Wende R. Reenstra

Complement Activation after Oxidative Stress : Role of the Lectin Complement Pathway

The complement system plays an important role in mediating tissue injury after oxidative stress. The role of mannose-binding lectin (MBL) and the lectin complement pathway (LCP) in mediating complement activation after endothelial oxidative stress was investigated. iC3b deposition on hypoxic (24 hours; 1% O(2))/reoxygenated (3 hours; 21% O(2)) human endothelial cells was attenuated by N-acetyl-D-glucosamine or D-mannose, but not L-mannose, in a dose-dependent manner. Endothelial iC3b deposition after oxidative stress was also attenuated in MBL-deficient serum. Novel, functionally inhibitory, anti-human MBL monoclonal antibodies attenuated MBL-dependent C3 deposition on mannan-coated plates in a dose-dependent manner. Treatment of human serum with anti-MBL monoclonal antibodies inhibited MBL and C3 deposition after endothelial oxidative stress. Consistent with our in vitro findings, C3 and MBL immunostaining throughout the ischemic area at risk increased during rat myocardial reperfusion in vivo. These data suggest that the LCP mediates complement activation after tissue oxidative stress. Inhibition of MBL may represent a novel therapeutic strategy for ischemia/reperfusion injury and other complement-mediated disease states.

Gastrointestinal Ischemia-Reperfusion Injury Is Lectin Complement Pathway Dependent without Involving C1q

Complement activation plays an important role in local and remote tissue injury associated with gastrointestinal ischemia-reperfusion (GI/R). The role of the classical and lectin complement pathways in GI/R injury was evaluated using C1q-deficient (C1q KO), MBL-A/C-deficient (MBL-null), complement factor 2- and factor B-deficient (C2/fB KO), and wild-type (WT) mice. Gastrointestinal ischemia (20 min), followed by 3-h reperfusion, induced intestinal and lung injury in C1q KO and WT mice, but not in C2/fB KO mice. Addition of human C2 to C2/fB KO mice significantly restored GI/R injury, demonstrating that GI/R injury is mediated via the lectin and/or classical pathway. Tissue C3 deposition in C1q KO and WT, but not C2/fB KO, mice after GI/R demonstrated that complement was activated in C1q KO mice. GI/R significantly increased serum alanine aminotransferase, gastrointestinal barrier dysfunction, and neutrophil infiltration into the lung and gut in C1q KO and WT, but not C2/fB KO, mice. MBL-null mice displayed little gut injury after GI/R, but lung injury was present. Addition of recombinant human MBL (rhuMBL) to MBL-null mice significantly increased injury compared with MBL-null mice after GI/R and was reversed by anti-MBL mAb treatment. However, MBL-null mice were not protected from secondary lung injury after GI/R. These data demonstrate that C2 and MBL, but not C1q, are necessary for gut injury after GI/R. Lung injury in mice after GI/R is MBL and C1q independent, but C2 dependent, suggesting a potential role for ficolins in this model.

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

Complement-Mediated Loss of Endothelium-Dependent Relaxation of Porcine Coronary Arteries: Role of the Terminal Membrane Attack Complex

Reperfusion of the ischemic myocardium results in the loss of endothelium-dependent relaxation. We have shown recently that the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium. We hypothesized that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary arteries. Bradykinin (BK) or substance P concentration-dependently relaxed precontracted (U46619, 50 nmol/L) left anterior descending coronary artery (LAD) rings in vitro. Addition of zymosan to human (10%) or porcine (10%) serum for 30 minutes significantly (P < 0.05) increased the EC50 of BK-induced LAD relaxation from 4 +/- 1 to 418 +/- 159 nmol/L (n = 8) and from 9 +/- 3 to 281 +/- 132 nmol/L (n = 7), respectively. Similarly, addition of zymosan to 10% human serum (HS) for 30 minutes increased the EC50 of substance P-induced LAD relaxation from 0.4 +/- 0.1 to 30 +/- 14 nmol/L (n = 9, P < .05). Basal release of nitric oxide was reduced significantly in LAD rings exposed to zymosan-activated HS compared with HS alone. Addition of soluble CR1 (sCR1, 10 nmol/L) to zymosan-activated HS preserved BK-induced relaxation (EC50) of the LAD rings (control, 4 +/- 1 nmol/L; sCR1 + zymosan+serum, 2 +/- 1 nmol/L; n = 6). Zymosan-activated C8-depleted HS (10%) did not attenuate the EC50 of BK-induced coronary artery relaxation (3 +/- 1 to 3 +/- 1 nmol/L, n = 7, P = NS). Zymosan-activated C8-depleted HS plus C8 (6 micrograms/mL) increased the EC50 of BK-induced coronary artery relaxation from 4 +/- 1 to 423 +/- 141 nmol/L (n = 12, P < .05). We have further demonstrated that C5b-9 complexes can be found on the luminal surface of LAD endothelial cells after 5 minutes of exposure to zymosan-activated HS by using C5b-9 reactive monoclonal antibody fluorescent immunohistochemistry and confocal microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial nuclear factor-κB translocation and vascular cell adhesion molecule-1 induction by complement inhibition with anti-human C5 therapy or cGMP analogues

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism.

