Chrysotile A affects YAC-1 cytolytic activity of spleen cells

Effect of short exposure of C57Bl/6 and F1/NZB x C57Bl/6/ mice to i.p. injected chrysotile A on YAC-1 cytolytic potential (NK cell function) of spleen cells was investigated. It was found that 3 days after injection, cytolytic activity of spleen cells was significantly abrogated and this was paralleled with an increase of alpha-naphtyl acetate esterase positive cells/macrophages in spleen cell population. In addition, effect of exposure of mice to i.p. injected chrysotile A coated with benzo(a)pyrene (BaP) was investigated. BaP did not modify the abrogative effect of chrysotile A on NK cell function. Asbestos fibres themselves were sufficient for depletion of NK cell function of spleen cells.     

Protective efficacy of a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

We reported previously that a detergent extract of the cell wall and cell membrane of Histoplasma capsulatum yeast cells contains antigens recognized by T cells. In T-cell immunoblot analysis, a region encompassing 62 kDa was stimulatory for an H. capsulatum-reactive T-cell line and T-cell clones derived from C57BL/6 mice. In this study, we isolated a 62-kDa band, termed HIS-62, from electrophoresed cell wall and cell membrane of H. capsulatum yeast cells and examined its antigenicity and immunogenicity. C57BL/6, BALB/c, and CBA/J mice that were immunized with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to HIS-62 that was stronger than that of normal controls. Spleen cells from each strain of mouse immunized with viable yeast cells proliferated vigorously in response to HIS-62; conversely, splenocytes from control animals did not recognize this antigen. A T-cell line and 5 of 5 T-cell clones from C57BL/6 mice, 10 of 15 BALB/c T-cell hybridomas, and 8 of 12 CBA/J T-cell hybridomas recognized HIS-62. A cutaneous delayed-type hypersensitivity response to the antigen was apparent in each strain of mouse that was injected with 80 micrograms of HIS-62 mixed with Freund adjuvant. In addition, spleen cells from HIS-62-immunized mice proliferated in vitro in response to this antigen. Vaccination of each strain of mouse with 80 micrograms of HIS-62 conferred protection against a lethal intravenous challenge with H. capsulatum yeast cells. Thus, HIS-62 appears to be an important target of the cellular immune response to H. capsulatum and induces a protective immune response in mice.     

Effects of gonadotrophin on resistance to Listeria monocytogenes infection in mice.

It was demonstrated that exogenous GH suppressed the resistance to L. monocytogenes infection in Listeria resistant C57Bl/6 and susceptible A/J mice. However, different parameters of the immunological reaction to Listeria were affected by GH treatment in these mouse strains. In C57Bl/6 mice GH decreased accumulation of macrophages at the inflammatory site. On the contrary, a depression of anti-listerial activity of the phagocytes and a reduction of DTH reaction to Listeria antigen was demonstrated in GH treated A/J mice.     

Characteristics of cell mediating spontaneous resistance to bone marrow allografts

Mice of most strains show a genetically determined ability to reject foreign bone marrow grafts even after lethal irradiation. The effector cells mediating this resistance are spleen localised, lymphoid in morphology, and rapidly renewing. Two additional aspects of these cells were characterized in the present study, i.e., their sensitivity to: 1) anti-T cell and -B cell antibodies, and 2) the immunoregulator PGE2. T and B cell depleted splenocytes, or splenocytes from PGE2 pre-treated, highly resistant mice (C57Bl/6J) were transferred into irradiated, non-resistant secondary host mice (129/J) to establish whether or not they would retain their normally strong ability to spontaneously reject a third party (DBA/2) bone marrow allograft. In all cases, bone marrow seeding was assayed by enumerating the spleen colonies present in the secondary host 7 days after bone marrow grafting. Irradiated 129/J mice injected with low numbers (20 x 10(6) of untreated C57Bl/6J spleen cells prior to DBA/2 bone marrow contained 27.8 +/- 1.9 colonies/spleen, while mice injected with the same number of T and B depleted C57Bl/6J spleen cells contained significantly fewer (16.3 +/- 1.7) colonies/spleen. On the other hand, when C57Bl/6J spleen cells were incubated overnight with PGE2 prior to injection (20 x 10(6) into the 129/J hosts, they were unable to prevent the development of numerous colonies upon challenge with the DBA/2 bone marrow allograft (31.5 +/- 1.5 colonies/spleen: PGE2 pre-treated vs 16.9 +/- 2.1 colonies/spleen: non-pre-treated, control). The results demonstrate firstly, that spleen cells depleted of both Thy-1- and Ig-bearing cells appear to have concentrated for the effector cells responsible for bone marrow allograft rejection, and secondly, such effector cells could be suppressed by short-term exposure to PGE2, resulting in successful "take" of the bone marrow allograft.     

