C3H/HeJ Mice Research Articles

Lipopolysaccharide (LPS)-induced IL-6 production by embryonic fibroblasts isolated and cloned from LPS-responsive and LPS-hyporesponsive mice

Fibroblasts participate in inflammatory processes and non-specific immunity by producing cytokines and mediators in response to bacterial lipopolysaccharide (LPS). The detailed mechanism of LPS-induced cytokine production by fibroblasts has not been sufficiently studied. We isolated murine embryonic fibroblasts (MEF) from LPS-responsive C3H HeN mice and LPS-hyporesponsive C3H HeJ mice and established MEF cell lines and MEF clones. Primarily cultured MEF, MEF cell lines and MEF clones from C3H HeN mice (MEF.He) expressed interleukin (IL)-6 mRNA and produced IL-6 molecules in response to even a very low dose (1 ng/ml) of LPS. By contrast, those from C3H HeJ mice (MEF.HeJ) neither expressed IL-6 mRNA nor produced IL-6 in response to 1 ng of LPS per ml, although they expressed IL-6 mRNA and produced IL-6 in response to high doses (more than 100 ng/ml) of LPS. The MEF.He clone, but not the MEF.HeJ clone, expressed IL-6 mRNA in response to taxol or ceramide, whereas MEF.HeJ clones as well as the MEF.He clone expressed IL-6 mRNA in response to IL-1α. These results indicate that in the responses to LPS, taxol and ceramide, MEF retain the same reactivity as that of the mouse strains from which the MEF were derived, and LPS shares the IL-6 signal transduction pathway with taxol and ceramide, but not with IL-1. CD14 is not relevant to the LPS-induced IL-6 production by MEF, since cloned MEF.He and MEF.HeJ were shown not to express CD14 mRNA by Northern blot analysis. No difference in LPS-specific binding capacity was shown between the MEF.He and MEF.HeJ clones. This finding, together with the fact that hyporesponsiveness of MEF.HeJ to LPS was shown at the level of IL-6 mRNA expression, suggests that the defect in the LPS-induced IL-6 signal transduction pathway in MEF from C3H HeJ mice is probably located at some site after the LPS-recognition site on the cell surface and before transcription of the IL-6 gene.

Roles of the Pas1 and Par2 genes in determination of the unique, intermediate susceptibility of BALB/cByJ mice to urethane-induction of lung carcinogenesis: Differential effects on tumor multiplicity, size and Kras2 mutations

The C3H/HeJ (C3H), A/J and BALB/cByJ (BALB) mouse strains are respectively resistant, sensitive and intermediate regarding the induction of lung tumors by urethane. The phenotypic difference between C3H and A/J is largely determined by the Pas1 (Pulmonary adenoma susceptibility 1) gene on chromosome 6, the A/J allele of which dominantly increases the tumor burden. We recently found that BALB mice possess a unique lung tumor resistance gene on chromosome 18, designated Par2 (Pulmonary adenoma resistance 2), which partially, but dominantly suppresses the sensitive phenotype of A/J mice (Oncogene 13: 1599-1604, 1996). It has, however, remained unclear why BALB mice carrying the Par2 gene are significantly more sensitive to urethane-induced lung carcinogenesis than C3H mice that have no dominant lung tumor resistance genes. In the present study, using (C3H x BALB)F1 x C3H backcross mice treated with urethane, we demonstrated that BALB mice possess the disease allele of the Pas1 gene despite their 15-fold more resistance relative to A/J mice (LOD = 22.6). The BALB Par2 allele only significantly reduced the mean lung tumor multiplicity (LOD = 4.4) in the backcross population carrying the BALB allele of Pas1, indicating that the intermediate BALB phenotype may at least in part be the result of interactions between these two dominant genes. While the BALB Pas1 allele increased both the mean multiplicity and size of lung tumors, the BALB Par2 allele affected only the mean tumor multiplicity, implying that they are involved in different stages of multi-step lung carcinogenesis. In addition, we found that 68% of lung tumors from the BALB Pas1-positive backcross mice contained activating point mutations of the Kras2 oncogene, tightly linked to the Pas1 locus, whereas these genetic alterations were absent in tumors from BALB Pas1-negative mice. The Par2 genotype exhibited no effect on this parameter. Since the activating point mutations were observed exclusively in the BALB allele as already reported with lung tumors in (C57BL/6J x BALB/cJ)F1 mice, BALB Pas1 or possibly Kras2 itself may confer selective growth advantage on the affected urethane-initiated lung lesions.

