Mast cells and sphingosine-1-phosphate underlie prelesional remodeling in a mouse model of eczema.

BACKGROUND: Atopic dermatitis (AD) is a chronic skin inflammation that affects children and adults worldwide, but its pathogenesis remains ill-understood. METHODS: We show that a single application of OVA to mouse skin initiates remodeling and cellular infiltration of the hypodermis measured by a newly developed computer-aided method. RESULTS: Importantly, we demonstrate that skin mast cell (MC) activation and local sphingosine-1-phosphate (S1P) are significantly augmented after OVA treatment in mice. Deficiency in sphingosine kinase (SphK)1, the S1P-producing enzyme, or in MC, remarkably mitigates all signs of OVA-mediated remodeling and MC activation. Furthermore, skin S1P levels remain unchanged in MC-deficient mice exposed to OVA. LPS-free OVA does not recapitulate any of the precursor signs of AD, supporting a triggering contribution of LPS in AD that, per se, suffice to activate local MC and elevate skin S1P. CONCLUSION: We describe MC and S1P as novel pathogenic effectors that initiate remodeling in AD prior to any skin lesions and reveal the significance of LPS in OVA used in most studies, thus mimicking natural antigen (Ag) exposure.

Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice.

Significant left-right (L-R) differences in tumor incidence and disease outcome occur for cancers of paired organs, including the breasts; however, the basis for this laterality is unknown. Here, we show that despite their morphologic symmetry, left versus right mammary glands in wild-type mice have baseline differences in gene expression that are L-R independently regulated during pubertal development, including genes that regulate luminal progenitor cell renewal, luminal cell differentiation, mammary tumorigenesis, tamoxifen sensitivity and chemotherapeutic resistance. In MMTV-cNeu(Tg/Tg) mice, which model HER2/Neu-amplified breast cancer, baseline L-R differences in mammary gene expression are amplified, sustained or inverted in a gene-specific manner and the mammary ductal epithelium undergoes L-R asymmetric growth and patterning. Comparative genomic analysis of mouse L-R mammary gene expression profiles with gene expression profiles of human breast tumors revealed significant linkage between right-sided gene expression and decreased breast cancer patient survival. Collectively, these findings are the first to demonstrate that mammary glands are lateralized organs, and, moreover, that mammary glands have L-R differential susceptibility to HER2/Neu oncogene-mediated effects on ductal epithelial growth and differentiation. We propose that intrinsic molecular laterality may have a role in L-R asymmetric breast tumor incidence and, furthermore, that interplay between the L-R molecular landscape and oncogene activity may contribute to the differential disease progression and patient outcome that are associated with tumor situs.

FK506 attenuates developing and established joint inflammation and suppresses interleukin 6 and nitric oxide expression in bacterial cell wall induced polyarthritis.

OBJECTIVE: To determine the efficacy of therapeutic administration of FK506 (Tacrolimus) in suppressing developing and established joint inflammation, proinflammatory cytokine expression, and nitric oxide (NO) production in peptidoglycan-polysaccharide (PG/PS) induced experimental polyarthritis in rats. METHODS: Chronic joint inflammation was induced by intraperitoneal injection of PG/PS, and joint inflammation was quantified using arthritis index and paw volume. Serum and joint levels of interleukin 6 (IL-6) were measured by bioassay and Western blot analysis respectively, and serum levels of NO production were determined by the Griess procedure and the expression of the inducible isoform of nitric oxide synthase (i-NOS) in the joints was determined by Western blot analysis. RESULTS: Arthritis induced by PG/PS is biphasic, progressing through an initial acute phase and a remission phase, which is followed by a persistent chronic phase. Daily administration of FK506 initiated during the remission phase significantly attenuated the onset and development of chronic joint inflammation. We observed a significant reduction in joint inflammation and swelling, an apparent suppression of pannus development, and minimal erosive damage to the articular cartilage and subchondral bone. Fully established chronic joint inflammation was also ameliorated by daily administration of FK506. Joint swelling and inflammation was significantly reduced by 5 days post-treatment with FK506 and the erosive activity associated with the pannus appeared diminished. The elevated expression of IL-6 and NO characteristic of chronic joint inflammation in the serum and in joint tissue was significantly reduced by FK506 treatment. CONCLUSION: Therapeutic administration of FK506 has a profound antiinflammatory effect on the development of the chronic, erosive arthritis induced by PG/PS. This attenuation in joint inflammation was associated with suppression of IL-6 and NO production systemically and locally in the joints. Our data suggest that FK506 may be effective in the treatment of chronic joint inflammation associated with rheumatoid arthritis.

