The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density

PURPOSE: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-beta-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction. The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms. METHODS: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions. Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes in E. coli) were added in concentrations ranging from 3.125 to 100 ng/ml. Cell numbers were determined after an incubation period of 6 days using a cell counter. RESULTS: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period. Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation. When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation. This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner. In contrast, PDGF BB was not effective. CONCLUSION: The results of the "high-density" assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high. The results of the "low-density" assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.     

Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines

Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF-beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF-alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.     

Neutrophil chemotaxis in response to TGF-beta isoforms (TGF-beta 1, TGF-beta 2, TGF-beta 3) is mediated by fibronectin

TGF-beta isoforms regulate numerous cellular functions including cell growth and differentiation, the cellular synthesis and secretion of extracellular matrix proteins, such as fibronectin (Fn), and the immune response. We have previously shown that TGF-beta 1 is the most potent chemoattractant described for human peripheral blood neutrophils (PMNs), suggesting that TGF-beta s may play a role in the recruitment of PMNs during the initial phase of the inflammatory response. In our current studies, we demonstrate that the maximal chemotactic response was attained near 40 fM for all mammalian TGF-beta isoforms. However, there was a statistically significant difference in migratory distance of the PMNs: TGF-beta 2 (556 microM) > TGF-beta 3 (463 microM) > TGF-beta 1 (380 microM) (beta 2: beta 3, p < or = 0.010; beta 3: beta 1, p < or = 0.04; beta 2: beta 1, p < or = 0.0012). A mAb to the cell binding domain (CBD) of Fn inhibited the chemotactic response to TGF-beta 1 and TGF-beta 3 by 63% and to TGF-beta 2 by 70%, whereas the response to FMLP, a classic chemoattractant, was only inhibited by 18%. In contrast, a mAb to a C-terminal epitope of Fn did not retard migration (< 1.5%). The Arg-gly-Asp-ser tetrapeptide inhibited chemotaxis by approximately the same extent as the anti-CBD (52 to 83%). Furthermore, a mAb against the VLA-5 integrin (VLA-5; Fn receptor) also inhibited TGF-beta-induced chemotaxis. These results indicate that chemotaxis of PMNs in response to TGF-beta isoforms is mediated by the interaction of the Arg-gly-Asp-ser sequence in the CBD of Fn with an integrin on the PMN cell surface, primarily the VLA-5 integrin. TGF-beta isoforms also elicited the release of cellular Fn from PMNs; we observed a 2.3-fold increase in Fn (389 to 401 ng/ml) in the supernatants of TGF-beta-stimulated PMNs compared with unstimulated cells (173.6 ng/ml). The concentration of TGF-beta required to cause maximal release of Fn from PMNs (4000 fM) is a concentration at which TGF-beta is no longer chemotactic, suggesting that PMNs only use Fn that is constitutively expressed for migration. At higher concentrations of TGF-beta, the Fn released may accumulate basal to the cell, ultimately retarding cellular migration and modulating the chemotactic response.     

Proliferation of chicken myoblasts is regulated by specific isoforms of platelet-derived growth factor: evidence for differences between myoblasts from mid and late stages of embryogenesis

In the present study we measured the level of PDGF receptor expression by chicken myoblasts and the effect of the three different PDGF isoforms (AA, AB, BB) on DNA synthesis by myoblasts. We examined PDGF receptor expression and function on clonally derived myoblasts in order to eliminate contaminating fibroblasts which are present in myogenic cultures and which bind PDGF. Furthermore, since we have previously shown that fetal myoblasts are replaced with adult myoblasts during late chicken embryogenesis, we compared PDGF receptor expression and function on myoblasts from Embryonic Day 10 (E10, mid development) and from Embryonic Day 19 (E19, late development). We found that all myogenic clones from late embryos (E19) express many receptors for PDGF-BB, far fewer receptors for PDGF-AB, and even fewer, if any, receptors for PDGF-AA. Myoblast clones derived from E10 were more heterogeneous in their PDGF binding pattern ranging from clones similar to E19 clones to clones having very few PDGF binding sites. We also found that both PDGF-AB and PDGF-BB can promote DNA synthesis by clonally derived chicken myoblasts maintained in 2.5% fetal bovine serum whereas PDGF-AA has no detectable effect. Finally, we observed that primary myogenic cultures from E10 and E19 differ strikingly in levels of PDGF binding; E19 cultures bind much more PDGF than do E10 cultures. We conclude that PDGF can enhance the proliferation of chicken myoblasts and that myoblasts responsive to PDGF are more frequent in late than in mid stages of development. We propose that PDGF may be a modulator of myogenesis of adult but not fetal myoblasts.     