Genetic modeling of susceptibility to nitrogen dioxide-induced lung injury in mice

We investigated the mode of inheritance of susceptibility to nitrogen dioxide (NO2)-induced lung injury in inbred mice. Susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) mice, as well as F1, F2, and backcross (BX) populations derived from them, were exposed to 15 parts per million NO2 for 3 h. Six hours after exposure, animals were lavaged, and differential cell counts and cell viability (cytotoxicity) were measured. Statistically significant (P < 0.05) differences in numbers of lavageable macrophages, epithelial cells, and dead cells were found between inbred strains. Distributions of cellular responses in F1 progeny overlapped both progenitors, and mean responses were intermediate. In C3:BX progeny, ranges of responses to NO2 closely resembled C3 mice, and means were not significantly different between populations. Ranges of cellular responses to NO2 in B6:BX and intercross progeny overlapped both progenitors; mean responses of both populations were intermediate to progenitors. Segregation analyses tested goodness of fit of phenotyping data with various inheritance models, and the highest likelihood for each cell response to NO2 was for the hypothesis two-unlinked loci general. We conclude that there are likely two major unlinked genes that account for differential susceptibility to acute NO2 exposure. The chromosomal location of the genes is not known.

Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio.

We have examined the effects of mildly oxidized LDL and atherosclerosis on the levels of two proteins associated with HDL; apolipoprotein J (apoJ), and paraoxonase (PON). On an atherogenic diet, PON activity decreased by 52%, and apoJ levels increased 2.8-fold in fatty streak susceptible mice, C57BL/6J (BL/6), but not in fatty streak resistant mice, C3H/HeJ (C3H). Plasma PON activity was also significantly decreased, and apoJ levels were markedly increased in apolipoprotein E knockout mice on the chow diet, resulting in a 9.2-fold increase in the apoJ/PON ratio as compared to controls. Furthermore, a dramatic increase in the apoJ/PON ratio (over 100-fold) was observed in LDL receptor knockout mice when they were fed a 0.15%-cholesterol-enriched diet. Injection of mildly oxidized LDL (but not native LDL) into BL/6 mice (but not in C3H mice) on a chow diet resulted in a 59% decrease in PON activity (P < 0.01) and a 3.6-fold increase in apoJ levels (P < 0.01). When an acute phase reaction was induced in rabbits, or the rabbits were placed on an atherogenic diet, hepatic mRNA for apoJ was increased by 2.7-fold and 2.8-fold, respectively. Treatment of HepG2 cells in culture with mildly oxidized LDL (but not native LDL) resulted in reduced mRNA levels for PON (3.0-fold decrease) and increased mRNA levels for apoJ (2.0-fold increase). In normolipidemic patients with angiographically documented coronary artery disease who did not have diabetes and were not on lipid-lowering medication (n = 14), the total cholesterol/HDL cholesterol ratio was 3.1+/-0.9 as compared to 2.9+/-0.4 in the controls (n = 19). This difference was not statistically significant. In contrast, the apoJ/PON ratio was 3.0+/-0.4 in the patients compared to 0.72+/-0.2 in the controls (P < 0.009). In a subset of these normolipidemic patients (n = 5), the PON activity was low (48+/-6.6 versus 98+/-17 U/ml for controls; P < 0.009), despite similar normal HDL levels, and the HDL from these patients failed to protect against LDL oxidation in co-cultures of human artery wall cells. We conclude that: (a) mildly oxidized LDL can induce an increased apoJ/PON ratio, and (b) the apoJ/PON ratio may prove to be a better predictor of atherosclerosis than the total cholesterol/HDL cholesterol ratio.

