 from day 5 onwards.     

Resistance to the induction of EAE in AO rats: its prevention by the pre-treatment with cyclophosphamide or low dose of irradiation

Susceptibility to the induction of EAE was compared in AO, DA and Lewis strain of rats. As evaluated by clinical and histological criteria, AO rats exhibited significantly lower susceptibility to EAE induced with guinea-pig spinal cord (GPSC) tissue and complete resistance to the encephalitogenic challenge with rat myelin basic protein (BP) irrespective of antigen dose and adjuvant used. AO rats pre-treated with BP + Freund's incomplete adjuvant became completely unresponsive to the induction of EAE with GPSC + Freund's complete adjuvant (FCA) indicating that they do possess cells sensitive to some antigenic determinants of rat BP. In order to test whether the resistance to EAE is due to an active suppression, low dose of irradiation (300 rad) and cyclophosphamide (20 mg/kg) was applied prior to the induction of EAE. Selective depletion of radiosensitive cells facilitated the induction of EAE. Similarly, cyclophosphamide given 2 days prior to BP + FCA completely abrogated the resistance to EAE induction. Thus, it appears that the inability of BP + FCA to produce EAE in AO rats is due to the disproportionate activation of suppressor cells.     

Role of thymus for N-acetyl muramyl-L-alanyl-D-isoglutamine-induced polyarthritis and granuloma formation in euthymic and athymic nude rats or in neonatally thymectomized rats.

A synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP), produced extremely severe polyarthritis with almost 100% incidence in Rowett euthymic rnu/+ rats, but the same dose of MDP (100 microgram) did not produce the disease in athymic rnu/rnu rats. Five hundred micrograms of MDP or 0.2 mg of heat-killed Mycobacterium bovis BCG, however, produced mild and transient polyarthritis in nude rats with very low incidence. We have not yet succeeded in reconstituting the disease susceptibility of nude rats by using thymus cells from normal rnu/+ rats. After intradermal inoculation of 100 microgram of MDP, nude rats developed small granulomas with a little necrosis and very few multinucleated giant cells only in the regional lymph nodes, whereas, in addition to the development of polyarthritis, euthymic rnu/+ rats developed typical granuloma with massive necrosis accompanied by numerous polymorphonuclear leukocytes and sparse multinucleated giant cells in the regional lymph nodes. Thymus cell-reconstituted rnu/rnu rats developed granuloma with sparse giant cells, relatively large areas of necrosis, and many polymorphonuclear leukocytes. Neonatal thymectomy may depress adjuvant-induced arthritis in the high-responder Lewis rats and enhance the disease development in the low-responder F344 rats. These findings suggested that (i) thymus plays an important role in promoting the development of MDP-induced arthritis; (ii) MDP-induced granuloma formation does not require thymus functions; (iii) the thymus functions may however be involved in the development of massive necrosis surrounded by considerable polymorphonuclear leukocyte infiltration, the mechanisms of which remain to be determined; and (iv) there is no direct correlation between granuloma formation and development of adjuvant arthritis.     

新型佐剂SWZY对弱免疫原性小鼠黑色素瘤瘤苗的免疫增强作用

目的:评价佐剂SWZY对弱免疫原性黑色素瘤瘤苗的免疫增强作用。方法:C57BL/6小鼠分为6组,实验组分别用5种不同配方的佐剂(FCA,FCA IL-2 GM-CSF,FIA IL-2 GM-CSF,FIA SWZY,FIA SWZY IL-2 GM-CSF)和照射灭活的小鼠黑色素瘤细胞株D5制成瘤苗免疫小鼠,对照组免疫用不加任何佐剂的灭活D5黑色素瘤细胞。末次免疫后3d各组取半数动物检测DTH反应、脾细胞的杀伤活性以及免疫小鼠血清及脾细胞培养上清中IFN-γ和IL-10的水平,剩余半数动物接种未灭活的D5黑色素瘤细胞,3周后再次检测上述各免疫学参数。结果:与对照组小鼠比较,各实验组小鼠的DTH反应及脾细胞的杀伤活性均明显升高(P<0.05),但成瘤后随着肿瘤的增大,则呈下降趋势。成瘤前各实验组小鼠血清及脾细胞培养上清中IFN-γ的水平均高于对照组小鼠(P<0.05),但IL-10的水平均低于对照组小鼠。成瘤后各实验组及对照组小鼠血清及脾细胞培养上清中IFN-γ的水平均下降,而IL-10的水平均明显上升,其中FCA瘤苗组和FIA SWZY瘤苗组免疫小鼠的血清及脾细胞培养上清中IFN-γ的水平仍高于对照组小鼠(P<0.05),IL-10的水平仍低于对照组小鼠(P<0.05)。结论:用5种佐剂配方制成的瘤苗免疫小鼠均能诱导对弱免疫原性肿瘤的细胞免疫应答,并增强Th1型细胞免疫的应答,但随着肿瘤的形成和逐渐进展,细胞免疫应答的效应逐渐减弱。其中佐剂SWZY与FCA的作用相当,但前者的毒副作用较小,有可能成为一种新型的人用肿瘤疫苗的佐剂。     

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 hyper– sensitivity (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.     