Interaction of fibronectin (FN) cell binding fragments and interleukin-8 (IL-8) in regulating neutrophil chemotaxis

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.     

Further evidence for the increased power of LOD scores compared with nonparametric methods

Platelet-derived growth factor (PDGF), a mitogen and chemoattractant for mesenchymal cells, occurs as cell-associated or released isoforms. To investigate their in vivo role, human keratinocytes, which normally synthesize both types of PDGF, were genetically modified to overexpress either wild-type PDGF-B (cell-associated) or the truncation mutant PDGF-B211 (released). Cells expressing the mutant isoform released 20 times more PDGF (145 ng/hour/10(7) cells) than cells expressing the wild-type isoform (6 ng/ hour/10(7) cells). When grafted as epithelial sheets onto athymic mice, modified cells formed a stratified epithelium and induced a connective tissue response that differed depending on the PDGF isoform expressed. Expression of PDGF-B211 induced a thick connective tissue with increased numbers of fibroblasts, mononuclear cells, and blood vessels evenly distributed throughout the connective tissue layer, whereas expression of PDGF-B induced a zone of fibroblasts and mononuclear cells localized to the interface of the epidermis and connective tissue, which often disrupted the continuity of the basement membrane. Immunostaining revealed that wild-type PDGF protein was deposited in the basement membrane region. These data suggest that the different binding properties of PDGF isoforms control the spatial organization of cellular events in regenerating mesenchymal tissue in vivo.     

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.     

Effect of antibiotics on the in vitro neutrophil chemotactic response

Polymorphonuclear neutrophilic leukocyte chemotaxis was examined in vitro in the presence of the following antibiotics: tetracycline, chloramphenicol, erythromycin, gentamicin, methicillin, sulfadiazine, cephalothin, oxacillin, nafcillin and clindamycin. Results indicate that high concentrations (1 mg/ml) of tetracycline, nafcillin, erythromycin and sulfadiazine inhibited the chemotactic response of neutrophils. Tetracycline produced an inhibition at concentrations as low as 1mug/ml. A stimulation of the neutrophil chemotactic response was observed in the presence of chloramphenicol in all concentrations. The results are discussed with particular emphasis on the implications to host immunity.     

Alteration of the neonatal pulmonary physiology after total cardiopulmonary bypass

PURPOSE: Smooth muscle cell (SMC) migration is an essential feature of the intimal hyperplastic process that so frequently limits the patency of vascular reconstructions. The purpose of this investigation was to evaluate the effect of a series of integrins, or cell surface receptors that mediate cellular attachment, on platelet-derived growth factor (PDGF) and extracellular matrix (ECM) protein-induced migration of human SMCs. METHODS: Immunofluorescence staining was used to search for various integrins and subunits on the surface of SMCs derived from human saphenous vein. Chemotaxis and haptotaxis of SMCs to various matrix proteins and PDGF were assayed using a 48-well microchemotaxis chamber in the presence or absence of antibodies that blocked the function of these integrins. RESULTS: Several subunits (beta 1, alpha 2, alpha 5) and one integrin (alpha v beta 3) were identified in saphenous vein SMCs. The beta 1 integrin antibody inhibited chemotaxis to collagen I and IV, laminin, and PDGF. The alpha 2 integrin antibody inhibited collagen I and IV, and laminin-induced chemotaxis. The alpha 5 integrin antibody had no effect on SMC migration. The alpha v beta 3 integrin antibody inhibited chemotaxis to PDGF but not to the ECM proteins. CONCLUSIONS: Integrins are necessary for SMC migration induced by PDGF and ECM proteins. The integrin or subunits responsible for facilitating migration varies with the stimulant. Agonists designed to inhibit integrin function might be used to suppress SMC migration and suppress the formation of intimal hyperplasia.     