The effect of immunomodulation of stimulator antigen presenting cells on subsequent responder T-cell function

In this study, we examined whether lipopolysaccharide (LPS)-, interferon-γ (IFN-γ) or 16,16-dimethyl prostaglandin E 2 (dmPGE 2)-pretreatment of stimulator spleen cells from C57BL6 (B6) mice affects effector function of responder T-lymphocyte from C3H HeJ mice. Stimulation of B6-derived splenic mononuclear cells (SMNCs) with LPS (10 μg/ml) prior to their utilization as stimulator cells in a mixed lymphocyte culture (MLC) resulted in an increase in responder T-lymphocyte proliferation compared to utilization of unstimulated SMNC ( P < 0.05). IFN-γ demonstrated similar effects in a dose dependent fashion with maximal stimulatory effect seen at 1000 U/ml. In contrast, pretreatment of stimulator SMNC with dmPGE 2 resulted in dose-dependent inhibition of the responder T-lymphocyte proliferation with maximum inhibitory effect seen using a concentration of dmPGE 2 of 10 −5 M. The presence of indomethacin in the MLC did not reverse this effect. These data demonstrate the effect of immunomodulation of stimulator spleen cells on subsequent T-lymphocyte function.

Genetic control of differential baseline breathing pattern.

The purpose of the present study was to determine the genetic control of baseline breathing pattern by examining the mode of inheritance between two inbred murine strains with differential breathing characteristics. Specifically, the rapid, shallow phenotype of the C57BL/6J (B6) strain is consistently distinct from the slow, deep phenotype of the C3H/HeJ (C3) strain. The response distributions of segregant and nonsegregant progeny were compared with the two progenitor strains to determine the mode of inheritance for each ventilatory characteristic. The BXH recombinant inbred (RI) strains derived from the B6 and C3 progenitors were examined to establish strain distribution patterns for each ventilatory trait. To establish the mode of inheritance, baseline breathing frequency (f), tidal volume, and inspiratory time (TI) were measured five times in each of 178 mature male animals from the two progenitor strains and their progeny by using whole body plethysmography. With respect to f and TI, the two progenitor strains were consistently distinct, and segregation analyses of the inheritance pattern suggest that the most parsimonious genetic model for response distributions of f and TI is a two-loci model. In similar experiments conducted on 82 mature male animals from 12 BXH RI strains, each parental phenotype was represented by one or more of the RI strains. Intermediate phenotypes emerged to confirm the likelihood that parental strain differences in f and TI were determined by more than one locus. Taken together, these studies suggest that the phenotypic difference in baseline respiratory timing between male B6 and C3 mice is best explained by a genetic model that considers at least two loci as major determinants.

Mouse strain differences in ozone dosimetry and body temperature changes.

Strain differences in susceptibility to inhaled ozone (O3) have been observed in mice, with C57BL/6J (B6) mice reported to be more sensitive than C3H/HEJ (C3) mice when exposed to equal concentrations of O3. To determine whether differences in the delivered dose of O3 to the lung could help explain these differences, C3 and B6 mice were exposed to 18O-labeled ozone (18O3), and the resulting 18O concentrations in pulmonary tissues were monitored as an indicator of O3 delivered dose. Body core temperatures (Tco) of similarly treated mice were measured during O3 exposures (using surgically implanted temperature probes) in an effort to correlate lung O3 dose to changes in basal metabolism. Immediately after exposure to 18O3, C3 mice had 46% less 18O (per mg dry wt) in lungs and 61% less in tracheas than B6 mice. Nasal 18O tended to be lower in the C3 mice, but these differences were not significant. Although both strains responded to the O3 exposure with significant decreases in Tco, C3 mice had a 70% greater mean temperature x time product decrease during the exposure than B6 mice. These results suggest that the strain differences in O3 susceptibility may be due to differences in O3 dose to the lung, which may be related to differences in the ability of the mice to lower their Tco in response to O3 exposure.