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.     

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 hyper- sensitivity (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.     

Regulation of immune functions by sperm-specific LDH and its differences with somatic isozyme in primary and secondary lymphocyte cultures

PROBLEM: Sperm-specific lactate dehydrogenase-C4 (LDH-C4) is an autoantigen that produces experimentally induced autoimmune orchitis in testes. In the present study, immunological functions of B and T cells have been examined and compared after immunization with sperm-specific LDH and the LDH from somatic cells. METHODS: Three sets of experiments were performed. In the first set, effects of Balb/C LDH isozymes at 10(-3) - 1 L microg/well were investigated: (i) by mixed lymphocyte cultures (MLC) using C-57 B1/6 female cells as responders and AKR lymphocytes (irradiated) as stimulators, (ii) for regulatory T cell activity in MLC co-cultured along with Con-A-induced AKR lymphoblasts and (iii) for modulation of lymphocyte activation by PHA in vitro. In the second set of experiments, female mice (C-57 B1/6) were distributed in six groups for various treatments: i) saline (as vehicle), ii) adjuvant, iii) LDH-B4 (20 x 3 microg), iv) LDH-B4 (40 x 3 microg), v) LDH-C4 (20 x 3 microg), and v) LDH-C4 (40 x 3 microg). Mice were hyperimmunized with -B4 or -C4 (Balb/c) with a primary dose of 20 or 40 microg of protein per mouse, emulsified in Freund's complete adjuvant (FCA) and two identical doses in Freund's incomplete adjuvant (s.c.) within 22 days. Saline (group i) or adjuvant treated dams (group ii) served as controls. One week after the second booster, sera were tested for IgG response and lymphocytes harvested for polyclonal activation in vitro using LPS and Con-A as mitogens. In the third set of experiments, female Balb/c mice were divided into six groups as in the second experiment and immunized with a single primary dose of isogenic LDH-B4 or LDH-C4 at 20 or 40 microg of protein in FCA. On day 5, after sensitization with LDH, lymphocytes were evaluated for mitogenesis and for IgM production in vitro using LPS and Con-A as mitogens. RESULTS: i) Primary MLC(s) were non-specifically suppressed in the presence of 10(-3)- 1 L x microg allogenic LDH-C4 or -B4, although LDH-C4 tended to abolish MLC completely. But MLC co-cultured with blast cells was suppressed by LDH-C4 alone, indicating that sperm LDH suppresses induced formation of regulatory T cells. ii) FCA primed lymphocytes in situ were significantly inhibited for Con-A stimulation in vitro. Since LPS stimulation remained unaffected, it appeared that FCA is immunosuppressive for T cell proliferation alone. iii) Cells primed with LDH increased mitogenic activity of LPS several fold, although LDH-C4 was less effective than LDH-B4 in sensitization of B lymphocytes. iv) However, effect of Con-A in mitogenesis was dose-dependent, viz. cells primed at 20 x 3 microg of each isozyme overcame the immunosuppressive nature of FCA by bringing back the SI ( x 25) equivalent to saline primed cells, while pre-treatment of cells with 40 x 3 microg LDH-C4 abolished SI completely, indicating that -C4 primed cells were immunologically suppressed for Con-A stimulation. Such a response was markedly visible when allogenic LDH-C4 was used for hyperimmunization; lymphocytes challenged with somatic LDH under similar conditions did not react. Loss of T cell functions by LDH-C4 was confirmed in the presence of PHA in primary cultures. v) For antibody responses, although sperm LDH was highly reactive and dose-dependent, somatic LDH was also immunogenic for IgG production in serum to a lesser degree. Besides, IgM antibody was also discernible by two isozymes in LPS-induced cultures. Significantly, -C4 primed cells at the higher dose, in comparison with the lower dose, were less responsive for IgM production. CONCLUSIONS: It is concluded that LDH(s) from sperm and somatic cells share functionally related antigenic epitopes that can generate/modify immune responses in vivo and in vitro with qualitative differences. However, immunosuppressive determinant of LDH-C4 is cell specific and dose selective.     