Experience with the rectus abdominis myocutaneous flap with vascularized hard tissue for immediate orbitofacial reconstruction

We have previously reported (Badolato, R., J.M. Wang, W.J. Murphy, A. R. Lloyd, D.F. Michiel, L.L. Bausserman, D.J. Kelvin, and J.J. Oppenheim. 1994. J. Exp. Med. 180:203; Xu, L., R. Badolato, W.J. Murphy, D.L. Longo, M. Anver, S. Hale, J.J. Oppenheim, and J.M. Wang. 1995. J. Immunol. 155:1184.) that the acute phase protein serum amyloid A (SAA) is a potent chemoattractant for human leukocytes in vitro and mouse phagocytes in vivo. To identify the signaling mechanisms, we evaluated patterns of cross-desensitization between SAA and other leukocyte chemoattractants. We found that the chemotactic bacterial peptide, N-formyl- methionyl-leucyl-phenylalanine (fMLP), was able to specifically attenuate Ca2+ mobilization in human phagocytes induced by SAA, but only at very high concentrations, suggesting that SAA uses a low affinity fMLP receptor. Here we demonstrate that SAA selectively induced Ca2+ mobilization and migration of HEK cells expressing FPRL1, a human seven-transmembrane domain phagocyte receptor with low affinity for fMLP, and high affinity for lipoxin A4. Furthermore, radiolabeled SAA specifically bound to human phagocytes and FPRL1-transfected 293 cells. In contrast, SAA was not a ligand or agonist for FPR, the high affinity fMLP receptor. Thus, SAA is the first chemotactic ligand identified for FPRL1. Our results suggest that FPRL1 mediates phagocyte migration in response to SAA.     

Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors

The interactions of polymorphonuclear leukocytes, spermatozoa, seminal plasma, and serum have been examined in an in vitro leukocyte chemotaxis system. Bovine or human spermatozoa have no neutrophil chemotactic activity alone, but generate potent activity when incubated with serum. Generation of this activity requires magnesium, but not calcium ions and is blocked by pre-heating the serum. Chemotactic activity is generated normally in serum deficient in immunoglobulins or C4, but is not produced in serum deficient in C3. Thus, spermatozoa activate complement via the alternative pathway. The chemotactically active agent has been identified as the low m.w. C5 cleavage product (C5a) on the basis of heat stability, gel filtration characteristics, and specific inhibition by anti-serum to C5. The spermatozoal constituents responsible for complement activation are largely heat stable, resistant to diisopropyfluorophosphate and on gel filtration compise a heterogeneous group of large m.w. materials. Bovine seminal plasma has no neutrophil chemotactic activity alone; considerable activity, however, is generated by incubating low, but not high concentrations of seminal plasma with serum. The chemotactic agent produced in serum is C5a. Bovine seminal plasma also contains potent heat labile inhibitors of chemotaxis. Two distinct inhibitors differing in m.w. (50 to 100,000 and 15,000) and heat stability have been isolated. The inhibitors appear to act directly on the neutrophil rather than on the chemotactic factor. Human seminal plasma exhibits slight chemotactic activity alone but does not generate significant additional activity on incubation with serum. In contrast to the inhibitory activity of bovine seminal plasma, the human material contains heat-stable, nondialyzable factors which enhance the neutrophil chemotactic response. These studies document a specific mechanism for the directed migration of polymorphonuclear leukocytes toward spermatozoa. The findings may provide an explanation for the observed in vivo leukocyte accumulation in the female genital tract in association with spermatozoa and seminal plasma. The biologic significance of this phenomenon relates to potential leukocyte-spermatozoa interaction which may influence the likelihood of fertilization.     