DIFFERENTIAL SUSCEPTIBILITY TO OXIDANT EXPOSURE IN INBRED STRAINS OF MICE: NITROGEN DIOXIDE VERSUS OZONE

The contribution of genetic background to the pathogenesis of airway responses to environmental agents including air pollutants is becoming increasingly clear. Characterization of genetic mechanisms of response to these agents may assist in the identification of susceptible individuals and populations. The primary objective of this investigation was to utilize inbred strains of mice to determine (1) whether there was significant genetic contribution in susceptibility to lung injury and inflammation induced by single and repeated acute exposures to nitrogen dioxide (NO2) and (2) whether similar genetic factors control sus- ceptibility to lung injury induced by NO2 and another oxidant, ozone (O3). Nine strains of inbred mice (male, 5-6 wk) were studied: 129/ J, A/ J, AKR/ J, BALB/ cJ, C3H/ HeJ, C57BL/6J, DBA/2J, SJL/J, and SWR/J. Each was exposed for 3 h to filtered air (controls) or 15 ppm NO2, and cellular inflammation, epithelial injury, and cytotoxicity were measured 2, 6, and 24 h thereafter. NO2 exposure caused significant increases in cytotoxicity and lavageable macrophages, epithelial cells, polymorphonuclear leukocytes, and protein in all strains. Interstrain variation in each of these effects indicated that genetic background contributed a significant portion of the variance in responses to this oxidant. Two strains that were differentially susceptible to 3-h exposure to 15 ppm NO2\\[C57BL/6J (B6), C3H/HeJ (C3)] were also exposed for 6 h/ day to 10 ppm NO2 on 5 consecutive days. Each of the responses to NO2 was completely adapted after 5 days in resistant C3 mice. Only the lavageable total protein response was adapted in susceptible B6 mice. To determine whether mechanisms of susceptibility to NO2 and O3 were the same, each strain was exposed for 3 h to filtered air or 2 ppm O3 and inflammation was assessed 6 and 24 h thereafter. Strain distribution patterns (SDPs) for responses to each oxidant were not significantly concordant and indicated that susceptibility mechanisms were different. Results of these studies suggest that there is a strong genetic component to NO2 susceptibility that is partially adaptable and significantly different from O3 susceptibility.

Ozone toxicity in the mouse: comparison and modeling of responses in susceptible and resistant strains.

Previous studies from this laboratory have demonstrated a concentration-related hypothermia and increases in bronchoalveolar lavage (BAL) fluid indexes of toxicity in the rat after exposure to environmentally relevant levels of ozone (O3). In similar studies with C57BL/6J (B6) and C3H/HeJ (C3) mice, other investigators have reported differential effects on BAL toxicity indexes between the two strains after O3 exposure. The present study investigated the relationship between the reported strain differences in BAL parameters in B6 and C3 mice exposed to O3 and the induced hypothermic response. Male 80-day-old mice (n = 94, 47/strain) were used for these studies. Subsets (n = 8/strain) of these animals were surgically implanted with radiotelemetry transmitters that permitted continuous monitoring of core body temperature and activity. All telemetry animals and an equal number of nontelemetry animals (n = 8/strain) were exposed to filtered air for 24 h followed by a 2-h exposure to 2 parts/million 16O3. With use of a similar protocol, groups of nontelemetry mice (n = 12/strain) were exposed to either filtered air or 2 parts/million 16O3 for 2 h. At 0 or 22 h postexposure, mice were anesthetized with halothane and intubated, and their lungs were lavaged with 37 degrees C saline. Although both strains of mice exhibited significant abrupt decreases in core body temperature on exposure to O3 and both recovered rapidly after cessation of the O3 exposure, the response of the C3 mice was more dynamic than that of the B6 mice. Similarly, both strains showed characteristic changes in biomarkers of O3 toxicity; however, the increases in BAL fluid protein and cells at 22 h postexposure were significantly greater and the percentage of neutrophils was significantly less in B6 mice than in C3 mice. It is possible that the strain differences in BAL constituents may be related to the differences in the hypothermic response.

Altering culture conditions reveals strain-related differences in activity and immunoreactive isoforms of 3β-hydroxysteroid dehydrogenase-isomerase in mouse Leydig cells

