Lipid peroxidation and acute lung injury after thermal trauma to skin. Evidence of a role for hydroxyl radical.

The authors have previously shown that thermal injury to the skin of rats results in the development of acute lung injury that is susceptible to systemic treatment of animals with catalase and dependent on the presence of neutrophils. The current studies have been expanded for exploration of the nature of the neutrophil-derived oxygen products responsible for the lung injury and have also focused on evidence of the appearance of products of lipid peroxidation (conjugated dienes). With respect to the former, treatment of rats with iron chelators (deferoxamine mesylate, 2,3-dihydroxybenzoic acid), with scavengers of hydroxyl radical (dimethyl sulfoxide, dimethyl thiourea, sodium benzoate), or with vitamin E affords a significant degree of protection from acute lung injury as assessed by changes in lung vascular permeability and by morphologic parameters. These data suggest that lung vascular injury after thermal trauma of the skin is related to the generation by neutrophils of the hydroxyl radical. Conjugated dienes have been demonstrated to appear sequentially both in the burned skin (at 1/4 hour) and in the lungs (at 2 hours), as well as in the plasma (with peaks at 1/2 and at 3 hours) after thermal injury. The appearance of the conjugated dienes in plasma at the two intervals of time is greatly diminished if animals are pretreated with the iron chelator deferoxamine, with catalase, or with scavengers of hydroxyl radical. Furthermore, the appearance of conjugated dienes in plasma at 30 minutes and 3 hours is significantly diminished if animals are depleted of neutrophils, complement-depleted, or the burned skin is excised immediately after thermal injury. These data indicate a linkage between thermal trauma of skin, secondary injury of lung, and appearance in plasma and tissues of products of lipid peroxidation.     

Role of cell adhesion molecules in leukocyte recruitment in the liver and gut

This article reviews the evidence that adhesion molecules are critical in leukocyte recirculation and pathogenesis of diseases affecting the closely related tissues of the liver and gut, which offer novel opportunities for treatment.     

Role of xanthine oxidase in thermal injury of skin.

These studies were designed to assess pathophysiologic factors responsible for increased vascular permeability occurring in rat skin that has been thermally injured in vivo. Under the conditions employed, permeability changes and edema formation progressed over time, with peak changes occurring 60 minutes after thermal trauma. The plasma of thermally injured rats showed dramatic increases in levels of xanthine oxidase activity, with peak values appearing as early as 15 minutes after thermal trauma. Excision of the burned skin immediately after thermal injury significantly diminished the increase in plasma xanthine oxidase activity. The skin permeability changes were attenuated by treatment of animals with antioxidants (catalase, superoxide dismutase [SOD], dimethyl sulfoxide [DMSO], dimethylthiourea [DMTU]) or an iron chelator (deferoxamine), supporting the role of oxygen radicals in the development of vascular injury as defined by greatly increased vascular permeability. Studies employing laser Doppler velocimetry in thermally injured skin revealed a pronounced and sustained decrease in blood flow after thermal trauma, a pattern not affected by protective interventions. The failure of neutrophil depletion to protect against the vascular permeability changes and the protective effects of the xanthine oxidase inhibitors (allopurinol and lodoxamide tromethamine) suggest that xanthine oxidase is the most likely source of the oxygen radicals involved in edema formation. Lodoxamide was found to have some hydroxyl radical (HO.) scavenging ability (greater than that of allopurinol) but no iron chelating activity. Some of the protective effects of lodoxamide and allopurinol may be linked to their HO. scavenging ability. These data suggest that, in this model of thermal trauma, vascular injury defined by increased vascular permeability is, in part, related to the activation of xanthine oxidase and the generation of toxic oxygen metabolites that damage microvascular endothelial cells.     

