Cellular mechanism of endotoxin unresponsiveness in C3H/HeJ mice.

B cells from C3H/HeJ mice fail to respond to an endotoxin (LPS K235) which is mitogenic for normal mice including the closely related C3H/HeN strain. The cellular basis for this unresponsive state has been investigated. The C3H/HeJ mice have normal numbers of B cells, which are capable of normal responses to other B cell mitogens, such as polyinosinic acid (Poly I). Addition of normal macrophages or spleen cells fails to reconstitute the normal response. Furthermore, neither macrophages nor spleen cells from the C3H/HeJ strain suppress the normal C3H/HeN spleen cells. Finally, spleen cells enriched for B cells by the removal of macrophages or T cells demonstrate the same differences in responsiveness to LPS. These results indicate that LPS unresponsiveness is a defect of the B cell itself and not due to suppressor cells or the absence of helper cells. When LPS is added to Poly I-stimulated cultures, there is additional enhancement of the response of normal C3H/HeN spleen cells. However, LPS causes a dose-dependent suppression of the Poly I response of C3H/HeJ spleen cells. This suppression is dependent on the time of addition of LPS to the Poly I-stimulated cultures. These data are interpreted as indicating that the binding of LPS to the membrane of C3H/HeJ B cells results in their inactivation or suppression, and that this is the basis of LPS unresponsiveness in this mouse strain.     

Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice.

Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.     

Macrophage stimulation by bacterial lipopolysaccharides. III. Selective unresponsiveness of C3H/HeJ macrophages to the lipid A differentiation signal.

Peritoneal macrophages from the endotoxin-unresponsive C3H/HeJ substrain of mice were entirely refractory to activation in vitro by protein-free LPS, a defect that was not overcome by co-culture of spleen cells from the responder C3H/St substrain with LPS resistant C3H/HeJ macrophages. The defect in responsiveness appears confined to the lipid A activation signal since C3H/HeJ macrophages were fully activated after in vitro treatment by lipid A protein (LAP)--LPS complex, isolated LAP, and BCG. Moreover, after exposure to allogeneic tumor cells in vivo, C3H/HeJ macrophages were cytotoxic for tumor target cells in vitro. By contrast, macrophages from the responder C3H/St strain were fully activated by protein-free LPS to become cytolytic for tumor cells in vitro. C3H/HeJ macrophages, therefore, exhibit a highly selective defect characterized by unresponsiveness to the lipid A activation signal of protein-free LPS and resistance to the toxic effects of high concentrations of LPS that were lethal to the responder C3H/St strain.     

The genetic mapping of a defective LPS response gene in C3H/HeJ mice.

The expression of a defective LPS response gene Lps and the major urinary protein (Mup-1) are concordantly inherited in backcross (C3H/HeJ x C57BL/6J)F1 x C3H/HeJ mice, indicating genetic linkage of these loci. Mup-1 is known to be linked to the brown coat color locus on chromosome 4 in mice; thus Lps can now be assigned to chromosome 4. A value of 0.06 +/- 0.02 has been estimated for the recombination frequency between Mup-1 and Lps. We have used the polysyndactyly (Ps) mutation further to localize Lps on chromosome 4. Lps is located between the Mup-1 and Ps loci.     

Restoration of LPS responsiveness of C3H/HeJ mouse lymphocytes by microinjection of cytoplasmic factor(s) from LPS-stimulated normal lymphocytes

Cytosol fractions of lymphocytes from LPS-responder mice (C3H/HeN) were prepared and injected into B lymphocytes of LPS-nonresponder mice (C3H/HeJ) by a microinjection technique utilizing polyethyleneglycol-mediated cell fusion. The B lymphocytes of C3H/HeJ mice microinjected with cytosol prepared from LPS-stimulated C3H/HeN cells became normally responsive to LPS. Microinjection of cytosol itself did not stimulate C3H/HeJ cells to proliferate or differentiate into immunoglobulin-producing cells, and the cells injected with cytosol had to be restimulated with LPS in order to proliferate and differentiate. These data suggested that C3H/HeJ B cells acquired LPS responsiveness by microinjection of cytoplasmic factor(s) from LPS-stimulated C3H/HeN cells and that these factor(s) may be one of the components involved in normal signal transmission from cell surface to nucleus in the early stages of the LPS response. The apparent m.w. of the cytoplasmic factor(s) is 100,000 by gel filtration. Chromatofocusing analysis suggested that these factors may consist of two components with the same m.w.     