We previously reported that 3β-hydroxysteroid dehydrogenase-isomerase (3βHSD) activity is higher in Leydig cells from C57BL 6J mice than those from C3H HeJ mice. This study examines whether the differences in 3βHSD activity in Leydig cells from the two strains of mice are due to the expression of different 3βHSD isoforms and if a specific isoform corresponds with the amount of 3βHSD activity under various culture conditions. Leydig cells were plated in Waymouth's + 15% horse serum (HS) medium. Some culture were terminated 24 h later after plating (day 1) and assayed for 3βHSD activity or immunoreactivity. The remaining cultures were maintained in HS or changed to serum-free medium. We demonstrate for the first time that two 3βHSD immunoreactive isoforms are expressed in freshly isolated Leydig cells and those cultured for 1 day. The same two 3βHSD isoforms are detectable in both strains. Thus, the previously reported strain-related differences in 3βHSD activity are not due to the expression of different isoforms. When cultured for 8 days, the higher molecular weight 3βHSD immunoreactive band is no longer detectable in Leydig cells from either strain. When maintained in HS, 3βHSD activity in C57BL 6J Leydig cells decreases significantly by day 8, while 3βHSD activity in C3H HeJ Leydig cells does not change through day 8. When Leydig cells were cultured in serum-free medium, 3βHSD activity is maintained in cultured Leydig cells from C57BL 6J and significantly increases in C3H HeJ 3βHSD by day 8. These data suggest that HS has a strain-specific inhibitory effect on 3βHSD activity, causing a significant decrease in C57BL 6J 3βHSD activity and preventing an increase in C3H HeJ . Densitometric analysis reveals a correspondence between changes in 3βHSD activity and the lower molecular weight isoform but not the higher molecular weight isoform. Treatment with cAMP induces the immunoreactive mass of the lower molecular isoform but not the higher molecular isoform of 3βHSD. Currently, it is unclear what the function of the higher molecular weight 3βHSD isoform is in mouse Leydig cells.

Experimental gut-derived endotoxaemia and bacteraemia are reduced by systemic administration of monoclonal anti-LPS antibodies

This study aimed to investigate the effects and mechanisms of action of systemic administration of monoclonal antibodies, anti-endotoxin (HA-1A), in an animal model of gut-origin sepsis. In the first experiment, Balb c mice were transfused with allogeneic blood ( C3H HeJ mice). Five days post-transfusion the animals were gavaged with 1 × 10 9 Escherichia coli and randomized into three groups ( n = 22 each) to receive a sham burn (SB group) or a 20 per cent TBSA thermal injury, immediately followed by the systemic administration of monoclonal antibodies (3 mg/kg) (HA-1A group) or aliquots of sterile saline (Control group). The animal survival rate was observed for 10 days poslburn. In the second experiment transfusion and burn injury were reproduced but the mice ( n = 8/group) were gavaged with 10 9 E.coli labelled with 111indium oxine. Four hours after the burn the mesenteric lymph nodes, liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of translocation of labelled bacteria measured by the residual radioactivity in the organs. Circulating endotoxin levels were determined by limulus assay. The mortality rate of the HA-1A group (9 per cent) was similar to the SB group (0 per cent) and significantly lower than the control group (59 per cent) ( P < 0.05). Both plasma endotoxin levels and degree of bacterial translocation in all extraintestinal tissues were significantly lower (by approximately 50 per cent) in the HA-1A group than in the control group ( P < 0.05). Systemic administration of HA-1A exerts a beneficial effect by reducing the circulating levels of endotoxin and by increasing the gut barrier function to translocating microorganisms.

Strain difference in jejunal crypt cell susceptibility to radiation-induced apoptosis.

Levels of radiation-induced jejunal crypt cell apoptosis were compared in C57BL/6J, C3Hf/Kam and C3H/HeJ mice. Apoptosis levels were consistently lower in the C3H strains than in C57BL/6J. Although other explanations are possible, the strain difference is most likely to have a genetic basis, and in fact a preliminary analysis of the F2 progeny of C3H/HeJ and C57BL/6J mice indicates that more than one gene is involved. Both C3H strains also had lower levels of radiation-induced thymocyte apoptosis than C57BL/6J mice. Jejunal crypt cell apoptosis levels did not co-segregate with thymocyte apoptosis levels in the F2 progeny of C57BL/6J and C3H/HeJ mice. These results imply that the genes responsible for the difference in radiation-induced thymocyte apoptosis levels between these two strains are not the same as those responsible for the strain difference in radiation-induced jejunal crypt cell apoptosis levels. The experiments reported here identify strain-specific differences in levels of radiation-induced crypt cell apoptosis and are a first step towards identifying genetic polymorphisms that influence sensitivity of the small intestine to damage from ionizing radiation.

Strain dependent effects of sex hormones on hepatocarcinogenesis in mice.