By-stander activation in autoimmune thyroiditis: studies on experimental autoimmune thyroiditis in the GFP+ fluorescent mouse

We have taken advantage of GFP+ fluorescent protein (GFP) tagged lymphocytes to examine by-stander activity in experimental autoimmune thyroiditis in the mouse. To generate GFP-positive EAT-susceptible CBA/J mice (H-2k) (GFP-CBA/J mice), we backcrossed CBA/J (H-2k) with heterozygous GFP+ transgenic mice (C57Bl/6; H-2b). I-Ak and GFP expression on peripheral lymphocytes was used to select the resulting progeny up to the N7 generation. Mixed lymphocyte reactions using spleen cells from N7 GFP-CBA/J mice showed negative responses to spleen cells from CBA/J confirming the inbreeding and with marked reactivity to cells from C57BL/6. Immunization with human thyroglobulin (hTg) in GFP-CBA/J mice induced thyroiditis in 50% of the animals and high titers of Tg antibodies in all the animals. In addition, priming of GFP+ spleen cells in vitro with hTg induced a marked proliferative response (mean stimulation index = 24.7), These proliferating spleen cells were then transferred to CBA/J recipients. Fourteen days after transferring 30 x 10(6) Tg-primed GFP+ spleen cells into irradiated (500 rad) normal syngeneic hosts, a GFP+ lymphocytic infiltration was seen within their thyroid glands along with a GFP- lymphocytic infiltration arising from the host. This suggested that the hTg-specific transferred cells had initiated by-stander activation of naive host lymphocytes. This model of bystander cell detection confirmed that such an effect occurs in EAT and adds weight to the importance of this phenomenon in the initiation of autoimmune thyroid disease.     

IL-21R expression on CD8+ T cells promotes CD8+ T cell activation in coxsackievirus B3 induced myocarditis

IL-21 is a multi-functional cytokine which can promote survival, proliferation and activation of T and B lymphocytes including CD8 T cells. Previous studies have shown that autoimmune CD8+ T cells are the primary pathogenic effector cell in coxsackievirus B3 (CVB3) induced myocarditis in C57Bl/6 mice. To evaluate the role of IL-21 in promoting CD8+ T cell mediated cardiac injury in myocarditis, C57Bl/6 and IL-21RKO mice were infected with CVB3. IL-21RKO mice developed significantly less myocarditis than C57Bl/6 animals although cardiac virus titers were equivalent between the mouse strains. Numbers of CD8+IFNγ+ cells were decreased in IL-21RKO mice but numbers of either CD4+IFNγ+ or CD4+IL-4+ cells were not significantly different from C57Bl/6 animals indicating a selective effect of IL-21 signaling on the CD8+ T cell response. To confirm that IL-21 signaling exclusively functions at the level of the CD8+ T cell in CVB3 induced myocarditis, purified CD8+ cells were isolated from either C57Bl/6 or IL-21RKO donors and adoptively transferred into CD8KO recipients prior to CVB3 infection. CD8KO recipients given either C57Bl/6 or IL-21RKO CD8+ cells showed equivalent reconstitution of the CD8+ cells in the spleen but the recipients given C57Bl/6 CD8+ cells showed significantly greater myocarditis than recipients of IL-21RKO CD8+ cells. These data demonstrate that IL-21 signaling directly in the CD8+ cell population is required for CVB3-induced myocarditis.     

