The JAK inhibitor ruxolitinib reduces inflammation in an ILC3-independent model of innate immune colitis.

Innate immunity contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms of IBD mediated by innate immunity are incompletely understood and there are limited models of spontaneous innate immune colitis to address this question. Here we describe a new robust model of colitis occurring in the absence of adaptive immunity. RAG1-deficient mice expressing TNFAIP3 in intestinal epithelial cells (TRAG mice) spontaneously developed 100% penetrant, early-onset colitis that was limited to the colon and dependent on intestinal microbes but was not transmissible to co-housed littermates. TRAG colitis was associated with increased mucosal numbers of innate lymphoid cells (ILCs) and depletion of ILC prevented colitis in TRAG mice. ILC depletion also therapeutically reversed established colitis in TRAG mice. The colitis in TRAG mice was not prevented by interbreeding to mice lacking group 3 ILC nor by depletion of TNF. Treatment with the JAK inhibitor ruxolitinib ameliorated colitis in TRAG mice. This new model of colitis, with its predictable onset and colon-specific inflammation, will have direct utility in developing a more complete understanding of innate immune mechanisms that can contribute to colitis and in pre-clinical studies for effects of therapeutic agents on innate immune-mediated IBD.

T cell-independent interleukin 15Ralpha signals are required for bystander proliferation.

Cytokine driven or "bystander" proliferation of T cells occurs in vivo independently of major histocompatibility complex-T cell receptor interactions. This process may be important for supporting T cell homeostasis and facilitating T cell responses to microbial antigens, and may involve the cytokine interleukin (IL)-15. In this study, we find that IL-15Ralpha-deficient (IL-15Ralpha(-/-)) mice fail to undergo poly I:C or IL-15 driven bystander proliferation of CD8(+) T cells. Surprisingly, IL-15Ralpha(-/-) CD8(+) T cells proliferate in response to poly I:C when adoptively transferred into normal mice, and normal CD8(+) T cells fail to proliferate in IL-15Ralpha(-/-) mice. Normal mice reconstituted with IL-15Ralpha(-/-) bone marrow cells also fail to exhibit bystander responses. Thus, CD8(+) T cell independent IL-15Ralpha signals from radiation sensitive hematopoietic cells are likely required for bystander responses. Moreover, normal CD8(+) T cells proliferate in IL-15Ralpha(-/-) mice after treatment with IL-15. Therefore, IL-15Ralpha signals may mediate a positive feedback loop involving the further physiological production of IL-15. These findings provide new insights into how IL-15Ralpha supports memory phenotype CD8(+) T cell proliferation, and suggest novel mechanisms by which memory CD8(+) T cells are maintained in vivo.

Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice.

A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappaB (NF-kappaB) activity and tumor necrosis factor (TNF)-mediated programmed cell death (PCD). TNF dramatically increases A20 messenger RNA expression in all tissues. Mice deficient for A20 develop severe inflammation and cachexia, are hypersensitive to both lipopolysaccharide and TNF, and die prematurely. A20-deficient cells fail to terminate TNF-induced NF-kappaB responses. These cells are also more susceptible than control cells to undergo TNF-mediated PCD. Thus, A20 is critical for limiting inflammation by terminating TNF-induced NF-kappaB responses in vivo.

IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation.

The IL-15 receptor alpha subunit (IL-15Ralpha) mediates high-affinity binding of IL-15, a pleiotropic cytokine implicated in the development of innate immune cells. We have generated IL-15Ralpha null (IL-15Ralpha-/-) mice to understand the role of IL-15Ralpha in immune development and function. IL-15Ralpha-/- mice are markedly lymphopenic despite grossly normal T and B lymphocyte development. This lymphopenia is due to decreased proliferation and decreased homing of IL-15Ralpha-/- lymphocytes to peripheral lymph nodes. These mice are also deficient in natural killer cells, natural killer T cells, CD8+ T lymphocytes, and TCRgammadelta intraepithelial lymphocytes. In addition, memory phenotype CD8+ T cells are selectively reduced in number. Thus, IL-15Ralpha has pleiotropic roles in immune development and function, including the positive maintenance of lymphocyte homeostasis.

The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation.