Expression of leukocyte adhesion molecules in human endometrium

In the present investigation the distribution of molecules that are involved in the leukocyte binding was studied in human endometrium. The expression of intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA-1), and HLA-DR molecules was studied in 13 proliferative and 10 secretory endometria as well as cultures of endometrial glands and stroma by avidin-biotin complex (ABC) procedure using monoclonal antibodies. The ICAM-1 and HLA-DR molecules were both strongly expressed in the lymphoid and endothelial cells. ICAM-1 expression was uniform in the epithelium, whereas the HLA-DR molecules were preferentially expressed in the epithelial cells in the basalis. Expression of both epithelial HLA-DR and ICAM-1 molecules was enhanced adjacent to lymphoid aggregates. ICAM-1 molecule was uniformly expressed in the stromal cells in the basalis and the functionalis, whereas the HLA-DR molecules were expressed exclusively in the stromal cells surrounding the lymphoid cells. ICAM-1 expression in the epithelial and stromal cells was confirmed in the isolated intact stromal cells and glands by immunohistochemistry. Although stromal and epithelial cells propagated in vitro expressed ICAM-1, they were rarely HLA-DR positive. The expression of LFA-1 was confined to the lymphoid cells. The high level of expression of ICAM-1, LFA-1, and HLA-DR molecules in human endometrial constituents may contribute to the presence, aggregation, and preferential distribution of lymphoid cells in human endometrium.     

Fibronectin fragments in lung lymph after thrombin-induced lung vascular injury

Fibronectin is an adhesive glycoprotein found in plasma and lymph as well as between lung endothelial cells and their collagenous basement membranes. Fibronectin is highly sensitive to proteolytic cleavage. We determined if fragments of fibronectin appear in lung lymph in association with increased lung protein clearance after thrombin-induced intravascular coagulation. Thrombin was infused intravenously, (80 units/kg for 30 minutes) into sheep (n = 8) surgically prepared with chronic lung lymph fistulas. Plasma and lymph fibronectin was assayed by electroimmunoassay. Fibronectin fragments were detected using Western blot analysis. After thrombin infusion, lymph flow increased 650% above baseline within 1-2 hours in association with a 35% decline in lymph-to-plasma total protein concentration ratio. This was followed by a second phase (3.5-6 hours) of normalized lymph-to-plasma ratios coupled with sustained elevation of lymph flow. Lung protein clearance remained elevated (p less than 0.10) for 5.5 hours. Plasma fibronectin levels declined slightly over 1-5 hours (zero time = 597 +/- 64 micrograms/ml; 1.5 hours = 478 +/- 59 micrograms/ml) and then increased significantly (p less than 0.05) over 24-48 hours (760 +/- 85 micrograms/ml). The amount of low molecular weight fibronectin fragments in lung lymph increased over the 1.5-6 hours post-thrombin and then declined over 12-48 hours. Thus after thrombin infusion, fragments of fibronectin were usually detected in increased amounts of lung lymph in association with an elevation of lung protein clearance.     

Reduction in cellular and vascular rejection by blocking leukocyte adhesion molecule receptors.

Whether antibody blockage of leukocyte receptors for intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 would prevent cardiac graft rejection was studied in a rabbit heterotopic transplant model. Monoclonal antibody 60.3, anti-CD18 (intercellular adhesion molecule-1 receptor, Group 1, n = 10) and monoclonal antibody HP1/2, anti-VLA-alpha 4 (vascular cell adhesion molecule-1 receptor, Group 2, n = 10) were administered to transplanted unimmunosuppressed animals. At 7 days, donor heart histology was compared to transplanted untreated controls (Group 3, n = 11). Peripheral white blood cell counts on postoperative day 2 were significantly higher in both treatment groups than controls. Significant increases in circulating neutrophils occurred in Group 1 (P < or = 0.05); lymphocytes predominated in Group 2 (P < or = 0.05). A significant reduction in cellular rejection was seen in Group 1 (P < or = 0.05) but not Group 2 hearts. Group 1 hearts demonstrated localization of lymphocytes to perivenular collections, whereas Group 2 hearts evidenced diffuse interstitial infiltration. Both treatment groups demonstrated a reduction in transplant arteritis compared to controls. Results suggest that monoclonal antibody 60.3 (anti-CD18) may hold promise as a therapeutic agent for both cellular and vascular rejection. Monoclonal antibody HP1/2 (anti-VLA-alpha 4) may reduce vascular rejection disproportionate to cellular rejection.     