Stimulation of the mouse monocytes by Listeria monocytogenes

The bacteria of Wellshimer's strain L. monocytogenes and their extract (LMA) showed in vitro the mitogenic activity which was demonstrated by increased proliferation of normal bone marrow cells and blood monocytes of the mouse strains resistant (C57Bl/6) and susceptible (DBA/2) to listeriosis. In the same experimental conditions the proliferation of spleen macrophages and resident peritoneal macrophages was not influenced. Besides, C57Bl/6 bone marrow cells, activated by live or killed L. monocytogenes, produced some growth factor for secondary bone marrow cell cultures.     

In vitro and in vivo autoimmune response to enamel matrix proteins.

Enamel matrix proteins taken from neonatal C57Bl/6 mice were shown to be able to elicit in vitro proliferative responses in C57Bl/6 splenic lymphocytes taken from animals which had not been exposed to exogenous enamel proteins. Animals which had been injected with enamel matrix proteins in Freund's complete adjuvant made IgG antibodies against enamel proteins which were detected by indirect immunofluorescence. Spleen cells from the immune animals gave an augmented in vitro proliferative response to enamel proteins, while spleen cells from C57Bl/6 nu/nu mice or anti-Thy 1 and complement-treated normal C57Bl/6 spleen cells were incapable of responding to enamel proteins in vitro. Thus, enamel matrix proteins appear to be T-cell dependent autoantigens. The natural history of enamel matrix proteins is reviewed, and it is suggested, based on the anatomic details of enamel synthesis, secretion, and maturation, that enamel matrix proteins are autoantigenic because they are anatomically sequestered.     

Endogenous dipeptide cycloprolylglycine shows selective anxiolytic activity in animals with manifest fear reaction

Experiments on two mouse strains with opposite reactions to emotional stress showed selectivity of the anxiolytic effect of endogenous dipeptide cycloprolylglycine. In the open field test cycloprolylglycine (0.01-0.10 mg/kg intraperitoneally) dose-dependently (1.8-2.1-fold) increased motor activity of BALB/c mice with manifest fear reaction and had no effect on C57Bl/6 mice with active behavior. The content of endogenous cycloprolylglycine in mouse brain correlated with the type of emotional stress reaction: its content in the brain of C57Bl/6 mice 1.5 times surpassed that in BALB/c mice. It is concluded that cycloprolylglycine is involved in the endogenous regulation of fear reaction.     

Immune reactions in different mouse strains

The responses of immunocompetent cells to thymus-dependent antigen differ in mice of different strains. Immunization stimulated phagocytic activity of peritoneal macrophages in CBA/CaLac, DBA/2, and BALB/c mice and suppressed it in CC57W mice. By the formation of antibody-producing cells in the spleen in response to thymus-dependent antigen DBA/2 and CBA/CaLac mice can be classified as high responders, BALB/c mice as medium-responders, and C57Bl/6 and CC57W mice as low responders.     