In order to study the interaction of genetic and hormonal factors during murine hepatocarcinogenesis, we compared the number of liver tumors induced by treatment of 12-day-old mice with N,N-diethylnitrosamine (DEN) (0.05 mumol/g body wt) in intact mice and animals gonadectomized at 8 weeks of age from the three inbred strains, C3H/HeJ (C3H), C57BL/6J (B6), and C57BR/cdJ (BR). At 50 weeks of age, the mean liver tumor multiplicity in intact BR females was 28 +/- 13, while that for intact female C3H and B6 mice was 1.4 +/- 4.7 and 0.5 +/- 1.0, respectively. In ovariectomized mice, the yield of liver tumors was approximately 8-fold higher than in intact C3H (10.3 +/- 7.5) and B6 (4.1 +/- 6.6) females. Only a slight increase (35 +/- 14) was seen in ovariectomized BR females compared to intact BR females. Castration resulted in lower mean tumor multiplicities at 32 weeks of age in the males of all three strains. Intact male C3H, B6, and BR mice had mean liver tumor multiplicities of 61 +/- 34, 7.4 +/- 13, and 26 +/- 18, respectively, while the mean tumor multiplicities in castrated C3H, B6, and BR mice were 24 +/- 14, 0.5 +/- 0.9, and 6.1 +/- 10 tumors per mouse, respectively. The apparent rate of growth of glucose-6-phosphatase-deficient, preneoplastic foci in DEN-treated BR females was significantly higher than in B6 females. The growth rates of hepatic foci in BR and B6 males were similar but foci in BR males were 5-fold more numerous than in B6 males. The high sensitivity of BR females may be due, at least in part, to the failure of ovarian hormones to inhibit the growth of preneoplastic foci and the subsequent development of liver tumors. Since BR males had a larger number of hepatic foci, it is likely that androgens increase the rate of focus formation in BR males.

Strain‐dependent differences in DNA synthesis and gene expression in the regenerating livers of C57BL/6J and C3H/HeJ mice

C3H/HeJ (C3H) mice are approximately 50-fold more susceptible to liver-tumor induction than C57BL/6J (B6) mice. This difference is susceptibility is a consequence of allelic differences in hepatocarcinogen sensitivity (Hcs) genes that control the growth of preneoplastic lesions in the liver. We have shown previously that these two strains differ in their responses to partial hepatectomy, which acts as a promoter of hepatocarcinogenesis in B6 mice but not in C3H mice. To determine whether there are also strain-specific differences in normal regulation of hepatic growth, we compared liver regeneration in C3H and B6 mice at the levels of DNA synthesis and gene expression. Partial hepatectomy induced a cascade of controlled events resulting in the regeneration of the liver to its original mass 11 d after surgery. We observed a two-fold greater level of DNA synthesis in C3H mice relative to B6 mice during the first peak of DNA synthesis, which occurred 35 h after hepatectomy in both strains. While the c-fos transcript was readily induced in both strains, there was a reduction in the expression of the late response genes E2F1 and dihydrofolate reductase in the livers of B6 mice when compared with the expression of these transcripts in the livers of C3H mice. The differential regulation of E2F1 between B6 and C3H mice may indicate that the Hcs genes and E2F1 function in the same signal transduction pathway of normal growth control.

The hematopoietic cytokine, colony-stimulating factor 1, is also a growth factor in the CNS: Congenital absence of CSF-1 in mice results in abnormal microglial response and increased neuron vulnerability to injury

In this study we used opop mice, which are deficient in the hematopoietic cytokine, colony-stimulating factor 1 (CSF-1), to determine the effect of CSF-1 on neuronal survival and microglial response in injury. In normal mice microglia express the CSF-1 receptor and are primarily regulated by CSF-1, produced mainly by astrocytes. The CSF-1 deficiency in opop mice results in a depletion in the number of monocytes and macrophages but does not affect the number of morphology of microglia. We produced an ischemic lesion in the cerebral cortex of mice by disrupting the pia-arachnoid blood vessels in a defined area. Using Nissl stain and strocyte- and microglia-specific antibodies, we determined the number of viable neurons in such injury and the intensity of glial reaction. The cellular response to injury on the operated side of opop mice was compared to that on the non-operated contralateral side and to the cellular response in similar lesions in CSF-1 producing C3HHeJ mice. We found that the systemic lack of CSF-1 in opop mice results in a significant increase in neuron vulnerability to ischemic injury and considerably reduced microglial response to neuron injury. Remedying the CSF-1 deficiency, either by grafting CSF-1 secreting astroglia into the brain or by implanting encapsulated CSF-1 secreting fibroblast-like cells into the peritoneum, partially restores the microglial response to neuron injury and significantly potentiates neuronal survival in cerebral cortex ischemic lesions. Astroglial reaction was approximately the same in the lesions in opop mice, grafted annd implanted opop mice and C3HHeJ mice, indicating that CSF-1 modulates microglia, but not the response of astrocytes to injury. The degree of neuronal survival was not correlated to the degree of microglial proliferation and intensity of their reaction. We report some indications that CSF-1, in addition to modulation of microglia, may also act directly on neurons.