OBJECTIVE: Little is known about the regulation of apoptosis in the adipocyte. Using the 3T3-L1 cell model of adipogenesis, we investigated whether induction of apoptosis by serum-starvation is differentiation-specific. METHODS: Apoptosis was assessed by phase-contrast microscopy, Hoechst staining and DNA fragmentation. Protein expression levels were evaluated by immunoblot analysis. RESULTS: Following serum deprivation, Hoechst staining revealed chromatin condensation and formation of apoptotic bodies in 3T3-L1 preadipocytes, but not in differentiated adipocytes. Similarly, although cell cultures of serum-starved 3T3-L1 preadipocytes displayed extensive apoptotic DNA fragmentation, this was barely detectable for cell cultures subjected to differentiation medium, in which 70-90% of the cells had assumed the mature phenotype. Consistent with the ability of the adipocytes to resist apoptosis, immunoblot analysis revealed that differentiated cell cultures expressed Bcl-2 and the endonuclease DNase I at higher and lower levels, respectively. CONCLUSION: Expression of cell survival genes is modulated during 3T3-L1 adipocyte differentiation, potentially contributing to the state of resistance to apoptosis observed for mature 3T3-L1 adipocytes upon growth factor deprivation.

Caspase-3 in the rat ovary: localization and possible role in follicular atresia and luteal regression.

Apoptosis, the cellular mechanism of ovarian follicular atresia and luteal regression, is triggered by the activation of a proteolytic cascade of cysteine aspartate-specific proteases (caspases). The principle downstream effector of cell death is caspase-3, but little is known about the role or regulation of this enzyme in ovarian apoptosis. Two substrates of caspase-3, actin and poly(ADP-ribose) polymerase (PARP), are inhibitors of DNase I, which is the endonuclease responsible for ovarian apoptotic DNA degradation. We therefore investigated the proteolytic cleavage of actin and PARP as well as the localization of caspase-3 during follicular atresia (induced by gonadotropin withdrawal) and luteal regression (induced by prostaglandin F2alpha) in the rat ovary. Apoptotic DNA degradation was evident during both follicular atresia and luteal regression, but cleavage of PARP and actin was observed only during luteal regression. Caspase-3 was localized in luteal cells of healthy corpora lutea (CL) and in theca, but not in granulosa cells of healthy follicles. However, caspase-3 immunostaining was evident in granulosa cells of atretic follicles in a pattern similar to that of the localization of granulosa cell death. There was no difference between healthy and apoptotic CL in the distribution or intensity of caspase-3 staining. These results demonstrate that the cleavage of actin and PARP are not necessary for activation of apoptotic DNA degradation during ovarian apoptosis. In addition, the presence of caspase-3 in granulosa cells of atretic, but not healthy, follicles suggests that the expression of this enzyme is regulated by gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells.

Granulosa cell apoptosis induced at the penultimate stage of follicular development is associated with increased levels of Fas and Fas ligand in the rat ovary.

Apoptosis of granulosa cells is the cellular mechanism of ovarian follicular atresia, and cytokines have been implicated as potential atretogenic factors. We therefore investigated the possible role of the cytokine Fas ligand (FasL) and its receptor Fas in apoptosis during ovarian follicular atresia induced by gonadotropin withdrawal. Immature rats pretreated with eCG were injected 24 h later with an antiserum generated against eCG (antibody group) or preimmune rabbit serum (control), and ovaries were removed 1 and 24 h after treatment. The eCG antiserum caused a dose-dependent inhibition of the significant increase in ovarian weight observed between 24 and 48 h after eCG treatment. In situ detection of fragmented DNA in histological sections identified cell death in atretic but not healthy small and medium-sized antral follicles of the antibody group. Cell death was distributed in a scattered pattern throughout the granulosa cell layer of small atretic follicles but was localized primarily in granulosa cells lining the antral cavity of atretic medium antral follicles. Immunohistochemistry of adjacent histological sections revealed intense positive immunostaining for Fas and FasL in granulosa cells of atretic small and medium antral follicles in a pattern coincidental to the localization of cell death. Intense FasL staining was evident in the theca cells of healthy small antral follicles. An increase in low molecular weight DNA (DNA "ladders") indicative of apoptosis was evident in granulosa cells of the antibody group. Western analysis demonstrated increased levels of both Fas and FasL in the granulosa cells of the antibody group. These results demonstrate that both Fas and FasL are present in ovarian granulosa cells and that FasL may be the signal that induces granulosa cell apoptosis during atresia at the penultimate stage of ovarian follicular development.