Serum from LPS nonresponder C3H/HeJ mice does not support the formation of functional EAC reagents.

Serum from C3H/HeJ mice in contrast to serum from other mouse strains does not convert EA into EAC. A factor in supportive serum permits nonsupportive C3H/HeJ serum to produce a functional EAC-rosetting reagent. This factor is heat stable. Its concentration in serum parallels the sensitivity of mice to LPS. It is absent or inoperative when sensitivity is reduced on a genetic basis and increased when sensitivity is increased by treatment with BCG.     

Structural features involved in the mitogenic activity of bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice

Abstract Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lps^d at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant ‘rough’ strain (A100) of Bordetella pertussis . Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lps^d spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lps^d splenocytes, whereas lipid A alone can stimulate Lpsⁿ spleen cells.     

The effects of LPS on the cellular composition of the splenic white pulp in responder C3H/He and non-responder C3H/HeJ mice

The aim of the present study was to compare the effects of LPS on the cellular composition of the splenic white pulp in responder C3H/He and non-responder C3H/HeJ mice. The present results show that an intravenous injection of LPS in C3H/He mice results in a number of prominent changes in the histology of the spleen, but none of these histological changes could be demonstrated in the unresponsive C3H/HeJ mice. However, the present study shows that LPS administration resulted in the disappearance of previously trapped immune complexes from the follicles in both responder C3H/He and non-responder C3H/HeJ mice. The significance of this phenomenon is discussed. The localization of intravenously injected LPS in both mouse strains was compared using an immunoperoxidase technique. Most of the injected LPS was taken up by marginal zone macrophages at 2 h after administration. No major differences could be detected in the localization pattern of LPS between C3H/He and C3H/HeJ mice. The present results support the suggestion that the genetically based unresponsiveness of C3H/HeJ mice could be due to an intracellular defect in their response to LPS.     

Heat treatment increases the incidence of alopecia areata in the C3H/HeJ mouse model

Alopecia areata (AA) is a common autoimmune disease characterized by non-scarring hair loss. Previous studies have demonstrated an association between AA and physiological/psychological stress. In this study, we investigated the effects of heat treatment, a physiological stress, on AA development in C3H/HeJ mice. Whereas this strain of mice are predisposed to AA at low incidence by 18 months of age, we observed a significant increase in the incidence of hair loss in heat-treated 8-month-old C3H/HeJ mice compared with sham-treated mice. Histological analysis detected mononuclear cell infiltration in anagen hair follicles, a characteristic of AA, in heat-treated mouse skin. As expected, increased expression of induced HSPA1A/B (formerly called HSP70i) was detected in skin samples from heat-treated mice. Importantly, increased HSPA1A/B expression was also detected in skin samples from C3H/HeJ mice that developed AA spontaneously. Our results suggest that induction of HSPA1A/B may precipitate the development of AA in C3H/HeJ mice. For future studies, the C3H/HeJ mice with heat treatment may prove a useful model to investigate stress response in AA.     

Limiting dilution analysis of TNF producing cells in C3H/HeJ mice

A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.     