Objective:This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)–induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. Design:Prospective, experimental study. Setting:University experimental research laboratory. Subjects:C57BL/6 and C57BL/6 IL-10−/− mice. Interventions:Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b+ peritoneal macrophages. Measurements and Main Results:Survival was monitored for 100 hrs after CLP ± HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. Conclusion:HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10−/− mice were not protected by HBO2 treatment.

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

ECM-stimulated actin bundle formation in embryonic corneal epithelia is tyrosine phosphorylation dependent.

Previous studies demonstrated that corneal epithelial cells isolated without basal lamina respond to extracellular matrix (ECM) in an actin dependent manner; the basal cell surface flattens and the actin cortical mat reorganizes. We hypothesize that the actin reorganization is initiated by intracellular signaling mechanisms that includes tyrosine phoshporylation and activation of the Rho, MAP kinase, and PI3 kinase signal transduction pathways. Our goals were to develop a morphological assay to test this hypothesis by answering the following questions: 1) Do the actin bundle formations in the cortical mat have the same configuration in response to different ECM molecules? 2) What is the minimum time ECM molecules need to be in contact with the tissue for the actin to reorganize? 3) Will blocking tyrosine phosphorylation inhibit reorganization of the actin? 4) Are known signal transduction proteins phosphorylated in response to soluble matrix molecules? The actin cortical mat demonstrated distinct bundle configurations in the presence of different ECM molecules. Soluble fibronectin accumulated at the basal cell surfaces 75‐fold over 30 min in a clustered pattern. The cells need contact with ECM for a minimum of 10 min to reform the actin bundles at 2 hr. In contrast, two substances that bind to heptahelical receptors to stimulate the Rho pathway, bombesin and lysophosphatidic acid, reorganized the actin bundles in 15–30 min. Focal adhesion kinase, p190 Rho‐GAP, tensin, and paxillin were tyrosine phosphorylated in response to soluble fibronectin, type I collagen, or laminin 1. Erk‐1, erk‐2, and PI3 kinase were activated after 1 hr stimulation by type I collagen. Herbimycin A blocked actin reorganization induced by ECM molecules. In conclusion, we have developed two morphological assays to examine the response of corneal epithelial cells to ECM molecules. In addition, actin bundle reorganization involved tyrosine phosphorylation, MAP kinase, and PI3 kinase activation. Anat Rec 254:348–359, 1999.

Factors influencing fetal macrophage development: III. Immunocytochemical localization of cytokines and time‐resolved expression of differentiation markers in organ‐cultured rat lungs

Exogenous TNFα, IL‐1β, M‐CSF, and GM‐CSF all stimulate growth of macrophages arising in explanted fetal rat lungs. The present study examines the intrinsic availability of these factors in intact and organ‐cultured lungs and utilizes expression of cytokines and marker proteins to explore the differentiation pathway followed by phagocytes in vitro.

Keratinocytes and dermal factors activate CRABP‐I in melanocytes

Abstract: Recognition that cellular retinoic acid binding protein (CRABP)‐I and CRABP‐II are found in different cell types has provided additional support for the presumably divergent roles of these two proteins in mediating retinoic acid (RA) effects in human skin. CRABP‐II is expressed in fibroblasts and keratinocytes, and CRABP‐I in as yet unidentified cells, possibly epidermal melanocytes. Recently, we demonstrated that each of these RA‐binding proteins in human skin possesses two classes of binding sites, possibly related to the state of phosphorylation of the proteins. We now characterize the cutaneous origin of CRABP‐I further using an anion‐exchange HPLC assay that allows effective separation of the two proteins in human skin, and a fluorescent in situ hybridization technique. We report that CRABP‐I is expressed in isolated melanocytes at the mRNA level, although under these circumstances the protein has minimal RA‐binding activity, and that keratinocytic and dermal influences are required for CRABP‐I activity in melanocytes. This melanocyte origin for CRABP‐I and the improvement by RA of the irregular hyperpigmentation associated with photoaging led us to examine the effects of RA using various cellular associations, from conventional pure cultures of melanocytes grown on plastic dishes to a pigmented skin equivalent consisting of melanocytes and keratinocytes grown on a dermal equivalent. We established that the inhibitory effects of RA on melanogenesis do not result from a direct effect on melanocytes alone but also involve keratinocytes and dermal influence. These data expand our understanding of cell‐to‐cell signaling in cutaneous pigmentation, and strongly suggest a role for CRABP‐I in mediating RA effects on melanogenesis.