Interleukin 1 stimulates endothelial cells to release multilineage human colony-stimulating activity

Cultured human umbilical vein endothelial cells, when exposed to soluble products of peripheral blood monocytes, elaborate granulocyte-macrophage colony-stimulating activity (GM-CSA) and erythroid burst-promoting activity (BPA). We have performed studies to determine if the monokine IL 1 can stimulate endothelial cells to release hematopoietic growth factors and whether such factors can also support human megakaryocyte (Meg) and mixed-cell colony growth. Various concentrations of recombinant human IL 1 beta (rIL 1) and media conditioned by monocytes (MCM), endothelial cells (ECM), and endothelial cells cultured for 3 days in 50% MCM (ECMM) or rIL 1 (ECMrIL 1) were added to marrow mononuclear cells cultured in methylcellulose. ECMM and ECMrIL 1 stimulated, in a dose-dependent fashion, the growth of Meg, mixed-cell, and GM colonies and erythroid bursts. In contrast, ECM, MCM, and rIL 1 displayed little or no activity in the colony-forming assays. Preincubation with specific antisera to native human IL 1 or rIL 1 reduced by 75 to 100% the activity of MCM in stimulating endothelial cell release of BPA, GM-CSA, Meg-CSA, and mixed-cell CSA. Meg-CSA, although readily detectable at ECMM and ECMrIL 1 concentrations in culture of 1 to 5%, was partially masked by lineage-specific inhibitors of Meg colony growth. When ECMM was analyzed by gel filtration chromatography, the megakaryocytopoietic inhibitory activity eluted in the high Mr fractions (greater than 75 kD). Meg-CSA co-eluted with GM-CSA and BPA in a single peak of 30 kD. We conclude that endothelial cells, in response to IL 1, produce one or more growth factors that probably act on multiple classes of progenitor cells.     

Lipoxygenase and cyclooxygenase blockade by BW755C prevents endotoxin-induced hypotension but not changes in glucose metabolism

Arachidonic acid metabolites are mediators of various pathophysiologic events following endotoxin administration. However, their role in the endotoxin-induced increase in glucose metabolism has not been examined. Rats were administered either saline or BW755C (an inhibitor of both the cyclooxygenase and lipoxygenase pathways) 30 min prior to injection of E. coli endotoxin and whole body glucose kinetics assessed using a constant iv infusion of [6-3H] glucose. Treatment with BW755C prevented the endotoxin-induced hypotension and tachycardia. Endotoxin produced characteristic increases in the plasma glucose (23-70%) and lactate (2- to 9-fold) concentrations, as well as elevations in the rate of glucose appearance (34-63%) and metabolic clearance (40-92%). In contrast to the amelioration in hemodynamics, pretreatment with BW755C did not prevent these alterations in glucose metabolism normally seen after endotoxin. BW755C markedly reduced the endotoxin-induced increase in plasma catecholamine concentrations, but levels were still elevated 2- to 4-fold compared to control values. The results suggest that arachidonic acid metabolites mediate the early hypotensive response following endotoxin, but are not by themselves responsible for the elevated rates of glucose production and utilization.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.     

Microbial oxidation of cumene by octane-grown cells

Previously, we reported that eight glucose-grown microbial cultures out of 1229 screened oxidize the alkyl side-chain of 2-phenylpropane (cumene) stereospecifically. Now, we have adapted these cultures to grow on n-octane and found that their cumene oxidation activities increased more than 30 times. We also found an additional 11 cultures (ten bacteria, one actinomycete) that oxidized cumene when grown on octane but not on glucose. In general, octane-grown cells were more active in cumene oxidation than glucose-grown cells. Rhodococcus rhodochrous NRRL B-2153 showed the best conversion yield (2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.5%) at 25°C, pH 8.0, 250 rpm, and 12 h of reaction. Structures of the reaction products were confirmed by gas chromatography (GC)/mass spectrometry and GC/infrared analyses. Products contained 84% ee (enantiomeric excess) of the R(–) isomer, as analyzed with a GC cyclodextrin chiral column. Strain B-2153 oxidized alkylbenzenes in the following order of reaction rate: ethylbenzene >amylbenzene > butylbenzene > cumene > propylbenzene > sec-butylbenzene. tert-Butylbenzene was not oxidized.     

Oleic acid transformations by selected strains of Sphingobacterium thalpophilum and Bacillus cereus from composted manure

In a survey of 186 randomly selected microbial strains isolated from composted manure, 63 transformed oleic acid into three types of products: hydroxy fatty acid, fatty amide, and less polar oleyl lipid. Selection of oleic acid-transforming microorganisms was enhanced in nutrient agar supplemented with 0.1% (vol/vol) oleic acid at pH 7.2. Most of the 63 diverse isolates elicited inconsistent and poorly reproduced transformations. However, strains 142b (NRRL B-14797) transformed oleic acid to 10-hydroxystearic acid consistently, and strain 229b (NRRL B-14812) produced an octadecenamide. Taxonomic studies indicated that NRRL strain B-14797, possessing 1,3-dihydroxy-2-amino-15-methylhexadecane and sphinganine bases, was closely related to Sphingobacterium thalpophilum, and NRRL B-14812 was identified as Bacillus cereus.     

Striated myofibrils in anti-myosin stained,isolated chicken gizzard smooth muscle cells

Summary Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM.Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 m. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5±0.2 m. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.     