Inhibition of Migration of Mouse Macrophages by Tuberculin-Sensitive Mouse Lymphocytes and by Mouse Migration Inhibitory Factor

The guinea pig migration inhibition technique, an accepted in vitro correlate of delayed hypersensitivity, has been adapted to a murine system. Peritoneal exudate cells from CF-1 mice vaccinated with viable cells of the H37Ra strain of Mycobacterium tuberculosis were inhibited in vitro by purified protein derivative (PPD) or whole H37Ra microorganisms. Peritoneal exudate cells from the inbred C57Bl/6 mice immunized with H37Ra cells also were inhibited in vitro by PPD or whole H37Ra microorganisms. Migration inhibitory factor (MIF) was produced by splenic lymphocytes from the H37Ra-immunized C57Bl/6 mice when incubated with either antigen. Intravenous injection of PPD or viable H37Ra organisms into H37Ra mice resulted in MIF production in vitro by splenic lymphocytes without further antigenic stimulation. Peritoneal exudate cells from nonimmunized C57Bl/6 mice and supernatant fluids from cultures of lymphocytes from nonimmunized C57Bl/6 mice were not inhibited in the presence of antigen. The production of MIF by splenic lymphocytes from immunized C57Bl/6 mice depended upon the conditions under which the lymphocytes were cultured, the time of exposure to antigen (3 days), the use of a higher concentration of PPD for stimulation of lymphocytes than that required for guinea pig cells, and also the use of cells from a highly inbred mouse strain.     

Alveolar hydatid cyst induced amyloid enhancing factor (AEF): physicochemical properties and abolition of AEF activity by serine protease inhibitors

Cell free supernatants prepared from amyloidotic liver, unfractionated and fractionated peritoneal and spleen cells from casein stimulated (16 h post-injection) or alveolar hydatid cyst-infected (7 or 12 weeks post-infection, p. i.) C57BL/6J mice were used to assess amyloid enhancing factor (AEF) activity and to determine its cell-source and physicochemical properties. Of the various supernatants used, the plastic adherent spleen cell lysate (95% macrophages) from 7 weeks p.i mice showed greater AEF activity, on a cell to cell basis, than the lysates prepared from whole spleen cells, peritoneal exudate cells or nonadherent (96% lymphocytes) spleen cells. Culture supernatants obtained from Con A, LPS or the parasite antigen stimulated amyloidotic spleen cells but not the normal mouse spleen cells contained AEF activity; the supernatant from unstimulated amyloidotic spleen cells was negative for AEF activity. AEF was precipitated with 25% and 50% saturation with (NH4)2SO4 and after gel-filtration the low molecular weight fraction contained the AEF activity which on SDS-PAGE resolved into three peptides ranging between mol. wts 15,000 and 31,000. Of the various specific and nonspecific protease inhibitors tested, AEF activity was completely abolished by 30 min preincubation with 10 mM phenylmethylsulphonyl fluoride. Taken together, these results indicate that AEF may be a small molecular weight lysosomal neutral protease of neutrophil/macrophage origin.     

Alveolar hydatid cyst induced amyloid enhancing factor (AEF): physicochemical properties and abolition of AEF activity by serine protease inhibitors.

Cell free supernatants prepared from amyloidotic liver, unfractionated and fractionated peritoneal and spleen cells from casein stimulated (16 h post-injection) or alveolar hydatid cyst-infected (7 or 12 weeks post-infection, p. i.) C57BL/6J mice were used to assess amyloid enhancing factor (AEF) activity and to determine its cell-source and physicochemical properties. Of the various supernatants used, the plastic adherent spleen cell lysate (95% macrophages) from 7 weeks p.i mice showed greater AEF activity, on a cell to cell basis, than the lysates prepared from whole spleen cells, peritoneal exudate cells or nonadherent (96% lymphocytes) spleen cells. Culture supernatants obtained from Con A, LPS or the parasite antigen stimulated amyloidotic spleen cells but not the normal mouse spleen cells contained AEF activity; the supernatant from unstimulated amyloidotic spleen cells was negative for AEF activity. AEF was precipitated with 25% and 50% saturation with (NH4)2SO4 and after gel-filtration the low molecular weight fraction contained the AEF activity which on SDS-PAGE resolved into three peptides ranging between mol. wts 15,000 and 31,000. Of the various specific and nonspecific protease inhibitors tested, AEF activity was completely abolished by 30 min preincubation with 10 mM phenylmethylsulphonyl fluoride. Taken together, these results indicate that AEF may be a small molecular weight lysosomal neutral protease of neutrophil/macrophage origin.     