Indomethacin induced gastropathy in CD18, intercellular adhesion molecule 1, or P-selectin deficient mice.

BACKGROUND: Neutrophil-endothelial cell interactions are thought to play a critical role in the pathophysiology of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy. AIMS: To optimise a mouse model of NSAID induced gastropathy and to evaluate the importance of adhesion molecules using adhesion molecule deficient mice. METHODS: Gastropathy was induced in C57BL/6 mice or their adhesion molecule deficient counterparts via oral administration of indomethacin (20 mg/kg). Lesion scores, mucosal permeability, and histopathology were used to assess gastric mucosal injury. RESULTS: Intragastric administration of indomethacin induced linear haemorrhagic mucosal lesions, primarily in the corpus of the stomach that were first observed at six hours. These lesions continued to develop over the next six hours with maximal lesion scores and mucosal permeabilities at 12 hours. When indomethacin was administered to mice deficient in CD18, intercellular adhesion molecule 1 (ICAM-1), or P-selectin, there were significant decreases in lesion scores compared with their C57BL/6 controls. In addition, mucosal permeabilities were found to be significantly lower in CD18 or ICAM-1 deficient mice observed at 12 hours. CONCLUSION: Certain leucocyte and endothelial cell adhesion molecules are important determinants for full expression of indomethacin induced gastropathy. It is proposed that this modification of the mouse model may be useful for the investigation of other pathophysiological mechanisms of NSAID induced gastropathy.

Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

ICAM-1 and P-selectin expression in a model of NSAID-induced gastropathy.

A growing body of experimental evidence suggests that neutrophilic polymorphonuclear leukocyte (PMN)-endothelial cell interactions play a critical role in the pathophysiology of nonsteroidal anti-inflammatory drug (NSAID)-induced gastropathy. The objective of this study was to directly determine whether the expression of endothelial cell adhesion molecules is enhanced in a model of NSAID-induced gastropathy. Gastropathy was induced in male Sprague-Dawley rats via oral administration of indomethacin (Indo, 20 mg/kg). Lesion scores, blood-to-lumen clearance of 51Cr-EDTA (mucosal permeability), and histological analysis (epithelial necrosis) were used as indexes of gastric mucosal injury. Gastric mucosal vascular expression of intercellular adhesion molecule 1 (ICAM-1) or P-selectin were determined at 1 and 3 h after Indo administration using the dual radiolabeled monoclonal antibody (MAb) technique. For some experiments, a blocking MAb directed at either ICAM-1 (1A29) or P-selectin (RMP-1) or their isotype-matched controls was injected intravenously 10 min before Indo administration. We found that P-selectin expression was significantly increased at 1 h but not 3 h after Indo administration, whereas ICAM-1 expression was significantly increased at both 1 and 3 h after Indo treatment. The blocking ICAM-1 and P-selectin MAbs both inhibited Indo-induced increases in lesion score, mucosal permeability, and epithelial cell necrosis. However, the Indo-induced gastropathy was not associated with significant PMN infiltration into the gastric mucosal interstitium, nor did Indo reduce gastric mucosal blood flow. We propose that NSAID-induced gastric mucosal injury may be related to the expression of P-selectin and ICAM-1; however, this mucosal injury does not appear to be dependent on the extravasation of inflammatory cells or mucosal ischemia.

Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis.

The objectives of this study were to (1) assess the role of the 26S proteasome complex in regulating the expression of the inducible isoform of nitric oxide synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in a model of chronic granulomatous colitis in vivo and (2) determine the role of the proteasome in regulating the inflammatory response observed in this model of chronic gut inflammation. The selective proteasome inhibitor MG-341 (0.3 mg/kg) was administered by gavage beginning immediately before the induction of colitis and continuing daily thereafter for the entire 14-day experimental period. We found that chronic proteasome inhibition using MG-341 significantly attenuated the peptidoglycan/polysaccharide (PG/PS)-induced up-regulation of iNOS in the colon and spleen and the consequent increase in plasma levels of nitrate and nitrite. Furthermore, we found that the proteasome inhibitor suppressed the up-regulation of the adhesion molecule VCAM-1 in the colon. We also found that MG-341 attenuated PG/PS-induced increases in macroscopic colonic inflammation, bowel wall thickness, colonic dry weight and colonic MPO activity. Treatment with MG-341 also significantly reduced PG/PS-induced increases in macroscopic spleen inflammation, spleen weight and spleen MPO activity. We conclude that the 26S proteasome complex plays an important role in regulating the PG/PS-induced up-regulation of iNOS and VCAM-1 in vivo and appears to be important in regulating colonic and splenic inflammation.