Expression of cartilage extracellular matrix and potential regulatory genes in a new human chondrosarcoma cell line

The ability of differentiating cells to migrate within the developing central nervous system (CNS) depends on extrinsic guidance signals, some of which are growth factors. In this study we have investigated the chemotactic response of cultured stem cells from the embryonic rat cortex to platelet-derived growth factor (PDGF). Nestin-positive stem cells from the developing CNS can be maintained and expanded in vitro under serum-free conditions in the presence of basic fibroblast growth factor (bFGF). Northern blot analysis of PDGF receptor expression revealed both alpha- and beta-receptors on bFGF-treated neural stem cells. Both PDGF-AA and PDGF-BB readily induced directed migration of cultured neuroepithelial cells as measured in a microchemotaxis assay. Blocking of the migratory response was achieved by incubation with PDGF isoform-specific antibodies. More than 90% of the migrating cells were nestin-positive and incorporation of BrdU was also seen suggesting the cells to be immature and not yet committed to a specific cell lineage. These findings suggest a role for PDGF in cell migration in the developing cortex.     

Molecular weight of cytoplasmic malate dehydrogenase, mitochondrial malate dehydrogenase and lactate dehydrogenase of a freshwater catfish

The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA and BB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues. However, the presence of a single band (LDH-1) of LDH in liver and double bands (LDH-1 and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87 kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines

Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3-primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic effector cells of allergic inflammation, depend largely on CCR3 for migration towards different chemokines into inflamed tissues.     

Role of cytokines in the modulation of neutrophil chemotaxis in localized juvenile periodontitis

Decreased neutrophil chemotaxis has been implicated in the pathophysiology of the disease, localized juvenile periodontitis (LJP). The biological basis for the altered neutrophil function in LJP has been suggested to be an intrinsic cellular defect, involving a decrease in the number of N-formyl-methionyl-leucyl-phenylalanine (FMLP) receptors on the cell surface. We have investigated the relative contribution of serum-borne factors in the modulation of neutrophil functions in LJP, in a large population of LJP patients and healthy control subjects (HS). Treatment of HS-neutrophils with LJP-sera, resulted in a decreased neutrophil chemotactic response, and down regulation of FMLP receptors on the cell surface. Pretreatment of LJP-sera with anti-TNF and anti-IL-1 antibodies effectively, although incompletely, neutralized the ability of LJP-sera to modulate chemotaxis and FMLP receptor levels in HS-neutrophils. The changes induced by LJP sera were specific and sustained and could not be reversed by placing LJP-serum treated neutrophils in HS-serum. Sera obtained from HS and patients with adult periodontitis (AP), both of which exhibit normal chemotaxis, and patients with clinically diagnosed LJP with normal neutrophil chemotaxis (LJP-nctx) did not modulate HS neutrophil chemotaxis or FMLP receptors. Furthermore, recombinant human TNF-alpha, rhIL-1 alpha and rhIL-1 beta, at very low concentrations (15 pg/ml to 150 pg/ml), modulated the chemotactic response as well as FMLP receptor numbers on HS-neutrophils, in a manner similar to those observed in LJP. The present findings demonstrate that the biologic basis for the altered neutrophil function may not be an intrinsic cellular defect in neutrophils, but at least in part due to quantitatively small but biologically significant elevations in the levels of TNF-alpha and IL-1 in the serum.     