Listeria antigen-binding cells in the spleens of normal and immunized mice resistant or susceptible to listeriosis

Normal mice of the Listeria-resistant C57Bl/6 strain contain in their spleens a higher number of cells that bind Listeria monocytogenes cell wall fraction antigen (LmA) than normal DBA/2 mice, which are more susceptible to infection. LmA-binding cells are probably B cells, nylon-wool adherent, and inhibited by anti-mouse immunoglobulin antibody but not sensitive to the action of monoclonal anti-mouse macrophage and anti-Thy.1.2 antibody. A single intraperitoneal injection of 10(8) Listeria monocytogenes causes a rapid increase in the number of LmA-binding cells in the spleens of C57Bl/6 mice, and this can be seen as early as 24 h. On the other hand, in DBA/2 mice an increase in these cells becomes evident only by the 4th day. Moreover, the increment in the number of LmA-binding cells in C57Bl/6 mice is more marked than in DBA/2 mice.     

Influenza virus host response of C57Bl/6 mice treated with TACI-Ig

TACI-Ig is a soluble glycoprotein comprised of a human IgG₁-Fc fused with the extracellular domain of the human TACI receptor. Chronic exposure to TACI-Ig is associated with reduced circulating B cells in mouse and non-human primates, and a concomitant decrease in circulating immunoglobulin. Because of these activities, TACI-Ig is in clinical evaluation for treatment of various autoimmune diseases and B cell malignancies. In this study, the effect of TACI-Ig treatment on the ability of C57Bl/6 mice to clear influenza virus was evaluated. C57Bl/6 mice were exposed to vehicle (negative control), dexamethasone (positive control), or TACI-Ig (0.05, 0.50, or 5.0 mg/kg, SC, thrice weekly) from within one week prior to viral exposure through 21 days thereafter. Dexamethasone treatment of influenza-infected mice prolonged the infection, and decreased survival, body weight, lymphoid organ weight, influenza-specific IgM and IgG, and viral clearance relative to control animals, consistent with its expected immunosuppressive activity. Animals treated with TACI-Ig (0.05, 0.50, and 5.0 mg/kg) demonstrated a dose-dependent decrease in spleen weight and influenza-specific IgG and IgM in both lung and serum relative to control animals. In addition, flow cytometric analyses showed a decrease in B cells, but not T cells, in peripheral blood in animals treated with TACI-Ig. However, neither viral clearance nor survival was affected by TACI-Ig treatment. These data demonstrate the expected B cell-specific pharmacological effects of TACI-Ig in influenza-challenged C57Bl/6 mice without apparent effect on influenza virus clearance. It is concluded that non-B cell related antiviral competence remains intact during TACI-Ig treatment.     

The relation between the T cells responsible for cell-mediated cytotoxic killing of mastocytoma cells and the helper-cell effect.

The relationship between T cells involved in cell-mediated immunity and antibody response of C57Bl/6J mice towards DBA/2 mastocytoma cells was investigated. Spleen cells primed with viable mastocytoma cells demonstrated marked cell-mediated cytotoxicity (CMC) in vitro, but antibody response of these cells to a hapten (TNP) conjugated to the allogeneic tumour cells in vitro was suppressed as compared with that of normal spleen cells. In contrast, spleen cells primed with frozen-thawed mastocytoma cells showed no CMC, but antibody response to the hapten in vitro of these cells was enhanced. C57Bl/6J mice primed with frozen-thawed mastocytoma cells produced more cytotoxic antibody than non-primed mice when immunized with viable mastocytoma cells. These results indicate that T cells involved in cell-mediated immunity and those involved in the antibody response to allogeneic mastocytoma cells are distinct.?222     

Effect of lipopolysaccharide (LPS) on the thymocyte response to PHA: strain difference.