Cytokine and nitric oxide production in the acute phase of bacterial cell wall-induced arthritis.

We have investigated the temporal relationship among proinflammatory cytokine expression, nitric oxide (NO) production and joint inflammation in the acute phase of bacterial cell wall-derived peptidoglycan polysaccharide (PG/PS)-induced arthritis. Acute joint inflammation was induced in female LEW/N rats by a single intraperitoneal injection of PG/PS. Arthritis index and paw volume were quantified and joint histopathology was evaluated during acute joint inflammation (0-10 days). Tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) were determined by bioassay whereas nitric oxide (NO) was quantified by measuring serum nitrate/nitrite levels via the Griess procedure. We found that serum levels of TNF and serum IL-1 preceded the increase in IL-6 and NO production. Furthermore, the production of these proinflammatory cytokines and NO preceded bone erosion and osteoclast activity. Erosion of subchondral bone preceded pannus formation and cellular synovitis in the acute phase of PG/PS-induced arthritis. The temporal expression of TNF, IL-1, IL-6 and NO suggest a cascade of inflammatory mediators in which monocytes and macrophages respond to PG/PS with enhanced synthesis of TNF and IL-1, which may in turn promote the synthesis of IL-6 and NO. We postulate that one or more of these inflammatory events are responsible for initiating the subchondral bone erosion observed in acute joint inflammation.

Modulation of endothelial cell permeability by lung carcinoma cells: a potential mechanism of malignant pleural effusion formation.

This study examined the hypothesis that tumor cells metastatic to the pleura secrete a soluble factor(s) that directly increases endothelial cell permeability. Nitrocellulose filters were endothelialized with bovine pulmonary artery endothelial cells and exposed to conditioned media from either human lung adenocarcinoma (Calu-3), human lung squamous cell carcinoma (SK-MES-1), or control media for 16 h. The diffusional permeability (Pd x 10(-5) cm/sec) to [14C]albumin was then determined for each monolayer with Ussing-type chambers. Both adenocarcinoma conditioned media (ACCM) and squamous cell carcinoma conditioned media (SCCM) caused a two- to threefold increase in endothelial monolayer permeability. The addition of indomethacin (10 micrograms/ml) blocked the observed permeability increase in ACCM but not in SCCM, suggesting that the increase in permeability by ACCM was secondary to the production of prostaglandins. To confirm this, a variety of prostanoids previously shown to be produced by the Calu-3 cell line were added directly to the endothelial monolayer. Prostaglandin F2 alpha (PGF2 alpha) in both low (10 ng/ml) and high (100 ng/ml) concentrations for 16 h resulted in a three- to fourfold increase in permeability. Prostaglandin E2 (PGE2) resulted in a small increase in [14C]albumin permeability but only at high concentrations (100 ng/ml). PGF2 alpha production by the two tumor cell lines was measured using radioimmunoassay. Baseline adenocarcinoma production of PGF2 alpha was 117.5 pmol/10(6) cells and fell to 24.2 pmol/10(6) cells hours following incubation with indomethacin. The decrease in PGF2 alpha occurred in parallel with the changes in permeability. Concomitant, reversible changes in cell shape and F-actin distribution were detected in endothelial cells exposed to ACCM. No significant production of PGF2 alpha by the squamous cell carcinoma cell line was detected. These results suggest that both adenocarcinoma and squamous cell carcinoma secrete a soluble factor(s) that directly increases endothelial cell permeability to albumin and that in the case of adenocarcinoma this soluble factor may be a prostanoid such as PGF2 alpha.

Maternal ethanol consumption induces transient compensatory hyperplasia of developing cardiac tissue in the neonatal rat.