Efficacy of Coxiella burnetii and its chloroform-methanol residue (CMR) fraction against Rift Valley fever virus infection in mice

Strains of Coxiella burnetii phase I and II whole cells (WC-I and WC-II) or whole cell fractions were assessed for their potential to induce long-lasting protection in endotoxin-non-responder C3H HeJ or CD-1 mice against Rift Valley fever (RVF) virus challenge. Among the whole cell fractions, only the chloroform-methanol residue (CMR), administered as a single dose (100 μg per mouse) 24 h before viral challenge, effectively protected 100% of the mice from RVF virus; the CMR of the Ohio strain of C. burnetii was not protective. Most of the RVF virus-infected mice treated with other C. burnetii cell fractions died, although their times to death varied. Lipopolysaccharide (LPS) associated with CMR preparations used in these studies, did not protect against RVF virus challenge. A single dose of 100 μg of CMR given 24 h before viral challenge completely eradicated 4-t logs of RVF virus in the serum, liver, spleen, and central nervous system. Compared to several other immunomodulators, CMR was an equally effective antiviral agent. Efficacy of CMR of both Henzerling and Ohio strains disappeared or was marginal when treatment was initiated 2–3 days before RVF viral challenge, even when a second or a third dose of CMR was administered after challenge. A single dose of liposome-encapsulated CMR to RVF virus-infected mice extended the range of therapeutic efficacy of this biologically active component of C. burnetii to 4 days before infection.

CD4+ T lymphocyte modulation of ozone-induced murine pulmonary inflammation.

Inhalation of elevated levels of ozone produces a potent inflammatory response in the lung. The magnitude of this response to ozone exposure in mice is inbred strain dependent with the susceptible phenotype being exemplified by the C57BL/6J (B6) strain and the resistant phenotype by the C3H/HeJ (C3) strain. To examine the role of T lymphocytes in the regulation of ozone-induced pulmonary inflammation, mice were pretreated by an intraperitoneal injection of anti-Thy1.2 monoclonal antibody (mAb), anti-CD4+ mAb, or isotype-matched control antibodies (0.5 mg each) and subsequently exposed for 72 h to either filtered air or ozone (0.3 ppm). Immediately after ozone exposure, the cellular profile in the bronchoalveolar lavage fluids (BALF) was assessed. In isotype-treated controls of both strains of mice, ozone exposure induced significant increases in the numbers of macrophages, neutrophils, lymphocytes, and epithelial cells recovered in the BALF; however, the magnitude of each cell type recovered was significantly greater in B6 mice as compared with C3 mice. Both anti-Thy1.2 and anti-CD4+ monoclonal antibody treatments decreased the number of each cell type recovered in the B6 mice and increased the number of cells in the C3 mice. To determine if the CD4+ T-cell-derived cytokine interleukin (IL)-4 was involved in the differential effect of T-cell depletion on the ozone-induced inflammatory responses of C3 and B6 mice, mice were pretreated with either 400 ng of recombinant mouse IL-4 or vehicle, or 5.0 mg anti-IL-4 receptor monoclonal antibody or an isotype-matched antibody before either air or ozone exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokines induced in vitro by Mycoplasma mycoides ssp. mycoides, large colony type