Thymocytes responded well to PHA in terms of [3H]-TdR incorporation if they were precultured in the presence of LPS, whereas fresh thymocytes or thymocytes pre-cultured in the absence of LPS responded poorly to PHA. The PHA response of thymocytes pre-cultured with LPS for several hours was similar to that of fresh spleen cells in the kinetics and dose-response profiles. The effect of LPS was found on thymocytes from BALB/c.Cr, AKR/Jms, and DDD/1 mice, but was not observed on those from C3H/HeJms and C57Bl/6J mice, indicating that there is a strain difference in the PHA responsiveness of LPS-pre-cultured thymocytes. In contrast to thymocytes, the response of spleen cells to PHA was enhanced in both C57Bl/6J and BALB/c.Cr mice by pre-culture with LPS.     

Influence of an M-Locus Difference on the Cellular and Humoral Immunological Reactivity Against H-2-Determined Antigens of the Mouse

Studies were initiated to determine whether an immune response to the Mls antigen of C3H mice could modify responses of CBA lymphocytes (H-2-compatible) to a foreign H-2 complex. CBA lymphocytes, partially tolerant to the C5H-determined Mls antigen, generated less effector cell activity against C57Bl cells (H-2-incompatible) Chan lymph node cells from normal CBA donors when infused into irradiated C.3H × C57Bl hosts. Effector cell activity was measured as the capacity of the cells in the irradiated spleens to inhibit CBA × C57Bl bone marrow proliferation. In contrast, immunization of CBA mice with C3H × C57Bl cells yielded lower antibody titers against C577Bl cells than immunization with CBA × C57Bl cells. Furthermore, preinjection of CBA mice with C3H × CBA cells strongly reduced the capacity of the animals to produce antibodies against C57Bl cells. Thus, these data support the conclusion that an immune response to a foreign Mls antigen may either enhance or suppress an immune response to H-2-incompatible cells.     

Morphofunctional characteristic of the immune system in BALB/c and C57BL/6 mice

Inbred animals serve as an adequate model to study the role of genetic factors in adaptive, disadaptive, and pathological processes. Morphofunctional study of the immune system was performed on intact BALB/c and C57Bl/6 mice. The structural and functional parameters of the immune system in BALB/c and C57Bl/6 mice differ under physiological conditions. In BALB/c mice, volume density of T zone in the spleen and production of IL-2, IL-3, IL-4, IL-10, and TNF-α were much higher than in C57Bl/6 mice. However, IL-12 production in BALB/c mice was lower than in C57Bl/6 mice. C57Bl/6 mice were characterized by higher cytostatic activity of splenic NK cells. The observed interstrain differences are genetically determined and contribute to the type of adaptive processes and different sensitivity of these mice to pathogenic agents.     

Experimental African trypanosomiasis: differences in cytokine and nitric oxide production by macrophages from resistant and susceptible mice.

Immunosuppression in experimental infections with Trypanosoma congolense is mediated by the synergistic action of macrophages and a novel lymphocyte(s), which involves the activity of IFN-gamma as well as IL-10. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant to T. congolense infections. Plasma and/or supernatants of spleen cell cultures of infected susceptible BALB/c mice have more IL-10 but less IL-12 than those of infected relatively resistant C57Bl/6 mice. Cells of a BALB/c macrophage cell line, when pulsed with T. congolense, produce more IL-10 and IL-6, but have less TNF-alpha mRNA, than equally treated cells of a C57Bl/6 cell line. Peritoneal and/or bone marrow-derived macrophages obtained from BALB/c mice, pulsed with T. congolense in culture, produce less nitric oxide, TNF-alpha and IL-12, but more IL-6 and IL-10 than equally treated macrophages isolated from C57Bl/6 mice. We suggest that genetic resistance to African trypanosomiasis is expressed at the level of the macrophage.