Developmental variability in expression and regulation of inducible nitric oxide synthase in rat intestine.

Inducible nitric oxide synthase (iNOS) may be a key mediator of intestinal injury, which varies with developmental age. One member of the mitogen-activated protein kinase (MAPK) family, p38, is involved in the lipopolysaccharide (LPS)-mediated iNOS induction. The involvement of p38 MAPK in basal and LPS-induced iNOS expression was examined in the rat intestine at two developmental ages. Neonatal (4 days postnatal) and adolescent (15 days postnatal) rats were injected with LPS (5 microg/g ip), a selective p38 inhibitor (SB 203580), or both. Tissue was removed after 4 h and 6 h for mRNA and protein analysis. iNOS mRNA and protein were markedly upregulated in the adolescent female following LPS exposure, whereas males had an attenuated response. Neonates had a minimal response. SB 203580 suppressed LPS-induced iNOS mRNA and protein in the ileum, more so in females than in males. Adolescent ileal p38 activation was constitutively high and nonresponsive to LPS. Basal and post-LPS p38 phosphorylation was low in neonatal ileum. We conclude that ileal iNOS expression is developmentally regulated and influenced by gender and that p38 is permissive for LPS effect. The developmental regulation of p38 may contribute to age-dependent variations of intestinal injury.

Increased mucosal B-lymphocyte apoptosis during polymicrobial sepsis is a Fas ligand but not an endotoxin-mediated process.

Sepsis is reported to induce an increase in the rate of apoptosis (Ao), in immature lymphoid cells residing in hematopoietic tissues such as the thymus and bone marrow. Alternatively, secondary lymphoid tissue, such as the spleen exhibit little innate (unstimulated) Ao. However, it is unknown whether or not polymicrobial sepsis has any effects on the frequency of Ao in mucosal lymphoid tissue and what, if any, are the functional consequences of such a change. To assess this, Peyer's patch cells were harvested from C3H/HeN (endotoxin-sensitive) mice killed 12 or 24 hours after the onset of polymicrobial sepsis (cecal ligation and puncture [CLP]). The results indicate that the percentage of cells that were Ao+ as determined by flow cytometry were markedly increased at 24 hours, but not at 12 hours post-CLP. This correlates well with evidence of increased DNA fragmentation as well as histological changes observed both at a light and transmission electron microscopic level of the Peyer's patch Ao. Phenotypically, these changes were restricted to the B220+ (B-cell) population that also exhibited a marked increase of Fas/Apo-1 antigen expression. The functional consequence of this increased apoptosis appears to be associated with the endogenous stimulation (activation) of IgA production by mucosal B lymphocytes and increased nuclear c-Rel expression. Furthermore, we found that Peyer's patch lymphocytes isolated from C3H/HeJ-Faslgld (endotoxin-tolerant/Fas ligand- [FasL] deficient) as opposed to C3H/HeJ (endotoxin-tolerant) inbred mice did not exhibit increased Ao after CLP. These findings indicate that increased B-cell Ao appears to be a FasL-Fas antigen-mediated process, but is not due to endotoxin sensitivity. In conclusion, we speculate that the increased Fas-associated apoptosis detected in mucosal B cells (as opposed to splenic or bone marrow B cells) may be due to increased luminal antigens other than endotoxin, released due to gut barrier integrity breakdown during sepsis.

Factors responsible for peritoneal granulocyte apoptosis during sepsis.

Apoptosis (Ao), is a process by which cells undergo a form of non-necrotic cellular suicide, the control of which may have significant impact on host immunoresponsiveness to a septic challenge. The aim of this study was to determine (1) if Ao is evident in granulocytes harvested from the blood or peritoneum of septic animals and to what extent this was associated with cell activation and (2) whether the in vivo administration of the TNF inhibitor (TNFbp) alters this process. To assess the first aim, C3H/HeN male mice were subjected to polymicrobial sepsis [cecal ligation and puncture (CLP)] or sham-CLP (Sham) and sacrificed at 4 or 24 hr following CLP. Blood leukocytes and peritoneal exudate cells were harvested, stained with monoclonal fluorochrome-conjugated antibodies to granulocytes (Gr1), the activation marker ICAM-1, and either the cell cycle dye, 4',6-diamidino-2-phenylindole, or TUNEL assay (to assess the %Ao+), was used for two- and three-color flow cytometric analysis. Peritoneal exudate cells exhibited increased %Ao+ cells at both 4 and 24 hr post-CLP, while blood leukocytes showed a decrease in %Ao+ only at 24 hr. The increase in Ao in the peritoneum was evident only in the Gr1- cell population at 4 hr but was present in both Gr1+ and Gr1- cells at 24 h. Furthermore, the increase in the %Ao+ cells was associated with an increased % of ICAM-1 positive cells. To the extent that TNF affects the 24 hr induction of Ao in peritoneal exudate cells, mice were treated with either 250 micrograms TNFbp/mouse (s.c.) or vehicle control immediately following CLP. The results indicate that administration of TNFbp markedly decreased Gr1+ but not Gr1- cell Ao. Thus, not only does polymicrobial sepsis induce a marked early rise in phagocyte Ao associated with cell activation, but the increase in peritoneal granulocyte Ao, unlike macrophage Ao, is mediated by TNF and/or an agent released by TNF.

Comparison of basic fibroblast growth factor levels in clone A human colon cancer cells in vitro with levels in xenografted tumours.

We measured levels of basic fibroblast growth factor (FGF-2) in human colon cancer cells (clone A) in vitro and in xenografted solid tumours using a commercial enzyme-linked immunoassay. In Vitro, levels in unfed plateau phase or exponentially growing cells were low, averaging respectively about 2 and 8 pg 10(-6) cells. However, when solid tumours (average volumes 787 mm3) were cut into halves and either enzymatically disaggregated to obtain a cellular fraction or extracted in toto, levels were much higher. In the cellular fraction, values averaged 110 pg 10(-6) cells, while in whole tumour extracts, average values were 24 pg mg-1 tumour tissue. These results indicate that growth factor levels in solid neoplasms may differ markedly from those predicted from in vitro measurements. We hypothesise that the apparent increase in FGF-2 levels in vivo results primarily from the presence of a significant fraction of host cells (in particular, macrophages, which may contain high levels of FGF-2) within xenografted clone A neoplasms.