Interaction between 'sensitized lymphocytes' and antigen in vitro. 3. Release of soluble mediators by particulate antigen

Guinea-pigs immunized with sheep red cells (SRC) in Freund''s complete adjuvant (FCA) developed delayed type hypersensitivity to a SRC urea extract 7 days after sensitization and showed combined Arthus and delayed reactivity from the 14th day on. Animals immunized by an intracardiac injection of SRC in saline and skin tested 4–5 days later with SRC urea extract, responded with an erythematous and indurated lesion reaching its peak 24 hr after testing and resembling a typical delayed hypersensitivity reaction. Lymphocytes from lymph nodes, peritoneal exudate and peripheral blood of guinea-pigs immunized with SRC in FCA released skin reactive factor (SRF) and macrophage migration inhibitory factor (MIF) when cultured for 24 hr with SRC. No SRF was formed in the absence of divalent cations in the cultured medium. No SRF or MIF was produced by lymphocytes of guinea-pigs immunized by the intracardiac route and cultured under identical conditions. SRF obtained after in vitro exposure of sensitized lymphocytes to SRC was eluted from Sephadex G-200 in the region of serum albumin. Lytic anti-SRC antibody of the IgM class was liberated into the supernatant of spleen cultures, after intracardiac immunization and antibody of the IgG class was produced by lymph node cultures after immunization with SRC in FCA, in both cases in the absence of antigenic stimulation in vitro. It is considered improbable that the latter antibody is involved in the release of soluble mediators.     

Modulatory effects of Freund's adjuvant treatment on mast cell histamine release and homocytotropic antibody synthesis

The present study examines the influence of Freund's complete adjuvant (FCA) injections on sensitized PVG rats with respect to serum levels of IgE and IgG2 alpha antibodies and total IgE (all assessed by radioimmunoassays) and the capacity of serosal mast cells to release histamine on challenge in vitro with 'immunological' secretagogues (specific antigen, anti-IgE, concanavalin A) or with compound 48/80. The rats were immunized with 10 micrograms ovalbumin (OA); alum, Bordetella pertussis vaccine, or silica gel were employed as adjuvants. Treatment with FCA was performed by single intraperitoneal injections 3, 2, or 1 week(s) before or 1 or 2 weeks after sensitization. Tests were conducted 3 weeks after sensitization. The results show that the effect of FCA treatment varied reproducibly with the adjuvant employed for sensitization and with the timing of the FCA administration. FCA treatment could either increase, fail to affect, or decrease total serum IgE and OA-IgG2 alpha antibody levels as well as serosal mast cell responsiveness, whereas OA-IgE antibody responses were decreased or not affected. Moreover, serum levels of OA-IgE and OA-IgG2 alpha antibodies and total IgE were affected by FCA treatment independently of each other. Finally, serosal mast cell responsiveness to a given secretagogue could be influenced by the FCA treatment apparently independently of that to other secretagogues. A salient finding was that effects of FCA treatment on mast cell responsiveness did not necessarily conform to effects on antibody synthesis. Collectively, these data support the opinion that the mechanisms of action of the IgE-promoting adjuvants employed differ and suggest that the expression of serosal mast cell responsiveness to each examined secretagogue can be regulated separately. They also suggest that the serosal mast cell sensitizing capacity of homocytotropic antibodies may not be adequately quantified by immunochemical methods employing reagents prepared against IgE and IgG2 alpha protein.     

Experimental production of pulmonary granulomas; II. Age dependency and immune modulation of granuloma production.

Endobronchial instillation of Freund''s complete adjuvant (FCA) induced epithelioid cell granulomas in the lungs of juvenile rabbits which had been kept free from contamination with the microbiol antigens. The granulomas were named "juvenile granulomas", because, unlike FCA granulomas in adult animals, prior immunization was unnecessary for their induction. The granulomas developed in several weeks with a peak at 14 weeks of age, after which the production decreased gradually. The rate of granuloma production seemed to vary with the acquisition of skin hypersensitivity to tuberculin (OT), suggesting that granuloma production, as well as skin hypersensitivity, is in the category of T-dependent immune reactions. In fact, T-generating lymphoid organs developed in parallel with the dermal and pulmonary reactions. Thus, juvenile rabbits at about 14 weeks of age are most susceptible to the microbial antigens. This susceptibility results in the unexpected production of immune granulomas in response to depot antigens at the site of instillation. The treatment of foetal or neonatal rabbits with FCA markedly suppressed granuloma production in juveniles but not in adults and did suppress but gradually enhanced the tuberculin skin reaction. It is suggested that generation of suppressor T cells is the cause of suppression of juvenile granuloma production.