Double-immunofluorescent staining of isolated smooth muscle cells

Summary Contractile proteins have been co-localized by double-immunofluorescent staining in several types of cultured cells. Since freshly isolated smooth muscle cells are more representative of the organization within smooth muscle cells in the intact tissue than cultured cells, the present study was undertaken to determine the feasibility of using double-staining techniques in freshly isolated cells. A new method of purifying -actinin from chicken gizzards was used to provide antigen for raising anti--actinin. Fluorescein isothiocyanate-labelled anti--actinin (FAA) was used in conjunction with tetramethyl rhodamine isothiocyanate-labelled anti-myosin (TRAM) Ouchterlony gels against myosin, tropomyosin, actin, and -actinin showed that antimyosin reacted only with myosin, anti--actinin only with -actinin. Anti--actinin stained only the Z-line of isolated chicken skeletal muscle myofibrils. FAA stained bright, discrete patches or strips on the plasma membrane, while TRAM was excluded from these areas. FAA stained myofibrils faintly in a striated pattern, while TRAM stained myofibrils heavily with less evident striations. Evidence for extramyofibrillar localization of -actinin within the cytoplasm was inconclusive. Although antibodies were quite specific in their labelling, resolution with double-staining was subject to the same limitations described for single labelling of whole cells (Bagby and Pepe 1978). Double-staining of whole cells is just as feasible as single-staining. Indeed, having a definite marker for myofibrils (TRAM) makes the localization of -actinin much easier to interpret.     

The fine structure of myocytes in the sponges microciona prolifera (Ellis and Solander) and tedania ignis (Duchassaing and Michelotti)

Myocytes are long, fusiform cells found in the osculum and other contractile areas of many sponges. Myocytes in the oscular sphincter of Tedania ignis and the osculum and dermal membrane of Microciona prolifera were studied with light- and electron-microscopes to compare their structure to that of muscles. Salient points of similarity between myocytes and smooth muscles were their long, fusiform shape, their red color after staining with Mallory's triple stain, and the presence of filaments running longitudinally in the cytoplasm. Microciona myocytes have thick filaments of 150–250 Å diameter and thin filament of 50–70 Å diameter, and in transverse sections the thin filaments occasionally appear as a ring of dots around a thick filament. Longitudinal sections of Tedania myocytes show only one type of filament, which varies from 100 Å to 200–300 Å diameter in thick regions of the filament. Although transverse sections show light material around the dense filaments, a distinct pattern of thick and thin filaments is not seen in Tedania. Due to infrequent contacts between cells, the large extra-cellular space observed with the electron microscope (49% in Tedania, 57% in Microciona), and the absence of nerves, each myocyte probably acts as an independent contractile unit.     

T cell requirement for development of chronic ulcerative dermatitis in E- and P-selectin-deficient mice

C57BL/6 mice deficient in E- and P-selectin (E(-/-)P(-/-)) kept under specific pathogen-free barrier conditions have high circulating neutrophil counts and develop hypercellular cervical lymph nodes with substantial plasma cell infiltrates, severe ulcerative dermatitis, conjunctivitis, and lung pathology, which eventually lead to premature death. To test the hypothesis that the pathology in E(-/-)P(-/-) mice may be caused by dysfunctional lymphocyte activity, we crossed E(-/-)P(-/-) mice with recombination activation gene (Rag)-1(-/-) mice to generate E(-/-)P(-/-)Rag-1(-/-) mice lacking mature T and B lymphocytes. E(-/-)P(-/-)Rag-1(-/-) mice had circulating neutrophil counts and plasma G-CSF levels similar to E(-/-)P(-/-) mice. Remarkably, none of the E(-/-)P(-/-)Rag-1(-/-) mice developed conjunctivitis or ulcerative dermatitis typical of E(-/-)P(-/-) mice. These mice were overall healthier in appearance than E(-/-)P(-/-) mice, and histopathologic changes in the lung were reduced. Cervical lymph nodes in E(-/-)P(-/-)Rag-1(-/-) mice were much smaller than those of E(-/-)P(-/-) mice, containing few mononuclear cells and no plasma cells. These data show that the severe disease phenotype of E(-/-)P(-/-) mice depends on lymphocyte function. We conclude that a dysregulated immune response in E(-/-)P(-/-) mice causes disease development, but is not necessary for elevated neutrophil counts.     

Differential flux of macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant from the lung after intrapulmonary delivery

Previously we showed that cytokine-induced neutrophil chemoattractant (CINC), but not macrophage inflammatory protein-2 (MIP-2), is detected in plasma after intratracheal challenge with LPS or the particular chemokines. To further understand the differences between CINC and MIP-2 flux from the lung, we attempted to detect the two chemokines in isolated erythrocytes and leukocytes in rats after intratracheal LPS challenge. In response to intratracheal LPS, we found both CINC and MIP-2 in isolated erythrocytes and leukocytes, suggesting that MIP-2 produced in the LPS-challenged lung entered the circulation like CINC. To assess the relative flux of CINC and MIP-2 from the intra-alveolar compartment into the blood, experiments were performed in rats implanted with vascular catheters in which both chemokines were either injected intratracheally (5 μg) or infused intravenously (20 ng/min) and subsequently measured in plasma or with the cellular elements. Both chemokines appeared in the blood following intratracheal injection, with CINC detected in plasma and cells but MIP-2 only detected in the cellular fraction of blood. Infusion of both chemokines allowed detection of MIP-2 and CINC in plasma and with the cellular elements, which allowed us to calculate clearance for each chemokine and to assess CINC and MIP-2 rates of appearance (Ra) following intratracheal injection. On the basis of plasma and whole blood clearance, CINC Ra was more than sevenfold and fourfold higher, respectively, than MIP-2 Ra. This analysis indicates that differences exist in the rate of flux of CINC and MIP-2 across the epithelial/endothelial barrier of the lung, despite similar molecular size.