Circulating leukocyte adhesion molecules in stable asthma and nonobstructive chronic bronchitis

Leukocyte adhesion molecules have been associated with airway inflammatory diseases such as asthma and obstructive chronic bronchitis. Lately, it has become possible to measure circulating forms of cell adhesion molecules 'cCAMs) in body fluids. Elevated serum levels have been found in acute asthma and in obstructive chronic bronchitis. We investigated whether the patterns of cICAM-1, cVCAM-1, and cE-selectin could serve as markers for airway inflammation in stable asthma and stable nonobstructive chronic bronchitis. Small-volume bronchial lavage 'BL) and serum from 15 controls, 13 asthmatics without steroid inhalation therapy, 11 asthmatics with regular steroid inhalation therapy, and 10 smokers with chronic bronchitis were analyzed. We found cICAM-1, cVCAM-1, and cE-selectin to be present in serum from patients with stable asthma and stable nonobstructive chronic bronchitis. Only cICAM-1 was found in BL fluid. No differences were seen between the subject groups for either cCAM, but levels of ECP were increased in the non-steroid-treated asthmatic group. Subject atopy or smoking did not increase the cCAM levels. In conclusion, the degree of airway inflammation in stable nonobstructive chronic bronchitis and stable asthma does not appear to be well associated with circulating ICAM-1, cVCAM-1, and cE-selectin.     

Vessel associated adhesion molecules in normal skin and acute graft-versus-host disease.

Immunohistological staining of skin from normal donors and bone marrow transplant recipients was undertaken using antibodies to two vessel associated adhesion molecules, endothelial leucocyte adhesion molecule-1 (ELAM-1). In normal skin ELAM-1 staining was restricted to a variable but generally small number of endothelial cells which were significantly increased in graft-versus-host disease (GvHD), but only when the fully developed histological picture of epidermal basal damage and leucocytic infiltration was present. All other biopsy specimens from marrow recipients taken before or after transplantation were similar to those of normal controls even in the presence of a clinical rash consistent with early GvHD. Although VCAM-1 positivity was seen on a few endothelial cells in normal skin, staining was mainly observed on dermal dendritic cells surrounding blood vessels and adnexal structures. In specimens with histological evidence of GvHD, positive perivascular dendritic cells were increased and were accompanied by the appearance of large numbers of similar cells dispersed throughout the upper dermis. Biopsy specimens from marrow recipients before and after transplantation resembled those from normal donors except for the presence of a rash after transplantation when some specimens, which lacked the leucocytic infiltrate diagnostic of GvHD, showed an increase in VCAM-1 positive cells, particularly in the upper dermis. The identification of these cells may therefore be useful in diagnosing early GvHD.     

Serum levels of adhesion molecules and thrombomodulin as indicators of vascular injury in severe Plasmodium falciparum malaria

Severe Plasmodium falciparum malaria is characterized by multiple organ involvment due to sequestration of infected erythrocytes in small vessels. Endothelial cell adhesion molecules play an important role in this interaction. During the course of a severe cerebral P. falciparum malaria infection we found very markedly elevated levels of the soluble adhesion molecules intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1, with a maximum increase of nine, seven, and eight times, respectively. These very high levels of soluble adhesion molecules point to an endothelial cell injury as an additional cause to physiological release or shedding due to receptor interactions. Soluble thrombomodulin (sTM) levels showed an extremely marked elevation up to 332 ng/ml (up to 13 times the normal value) as well. Malaria patients without severe organ involvement/cerebral manifestation showed only a mild elevation of sTM levels. TM is a parameter independent of the immunological system. It is regarded as a marker of vasculitis and endothelial cell destruction. Therefore, markedly elevated sTM levels document a substantial endothelial cell injury in severe malarial infection and may be of diagnostic and prognostic importance.Abbreviations VCAM-1 vascular cell adhesion molecule-1 (CD106) - ICAM-1 intercellular adhesion molecule-1 (CD54) - IL-2R interleukin-2 receptor (CD25) - TM thrombomodulin     