Identification and localization of deoxyribonuclease I in the rat ovary.

Our objective was to identify the endonuclease responsible for DNA degradation in the ovary and determine its localization relative to the developmental state of ovarian follicles. Immature rats were treated with diethylstilbestrol (DES; DES group), DES + eCG (eCG group) or DES + eCG + hCG (hCG group). Nuclei of the eCG and hCG but not the DES group contained a 32/34-kDA DNase I-like endonuclease activity that was Ca2+/Mg(2+)-dependent, stimulated by Mn2+, optimal at pH 7, and identified by anti-DNase I antibody. G-actin, Zn2+, dithiothreitol, aurintricarboxylic acid, and sodium aurothiomalate, but not iodoacetic acid, inhibited the activity. Addition of eCG nuclear protein extracts to nuclei from the DES group induced oligonucleosomal DNA fragmentation, which could be prevented by pretreatment of the extracts with anti-DNase I antibody. DNase I was immunolocalized in nuclei of healthy luteal cells, antral follicle granulosa cells, oocytes of preantral follicles, atretic preantral follicles, and testicular spermatogonia, but was not observed in granulosa cells of preantral follicles, theca cells, antral follicle oocytes, or testicular spermatocytes. Nuclear extracts of rat kidney, liver, and spleen, and bovine, chicken, and human ovaries displayed DNase I-like activity. These results suggest that an endonuclease indistinguishable from DNase I is responsible for ovarian apoptotic DNA degradation.

Induction of apoptosis in equine chorionic gonadotropin (eCG)-primed rat ovaries by anti-eCG antibody.

We have established a model to examine the early events of apoptosis in small antral follicles in vivo. Immature female rats injected with 15 IU eCG, and subsequently (24 h later) with an anti-eCG antibody to induce gonadotropin withdrawal, displayed a significantly lower ovarian weight and increased incidence of follicular atresia and granulosa cell death, especially in small- to medium-sized follicles. Evidence of apoptosis was apparent in a significantly higher proportion of granulosa cells from antibody-treated rats, which exhibited membrane blebbing, nuclear and cytoplasmic condensation, fragmentation, and phagocytosis. In addition, there was a loss of the regular organization of the lamina densa in the follicular basement membrane. Degradation of DNA was consistently found by 24 h in the antibody-treated group, and ladders could be observed as early as 1 h after treatment. Although cell death was observed after antibody treatment in some larger antral follicles, the occurrence of apoptosis was less frequent. These results demonstrate that gonadotropin withdrawal in vivo rapidly induces apoptosis in small- to medium-sized antral follicles at the critical stage of development when atresia is commonly observed, suggesting that this model is ideal for studying apoptosis in the ovary.

Identification of a deoxyribonuclease I-like endonuclease in rat granulosa and luteal cell nuclei.

Apoptosis, a process recently implicated as the cellular mechanism underlying ovarian follicular atresia and luteal regression, is characterized by the internucleosomal degradation of DNA by a Ca2+/Mg(2+)-dependent endonuclease. Although hormones and growth factors have been demonstrated to modulate the DNA degradation associated with ovarian follicular apoptosis, the nature and identity of the endonuclease involved is not known. Ca2+/Mg(2+)-dependent endonuclease activity has a developmental pattern of expression in rat granulosa and luteal cell nuclei. Thus, the present study was conducted to establish the presence of an endonuclease in the nuclei of ovarian granulosa and luteal cells and to examine the biochemical properties of the enzyme relevant to apoptosis. Nuclei from diethyl-stilbestrol (DES)-, eCG-, and hCG-primed rat ovaries were isolated and exposed to Ca2+ and Mg2+ in vitro. Nuclei from rat ovaries primed with eCG and hCG, but not DES, substantially degraded their DNA in an apoptotic fashion, and this DNA degradation was Ca2+/Mg(2+)-dependent and inhibited by Zn2+. Protein extracts from the nuclei of DES-, eCG-, and hCG-treated rat ovaries were tested for endonuclease activity by a plasmid degradation assay. The extracts were found to contain endonuclease activity with the same developmental pattern and cation dependency as found in intact nuclei. These protein extracts were assessed for nuclease activity by zymography, and three nuclease activities were identified depending on the type of DNA used in the gel and the electrophoresis conditions used for protein separation.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of progesterone secretion in dispersed cells of rat corpora lutea by antioxidants.