The effect of continuous exposure to ethanol in utero and postpartum on growth and cell division in developing cardiac tissue was studied in neonatal Fischer rats. Pregnant and lactating females were maintained on three dietary regimens; a control group fed rat chow ad libitum, an experimental group receiving an ethanol-containing (6% by volume) liquid diet, and a pair-fed control group, which received an isocaloric amount of control liquid diet. At days 1, 5, and 10 postpartum, five litters of pups from each control and experimental group were sacrificed and the body weights, heart weights, heart-to-body weight ratios, and mitotic frequency of the ventricular myocardium were measured. When compared to either group of controls, pups continuously exposed to dietary ethanol expressed significantly (P < 0.01) lower body weights. Pups maintained by the pair-fed females had significantly (P < 0.01) lower body weights at days 5 and 10 than pups maintained by the chow-fed females, indicating a pair-fed effect of suboptimal nutrition of the model. As the pups developed, the heart weights of pups maintained by the chow-fed females became progressively greater (P < 0.01) than the heart weights of pups maintained by the pair-fed and ethanol-fed females, which expressed no weight difference. The reduction of heart weight present in the ethanol-fed and pair-fed pups represents a pair-fed effect of suboptimal nutrition and not an obvious effect of exposure to dietary ethanol. The ratio of heart weight to body weight and mitotic frequency were significantly greater (P < 0.01) in 1- to 5-day-old pups exposed to ethanol. Following day 5, these parameters decreased and approached the control values. This indicates that growth of cardiac tissue is not suppressed in the 1- to 5-day-old rat pups exposed continuously to dietary ethanol. These observations further suggest the presence of a mechanism intrinsic to the heart which can provide stage-dependent protection from the adverse effects of ethanol during early development. The decline in heart weight to body weight ratios and mitotic frequency in pups of ethanol-fed females also suggests that ethanol may initiate suppression of the growth of cardiac tissue or may incur stage-dependent injury during the later stages of development. The possible mechanism of this stage-dependent protection during early neonatal development is an increased mitotic activity of the cardiac myocytes.

The effect of pulsed expansion of subfascially placed expanders on the extent and duration of mitosis in the capsule and rat integument.

This study investigated the mitotic activity in the subdermally induced capsule and the epithelium surrounding tissue expanders maintained by pulsed expansions. Tissue expanders were placed into the fascial plane beneath the panniculus muscle of the rat and expanded sequentially. The volume in the expander was rapidly increased to a constant level and maintained for 48 hours; this was followed by a total decrease of volume for 48 hours. The procedure was repeated with volumes of 20, 30, and 40 mL. At the conclusion of each cycle of expander inflation, a group of rats was killed, and biopsy specimens of the induced capsule and epithelium were obtained. The developing capsule exhibited a significant increase in the mitotic index in the layers adjacent to the expander. The highest mitotic index was obtained with an inflation of 30 mL. These observations suggest that cellular proliferation, growth, and development of the extracellular matrix in the induced subdermal capsule can be sustained by pulsed volume changes in the expander.

The effect of stepwise expansion on the mitotic activity and vascularity of subdermal tissue and induced capsule in the rat.

This study proposes that the mitotic activity and morphology of a subdermal induced capsule in response to a tissue expander can be regulated by the application of stepwise pressure increases in the expander. Tissue expanders were introduced into the fascial plane beneath the panniculus muscle of the rat and expanded in a stepwise fashion. Biopsies were taken at 48, 96, and 144 hours postexpansion at each increased pressure level. The developing capsule exhibited an increase in mitotic activity in its innermost layer, which reached a maximum 96 hours postexpansion. Application of constant pressure beyond 96 hours resulted in a progressive decrease in mitotic activity of the capsule. These observations suggest that cell proliferation and the resulting growth and differentiation of the extracellular matrix of the capsule may be controlled by appropriate pressure changes in the expander.

Responses of cultured cardiac myocytes to lysosomotropic compounds and methylated amino acids.