Expression of adhesion molecules in allergic lung diseases

Endothelial adherence and migration of leukocytes into tissue is mediated by different sets of adhesion molecules. The expression of these sets might not only preselect the types of leukocytes that enter the inflammatory sites, but also activate these leukocytes, induce adherence to epithelial cells, and cause the release of cytokines. Atopic asthma, extrinsic allergic alveolitis, and sarcoidosis as examples of immunologic lung diseases were investigated for the expression of adhesion molecules. Bronchial biopsies in chronic obstructive lung disease (COPD) and resected lung tissue of juvenile emphysema were chosen for controls. Immunohistochemistry was done on sections from bronchial and transbronchial biopsies and on smears from bronchoalveolar lavage cells. In all three types of immune disorders, lymphocytes expressed the integrins alpha4/beta1 (VLA4) and ICAM3, whereas lymphocytes in COPD bronchitis and in emphysema controls were unreactive. Eosinophils in atopic asthma bronchitis in contrast to COPD bronchitis also expressed both VLA4 and ICAM3. The expression of VCAM1 on endothelial cells was only seen in atopic asthma and was related to disease activity. The expression of other adhesion molecules was nonspecific. Expression of VCAM1 on endothelial cells and its ligand VLA4 on lymphocytes and eosinophils seems to be a specific event in atopic asthma. Expression of VLA4 and ICAM3 on lymphocytes, however, might be a specific event in all three immune reactions.     

Role of adhesion molecules in chronic allograft rejection

Endothelial adhesion molecules play an important role in T cell recruitment to an allograft site. Therefore, it could be expected that their blocking may be beneficial for allograft survival. In this report, we show that T cells from patients with chronic rejection have an up-regulated ability to adhere to inflamed endothelium in vitro. Furthermore, this enhanced T cell: endothelial interaction could be blocked by anti-VCAM and anti-E-selectin monoclonal antibodies.     

Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leukocyte adhesion.

Interleukin-1 (IL-1), an inflammatory/immune mediator, acts directly and selectively on cultured human vascular endothelial cells to alter two important functional properties. First, IL-1 induces endothelial cell biosynthesis and surface expression of a tissue factor-like procoagulant activity. Second, IL-1 dramatically increases the adhesiveness of the endothelial cell surface for human peripheral blood polymorphonuclear leukocytes (6-42-fold increase) and monocytes (2-5-fold increase), as well as the related leukocyte cell lines HL-60 and U937. These IL-1 effects are concentration-dependent (maximum, 5-10 U/ml), time-dependent (peak 4-6 hours), and reversible. Cycloheximide and actinomycin D block these IL-1 actions on endothelium, which suggests the requirement for de novo protein synthesis. Human-monocyte-derived IL-1, cell-line--derived IL-1, and recombinant IL-1 exhibited comparable biologic activities in our assays, whereas two other mediators, IL-2 and immune interferon, were without effect. IL-1 stimulated procoagulant activity and leukocyte adhesion in human endothelial cells cultured from both umbilical veins and adult saphenous veins but not in other cultured cell types, including SV-40-transformed human endothelial cells and human dermal fibroblasts. Similar actions of IL-1 on vascular endothelium in vivo may contribute to the development of intravascular coagulation and enhanced leukocyte--vessel wall adhesion at sites of inflammation.     