Transthoracic vertebrectomy for metastatic spinal tumors

Protein kinase C is an important second messenger system, which is translocated from the cytosol to the cell membrane upon cell stimulation. We used confocal microscopy to study the spatial distribution of protein kinase C isoforms after stimulation of cultured vascular smooth muscle cells with different agonists. First, we analysed the effects of angiotensin II and platelet-derived growth factor (PDGF). Confocal microscopy showed a rapid assembly of PKC alpha along cytosolic fibres followed by a translocation towards the nucleus with angiotensin II. PDGF engendered a similar, but much slower response; however, a cytoskeletal distribution was not observed. We then investigated the effects of thrombin and bFGF on nuclear translocation. bFGF induced a rapid translocation of the isoform towards the perinuclear region and into the nucleus. bFGF had a similar effect on PKC epsilon. In contrast, thrombin had a smaller effect on nuclear translocation of PKC alpha and did not influence PKC epsilon, but instead induced a rapid nuclear translocation of PKC zeta. Thus, tyrosine kinase receptor activation via bFGF induces a rapid association of PKC alpha and epsilon within nuclear structures. Our results show that agonists cause, not only a translocation of protein kinase C isoforms into the cell membrane but also into the cell nucleus. Lastly, we analyzed the nuclear immunoreactivity of the PKC isoforms, alpha, delta, epsilon and zeta in vascular smooth muscle cells during the cell cycle. Resting cells were stimulated with foetal calf serum (FCS, 10%), which translocated PKC alpha and epsilon to the perinuclear region and into the nucleus, while PKC delta and zeta showed no increase in nuclear immunoreactivity. After 4 h of FCS, the nuclear immunoreactivity for PKC alpha and epsilon was reduced to or below control values. At 8 h, increased nuclear expression of isoforms alpha, epsilon and zeta was observed, while isoform delta was not affected. Our results demonstrate a complex spatial and temporal regulation of PKC isoforms in response to vasoactive hormones and growth factors. We suggest that protein kinase C may be important for nuclear signaling and demonstrate that nuclear translocation of PKC isoforms is differentially regulated during the cell cycle.     

Alternative paths of zinc transepithelial transport in the small intestine

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.     

Dopamine D1/D2 antagonist combinations as antagonists of the discriminative stimulus effects of cocaine

Platelet-derived growth factor (PDGF) exists as a dimer composed of two homologous but distinct peptides termed PDGF-A and -B chains, and may exist as AA, AB, and BB isoforms. The PDGF-B chain has been implicated as a mediator of renal vascular rejection by virtue of up-regulated expression of its receptor, PDGF beta-receptor, in affected arteries. A role for PDGF-A chain in mediating intimal proliferation has been suggested in human atherosclerosis (Rekhter MD, Gordon D: Does platelet-derived growth factor-A chain stimulate proliferation of arterial mesenchymal cells in human atherosclerotic plaques? Circ Res 1994, 75:410), but no studies of this molecule in human renal allograft injury have been reported to date. We used two polyclonal antisera to detect expression of PDGF-A chain and one monoclonal antibody to detect PDGF-B chain by immunohistochemistry in fixed, paraffin-embedded tissue from 1) normal adult kidneys, 2) a series of renal transplant biopsies chosen to emphasize features of vascular rejection, and 3) allograft nephrectomies. Immunohistochemistry was correlated with in situ hybridization on adjacent, formalin fixed tissue sections from nephrectomies utilizing riboprobes made from PDGF-A and -B chain cDNA. PDGF-A chain is widely expressed by medial smooth muscle cells of normal and rejecting renal arterial vessels of all sizes by immunohistochemistry and in situ hybridization. PDGF-A chain is also expressed by a population of smooth muscle cells (shown by double immunolabeling with an antibody to alpha-smooth muscle actin) comprising the intima in chronic vascular rejection. In arteries demonstrating acute rejection, up-regulated expression of PDGF-A chain by endothelial cells was detected by both immunohistochemistry and in situ hybridization. In contrast, PDGF-B chain was identified principally in infiltrating monocytes within the rejecting arteries, similar to its localization in infiltrating monocytes in human atherosclerosis. Although less prominent than the case for PDGF-A chain, PDGF-B chain also was present in medial and intimal smooth muscle cells in both rejecting and nonrejecting renal arteries. PDGF-A and -B chains have now been localized at both the mRNA and protein levels to the intimal proliferative lesions of vascular rejection. These peptides, which are known stimuli for smooth muscle cell migration and proliferation in experimental vascular injury, may have similar stimulatory effects on smooth muscle cells in an autocrine and/or paracrine manner to promote further intimal expansion and lesion progression in this form of human vasculopathy.