Calcium ionophore A23187 does not stimulate lipopolysaccharide nonresponsive C3H/HeJ peritoneal macrophages to produce interleukin 1

C3H/HeJ mice are hyporesponsive to the biologic effects of bacterial lipopolysaccharide (LPS). The defect in the strain of mice is believed to be due to the expression of a mutant allele designated Lpsd at the chromosome four locus. The molecular basis of this hyporesponsiveness is not known, but it may result from some defective membrane signal transductions. To examine this possibility, we compared the abilities of interleukin 1 (IL-1) production by C3H/HeJ macrophages with those by C3H/He macrophages (LPS responsive) after stimulation with the calcium ionophore A23187 or phorbol myristate acetate (PMA). A23187 induced IL-1 production by C3H/He macrophages, but it did not induce IL-1 production by C3H/HeJ macrophages and neither did LPS. However, it had the ability to increase intracellular free Ca2+ in C3H/HeJ macrophages as well as in C3H/He macrophages, this being examined by the changes in cytosolic Ca2+ in the macrophages by using Quin 2. In contrast, PMA was able to induce IL-1 production by both C3H/He and C3H/HeJ macrophages without increasing intracellular Ca2+. Since polymyxin B did not inhibit A23187- or PMA-induced IL-1 production by C3H/He macrophages, these results are not due to the little amount of LPS in culture medium, but due to their own characteristics. A calmodulin antagonist W-7 effectively inhibited A23187-induced IL-1 production by C3H/He macrophages. However, it hardly inhibited LPS-induced IL-1 production except at high concentration, and it caused no inhibition of the PMA-stimulated one. These results suggest that the blocking sites expressed phenotypically by the Lpsd are shared by LPS- and A23187-stimulated cellular processes, although the actions of LPS and A23187 are different from each other. In addition to the direct study with LPS or lipid A, A23187 should provide another useful approach to clarify the molecular mechanisms of Lpsd defect in C3H/HeJ macrophages.     

Vascular alterations in response to air desiccation injury in the carotid artery of the C3H/HeJ mouse

The purpose of this study was to develop a model of vascular injury in 8-week-old C3H/HeJ mice (weight, 25 to 30 g) by using air desiccation. The carotid arteries were excised 1 to 8 weeks postinjury and evaluated by Verhoeff's stain and immunocytochemistry. In the first group of mice studied (n = 107), neointimal formation occurred and peaked at Day 14. In addition, medial cell division (measured by bromodeoxyuridine labeling) peaked at Day 3, whereas intimal cell proliferation increased gradually throughout the experimental period of 21 days. In addition, extensive thrombus formation occurred within 3 days after injury. The next experiment involved 124 mice and evaluated the effect of anticoagulants on the neointimal and thrombotic response. Mice received aspirin, heparin, or vehicle-only time-release pellets. Both anticoagulants significantly decreased the neointimal and thrombotic responses. The results of this study validate our animal model as being consistent with the Response to Injury Hypothesis of atherogenesis.     

Prevention and treatment of alopecia areata with quercetin in the C3H/HeJ mouse model

Alopecia areata (AA) is an autoimmune non-scarring hair loss disorder. AA can be acute, recurrent, or chronic. Current therapeutic options for AA are limited, and there is no effective prevention for recurrent AA. We have previously shown a correlation between the expression of HSP70 (HSPA1A/B), a heat shock protein involved in the inflammatory response, and the onset of AA in the C3H/HeJ mouse model. In this study, we tested the effects of quercetin, a bioflavonoid with anti-inflammatory properties, on AA development and HSP70 expression in the C3H/HeJ model. Mice with spontaneous AA were treated with subcutaneous quercetin or sham injections. Hair regrowth was observed in lesional areas in all the quercetin-treated mice, but in none of the sham-treated mice. In addition, non-alopecic C3H/HeJ mice were heat-treated to induce alopecia, along with quercetin or sham injections. Whereas 24% of the heat-treated mice with sham injections developed alopecia, none of the mice receiving quercetin injections did. As expected, the level of HSP70 expression in quercetin-treated areas was comparable to control. Furthermore, we showed that systemic delivery of quercetin by intraperitoneal injections prevented/reduced spontaneous onset of AA. Our results demonstrated that quercetin provided effective treatment for AA as well as prevention of onset of AA in the C3H/HeJ model, and warrant further clinical studies to determine whether quercetin may provide both treatment for preexisting AA and prevention of recurrent AA. The ready availability of quercetin as a dietary supplement may lead to increased patient compliance and positive outcomes for AA.