Antioxidants were used to investigate the role of free radicals in control of luteal steroidogenesis. Corpora lutea from pseudopregnant rats were enzymatically dispersed, the cells were incubated with antioxidants, and progesterone production was measured. Addition of the antioxidants nordihydroguaiaretic acid (NDGA), butylated hydroxytoluene (BHT), and the gonadotropin luteinizing hormone (LH) resulted in a dose-dependent increase in progesterone secretion. However, the response pattern to these treatments differed with the age of the corpora lutea, and unlike LH neither NDGA nor BHT treatment resulted in an increase in the intracellular second messenger cAMP. Nevertheless, LH and antioxidant-induced progesterone stimulation could be blocked by the addition of either aminoglutethimide or ketoconazole, cytochrome P450 side-chain cleavage (cytochrome P450 SCC) enzyme inhibitors, which prevent the conversion of cholesterol to pregnenolone and thus block steroid hormone synthesis. Also, unlike exposure to LH, exposure to antioxidants resulted in an additional increase in progesterone production in luteal tissue saturated with 25 hydroxycholesterol, a soluble cholesterol analog which serves as a substrate for cytochrome P450 SCC. This study suggests that the site of antioxidant action in affecting progesterone secretion may be at the cytochrome P450 SCC enzyme. Based on these results and on studies in other steroid hormone-producing cells, it appears that free radicals may be involved in regulating synthesis by modulating activity of cytochrome P450 SCC enzyme in rat luteal tissue.

Arachidonic acid and cell signalling in the ovary and placenta.

Arachidonic acid (AA) and its metabolites make up a diverse group of signalling molecules important to mediation of metabolic and endocrine function of ovarian and placental cell membranes. This paper reviews recent literature examining AA and eicosanoid involvement in the functional dynamics of follicular development, ovulation and corpus luteum function. The putative roles of AA metabolites in establishment and maintenance of pregnancy are reviewed with reference to decidualization, trophoblast invasion and implantation, maintenance of perfusion of the feto-placental unit and lipid transfer. Finally, recent evidence implicating AA metabolism in mediation of enzyme activity following hormone-receptor coupling within various cells types comprising the placental membranes is reviewed.

Supplemental insulin delivery from an implanted reservoir activated by external compression.

An implant made of insulin in palmitic acid provides a basal dose sufficient to reduce hyperglycemia for 42 +/- 12 days in rats with induced diabetes. For preprandial dose supplements another arrangement is required. In this study a device consisting of a reservoir is assembled by attaching a 1 cm diameter foam ring to a 5 mm diameter piece of the same material. A 6 mg piece of compressed insulin is inserted into a cut between the ring and the attachment, along with 2 mg tetracycline to hinder microbial growth. The assembly is then enclosed between two membranes and an annular external wall. The top membrane is pierced once, and the device is tested by implantation under the abdominal skin of diabetic rats. Serous fluid will enter the interior through the orifice and leach the solid insulin that does not leak out. Daily sidewise compression over the skinfold of the unanesthetized animal indicated that the insulin supply lasted for 24 +/- 4 days. The blood glucose was consistently maintained at 3.4 +/- 1.1 mmol/L for 6-8 hr each day. After depletion the device can be refilled percutaneously by injecting insulin suspended in phosphate-buffered saline.

Changes in the activities of protein kinase modulators in the cerebellum of mice due to ethanol, caffeine, or phenobarbital administration.

Decreased activities of both the inhibitory modulator of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (A-PK) as well as the stimulatory modulator of guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase (G-PK) from the mouse cerebellum were noted due to the administration of excessive doses of ethanol, caffeine, and phenobarbital for up to 28 days. The dose-dependent of the inhibition of A-PK or the stimulation of G-PK was observed as a function of the amount of protein kinase modulators in the cerebellum of mice receiving different doses of ethanol.