Cardiac myocytes whose lysosomes had been pre-labelled with acridine orange were exposed to either L-amino acid methyl esters (L-leucine or methionine methyl ester) or to 'lysosomotropic' weak bases (chloroquine, methylamine, and NH4Cl) for 1 h. Both types of interventions dilated lysosomes equally and inhibited proteolysis to varying degrees. The weak bases produced no apparent alterations in the acridine orange staining, whereas the methylated amino acids induced a marked redistribution of the fluorescent dye from lysosomes into the myoplasm, suggesting that they may have provoked a change in lysosomal membrane permeability. A brief exposure to weak bases failed to enhance acid proteinase secretion into the culture medium but apparently inactivated cellular cathepsin B activity. In contrast, methylated amino acids induced no alterations in acid proteinase activity or the cellular distribution of the two proteolytic enzymes. Lastly, weak bases markedly elevated intralysosomal pH as measured with fluorescein dextran, while only modest rises were observed after amino acid methyl ester treatment. The present observations imply that amino acid methyl esters represent a new class of reagents with actions distinctly different from those of chloroquine and NH4Cl, and they may provide a unique and valuable means of studying secondary lysosomal function in cell culture.

Alterations of cytoskeletal morphologies and growth patterns in human fibroblasts treated with polyethylene glycol.

Non-transformed human fibroblasts, strain PA-2, were treated with polyethylene glycol (PEG) in monolayer culture to produce multinucleate fibroblast homokaryons. Antibodies to tubulin or actin were used to monitor cytoplasmic microtubule and actin filament patterns immediately after cytoplasmic fusion, as well as after the fused cells had been in culture for varying amounts of time. The cytoplasmic microtubule complex as increased for a short time after cell fusion and then decreased to resemble the complex seen in control cells. The actin stress fibers were similarly enhanced for a comparable period of time. However, this initial enhancement of the actin stress fibers gradually diminished for approximately one month in culture after which the fibers were greatly reduced in both size and number. Concurrent with the changes in cytoplasmic microtubule and actin fiber complexes, the PEG-treated cells began to show alterations in growth parameters which progressively resembled those characteristic of transformed cell populations. Fusion of normal cells may be an initial step in the transformation of such cells to malignancy.

Cytoplasmic microtubules in transformed mouse x nontransformed human cell hybrids: correlation with in vitro growth.

Patterns of cytoplasmic microtubules in somatic cell hybrids between transformed mouse cells and nontransformed human skin fibroblasts were examined using antitubulin antibodies as an immunofluorescent probe. Nontransformed cells have been shown to exhibit an extensive cytoplasmic microtubule complex (CMTC), while in transformed cells, this complex is greatly diminished. The hybrid populations contained both types of cells. In addition, they contained cells with previously undescribed intermediate CMTC phenotypes. The percentage of each phenotype present in hybrid populations was determined for sixteen hybrid clones. Seven clones were found which appeared transformed on the basis of their CMTC pattern. The others were comprised of various proportions of all the cell types described. Repeated quantitation of the proportions of these types in the hybrid populations showed them to be stable with time in culture. Growth in vitro of the hybrid clones was assayed by determining their saturation densities, their plating efficiencies on plastic and their colony-forming abilities in soft agar. In vitro growth of a cell population was found to be directly proportional to the percentage of cells in the population which showed the greatly diminished CMTC pattern which has been described for transformed cells. This is strong evidence that a greatly reduced CMTC is associated with transformed behavior, especially the increased capacity of transformed cells for in vitro growth.

Temperature dependence of anaphase chromosome velocity and microtubule depolymerization.

The time course of chromosome movement and decay of half-spindle birefringence retardation in anaphase have been precisely determined in the endosperm cell of a plant Tilia americana and in the egg of an animal Asterias forbesi. For each species, the anaphase retardation decay rate constant and chromosome velocity are similar exponential functions of temperature. Over the temperature range at which these cells can complete anaphase, chromosome velocity and retardation rate constant yield a positive linear relationship when plotted against each other. At the higher temperatures where the chromosomes move faster, the spindle retardation decays faster, even though the absolute spindle retardation is greater. Chromosome velocity thus parallels the anaphase spindle retardation decay rate, or rate of spindle microtubule depolymerization, rather than absolute spindle retardation, or the amount of microtubules in the spindle. These observations suggest that a common mechanism exists for mitosis in plant and animal cells. The rate of anaphase chromosome movement is associated with an apparent first-order process of spindle fiber disassembly. This process irreversibly prevents spindle fiber subunits from participating in the polymerization equilibrium and removes microtubular subunits from chromosomal spindle fibers.