Septicemic disease in goats and sheep caused by the large colony type of Mycoplasma mycoides ssp. mycoides has clinical and pathological features in common with septic endotoxemia. We studied the ability of the mycoplasma to induce mediators of biological responses to endotoxin, such as TNFα, IL-1α, IL-6 and nitric oxide. Heat-killed suspensions of mycoplasmas in a concentration of 25 μg protein ml −1 induced all mediators in macrophages from peritoneal cavity and bone marrow of both C3H HeN (LPS responsive) and C3H HeJ (LPS low-responsive) mice. Lipopolysaccharide (LPS) in a concentration of 100 ng ml −1 induced the mediators in C3H HeN derived macrophages, only. Simultaneous stimulation of macrophages with interferon-γ enhanced the secretion of nitric oxide (measured as nitrite) but not the cytokines. We conclude that heat-killed Mycoplasma mycoides ssp. mycoides, large colony type, has cytokine inductive activity similar to bacterial endotoxin, but with an induction mechanism different from LPS.

Endotoxin and tumour necrosis factor do not cause mortality from caecal ligation and puncture

Macrophage tumour necrosis factor-alpha (TNF-α) production is thought to represent an important pathogenic mechanism by which Gram-negative sepsis is mediated. We compared the effects of caecal ligation and puncture (CLP) on endotoxin-sensitive ( C3H HeSnJ ) and endotoxin-resistant ( C3H HeJ ) mice. Mortality after CLP for C3H HeSnJ mice compared with C3H HeJ mice was not significantly different (32% and 55%, respectively). When survivors were injected with lipopolysaccharide intraperitoneally on the 7th day after CLP, the mortality rate was 82% for C3H HeSnJ mice versus 0% for C3H HeJ mice ( P < 0.0001). Serum endotoxin levels at 24 h after CLP were only slightly elevated. Serum TNF levels and peritoneal macrophage TNF production were undetectable in C3H HeJ mice and were only slightly elevated in C3H HeSnJ mice by 24 h after CLP. Peritoneal macrophage mRNA levels for TNF-α, IL-1β, and I-Aα displayed a similar pattern in the two strains of mice, with a 2- to 3-fold increase in TNF-α and IL-1β mRNA levels by 24 h and a sharp decrease in I-Aα mRNA by 24 h. The cause of mortality in mice that undergo CLP cannot be attributed to overwhelming endotoxemia and/or TNF production.

Differential effects of iron status on lymphocyte subsets in lupus and non-lupus strains of mice

The MRL/MPJ-lpr/lpr (MRL) mouse is a model of human systemic lupus erythematosus (SLE) and develops autoimmunity accompanied by lymphadenopathy and accumulation of cells bearing CD3+L3T4-Lyt2-markers. Since iron status was previously shown to alter disease manifestation in these mice, the effect of iron status on lymphocyte subsets was studied. Weanling female MRL mice were divided into four groups (n=9–10) and fed purified diets containing varying levels of iron: 4 mg/kg diet-severely deficient (SD); 35 mg/kg diet-iron-adequate controls (C); and 250 mg/kg diet-iron supplemented (IS). A fourth group was fed C diet in amounts eaten by the SD group-pair-fed controls (PF). C3H/Hej (C3H) mice were fed the same diets and served as non-SLE controls. At 18 weeks of age, both MRL and C3H SD mice were anemic; however, MRL SD mice were significantly more anemic as reflected in lower hemoglobin and hematocrit levels. Spleen and lymph node weights were lowest in MRL SD and PF mice, and spleen cell number was significantly reduced in SD mice. Iron deficiency in C3H mice resulted in reduced percentages of total T lymphocytes, T helper/inducer and T suppressor/cytotoxic, and B lymphocytes. The T helper/T suppressor cell ratio was not affected. The percentage of B cells was also reduced in C3H IS mice compared to C mice. In MRL mice, iron status had no effect on the percentages of T cells, T helper cells, or B cells, nor the estimated percentage of cells expressing the double negative (CD3+L3T4-Lyt 2-) phenotype. T supressor/cytotoxic cells were significantly higher in the lymph nodes of SD MRL mice. No statistical differences were found in the T helper/T suppressor ratio. This study has demonstrated differential effects of iron status on lymphocyte subsets of SLE and non-SLE strains of mice. Altered disease manifestation by iron status in MRL-lpr mice is not related to reductions in T and B lymphocytes or the CD3+L3T4- Lyt 2-population