Role of dermal fibroblasts in rat skin tissue biomechanics

There are few studies on the effect of in situ fibroblast viability on the mechanical properties of skin. This study examines the effect of poison (2-deoxy-D-glucose) and strain on fibroblast viability and stress-relaxation in skin samples from the backs of 20 same-age male rats. Skin samples were either soaked in Kreb's solution or in poison and was then either strained or left unstrained, for a total of four different treatment groups. All samples were fixed and processed for apoptosis assay and light microscopy. The viability study showed that strained tissues soaked in Kreb's solution had significantly more apoptotic cells compared to unstrained tissues soaked in the same solution. For tissues soaked and strained in poison the increase in apoptotic cells was negligible. Samples strained in Kreb's solution were found to have greater stress relaxation compared to samples strained in Kreb's solution with poison. The amount of stress relaxation was found to correlate with the number of viable fibroblasts in the tissues; tissues with more viable fibroblasts have lower stress relaxation. According to the relationships observed, fibroblasts do play an important role in the mechanical properties of rat skin tissues.     

Effects of mixed leukocyte reaction,hydrocortisone and cyclosporine on expression of leukocyte adhesion molecules by endothelial and mesangial cells.

We investigated the effects of mixed leukocyte reaction (MLR), hydrocortisone (HC) and cyclosporine A (CsA) on the expression of leukocyte adhesion molecules on the mesangial (MC) and endothelial cells (EnC). Cell surface enzyme immunoassay showed that INFnu, IL-1beta, or TNF alpha stimulated expression of ICAM-1, or VCAM-1 on MC after 24 hours. Flow cytometric analysis demonstrated that MLR supernatant induced a marked increase in mean fluorescence of or % of cells highly expressing intercellular adhesion molecule(ICAM)-1 or vascular cell adhesion molecule (VCAM)-1 on both cells after 24 hours (p<0.001). HC treatment(300 ng/ml) during MLR effectively inhibited MLR-induced upregulation of ICAM-1 and VCAM-1 on both cells (p<0.005). When MLR supernatant with HC was added to adhesion molecule assay, there was no inhibitory effect of HC on VCAM-1. CsA treatment (500 ng/ml) during MLR caused a modest decrease in upregulation of VCAM-1 on EnC (p<0.05), but had no effects on ICAM-1 on both cells. CsA directly decreased expression of VCAM-1 on MC. In conclusion, alloreactive lymphocytes and monocytes upregulate the expression of VCAM-1 and ICAM-1 on target cells probably by the mediation of cytokines. HC effectively prevents MLR-induced upregulation of VCAM-1 and ICAM-1. CsA does not increase the expression of VCAM-1 and ICAM-1.     

Expression of vascular adhesion molecules on human endothelia in autoimmune thyroid disorders.

Cellular activation and expression of certain adhesion molecules within vascular endothelium is a critical event in leucocyte recruitment and emigration. A wide array of different adhesion receptors has been identified to mediate the interaction between endothelial cells (EC) and leucocyte subpopulations. In this study, the tissue expression of E-selectin, P-selectin, CD31, and endoglin endothelial cell adhesion molecules was studied on thyroid tissue from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). We found an up-regulated expression of E-selectin in EC in GD and HT thyroids, specifically in those areas more severely inflamed, with no reactivity in control thyroids. P-selectin was basally expressed in postcapillary venules in control glands, with an increased expression in HT and GD glands. On the other hand, increased CD31 expression was found on perifollicular, small and large venule EC from GD and HT glands, that correlated with the severity of mononuclear infiltration. In addition, CD31 expression was observed in some intrathyroidal macrophages and T cells in close proximity to CD31+ EC. Furthermore, a markedly enhanced expression of endoglin, a transforming growth factor-beta binding protein, was mainly located on perifollicular EC and EC from small venules as well as in adjacent macrophages from GD and HT thyroid glands. This enhanced expression of E- and P-selectins, CD31 and endoglin by thyroid EC in GD and HT may reflect their ability to regulate leucocyte trafficking and activation.