Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency.

AIM: To investigate the expression of p63 and cytokeratins throughout the course of producing a cultivated autograft of limbal epithelial cells. METHODS: A 75 year old male with a severe alkali burn to his right eye received two cultivated autografts of limbal epithelial cells on amniotic membrane followed by a corneal allograft. Immunostaining for p63 and cytokeratins was performed during ex vivo expansion with 3T3 fibroblasts, following subcultivation on amniotic membrane, and on the excised corneal button. RESULTS: Cultures grown in the presence of 3T3 fibroblasts or on amniotic membrane displayed positive staining for keratins 14 and 19, and p63, but poor staining for keratin 3 (K3). The excised corneal button possessed a stratified epithelium of K3 positive cells residing on amniotic membrane. CONCLUSIONS: Our results document for the first time the co-expression of cytokeratins 14 and 19 with p63 in a cultivated limbal graft. These data support the conclusion that cultivated grafts of limbal epithelium contain predominantly undifferentiated cells with the potential to regenerate a normal corneal epithelium.

Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.

Inhibition of C5a-induced neutrophil chemotaxis and macrophage cytokine production in vitro by a new C5a receptor antagonist.

A cyclic peptide, Phe-[Orn-Pro-D-Cyclohexylalanine-Trp-Arg] (F-[OPdChaWR]), was recently shown in vitro to antagonise the binding of C5a to its receptor (CD88) on human polymorphonuclear leukocytes (PMNs) and in vivo to inhibit the neutropenia associated with septic shock induced by lipopolysaccharide (LPS) in rats. The aim of this study was to investigate whether F-[OPdChaWR] inhibits C5a-mediated chemotaxis of human PMNs using a modified Boyden chamber and C5a-stimulated release of cytokines from human monocytes in vitro. Approximately 50% of the chemotactic activity induced by 10 nM C5a was inhibited by 76 nM F-[OPdChaWR]. This correlated with inhibition of C5a-induced polarisation of PMNs by F-[OPdChaWR]. C5a alone failed to induce release of the inflammatory cytokines interleukin(IL)-1beta, tumour necrosis factor (TNF)-alpha, and IL-6 from human monocytes at concentrations up to 100 nM. However, in the presence of low concentrations of LPS (50 ng/mL), both IL-1beta and TNF-alpha were stimulated by 1 nM C5a. This co-stimulation was inhibited by F-[OPdChaWR] with IC(50)s of 0.8 and 6.9 nM for release of TNF-alpha and IL-1beta, respectively. No agonist activity was detected for F-[OPdChaWR] in either the chemotaxis or cytokine release assays at concentrations up to 50 microM. These results show that F-[OPdChaWR] inhibits several important inflammatory activities of C5a and suggest that C5a receptor antagonists may be effective in the treatment of inflammatory diseases mediated by C5a.

A novel approach to studying the migratory morphology of embryonic mesenchymal cells.

A polarized morphology, defined by extension of an anterior pseudopod, is essential for the amoeboid migration of embryonic mesenchymal cells. Leukocytes adopt a similar morphology immediately following suspension in simple buffers containing chemotactic factors. Polarization in suspension therefore provides a rapid and sensitive screening assay for putative regulators of leukocyte migration. The aim of the present study was to investigate whether this assay might also be used to study the motile behaviour of embryonic mesenchymal cells. Primary cultures of mesenchymal cells were established from explants of stage 28 chick embryo corneal-limbal stroma. Serum-starved, subconfluent cultures were harvested using ethylene-diamine tetra-acetic acid and resuspended in Hanks' solution for up to 15 min at 37 degrees C. A variety of cell shapes, including spherical cells, blebbed cells, and cells with either non-polarized or polarized pseudopodia were observed. The proportions of cells with pseudopodia increased significantly over time. Treatment of cells with the chemotactic mitogen platelet derived growth factor-BB (PDGF-BB, homodimer isoform) suppressed blebbing and increased both pseudopod formation and polarization. Optimal polarization occurred in concentrations of PDGF-BB that are similar to those required for optimal chemotaxis (10 ng x mL(-1)). The polarization observed in the absence of PDGF-BB suggests that the migration of cells examined in this study might be controlled at least in part by some intrinsic mechanism. In addition, the strong polarization response to PDGF-BB confirms the role for this growth factor during corneal development. Observations of mesenchymal cell morphology in suspension, therefore provide novel data regarding the motile behaviour of embryonic cells.

Imaging of renal medullary interstitial cells in situ by confocal fluorescence microscopy.

Renal medullary interstitial cells are a prevalent and characteristic feature of the inner medulla of the kidney, but the physiological significance of this is unclear. We have developed a method for imaging renal medullary interstitial cells in situ by loading the cells with fluorescent dyes and monitoring their distribution using confocal microscopy. The pH-sensitive probe 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester was used as a marker of cytoplasmic volume and therefore of cell morphology. Nile Red was used to demonstrate the presence of renal medullary interstitial cell lipid droplets. Papillae were excised from 100 g Sprague-Dawley rats and loaded with the appropriate dye. The papillae were then examined using a Leica TCS 4D confocal microscope and oil immersion lenses. Fluorescence was excited (488 nm) using an argon laser and emission wavelengths above 515 nm collected using a long pass filter. Images of papillae loaded with 2'7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester clearly demonstrate a ladder-like arrangement of renal medullary interstitial cells. More detailed examination revealed the presence of cytoplasmic extensions that appear to make close contact with adjacent loops of Henle. Three-dimensional reconstructions of serial sections revealed spiral arrangements in some ladders of renal medullary interstitial cells. Nile Red-labelled lipid droplets of 0.5-1.0 microm diameter were located throughout the cytoplasm of renal medullary interstitial cells and especially within the cytoplasmic extensions. These experiments highlight the ability of confocal microscopy to allow investigation of renal medullary interstitial cells in situ.

Eosinophilic interleukin 5 (IL-5) transgenic mice: eosinophil activity and impaired clearance of Schistosoma mansoni.

Eosinophilia is a feature common to many invasive helminth infections and eosinophils are often considered to be effector cells in immunity to helminths. This study examined the possible influence of constitutive eosinophilia on the clearance of Schistosoma mansoni infections in mice. Eosinophils from interleukin-5 transgenic mice exhibit normal ultrastructure and function with regard to phagocytosis and killing of bacteria and responses to chemotactic stimuli. IL-5 transgenics and non-transgenic littermates were immunized once or four (hyperimmunization) times with irradiated cercariae of S. mansoni. Animals were challenged percutaneously with unirradiated cercariae one month after their last exposure to irradiated parasites. One month after challenge transgenic animals, whether unimmunized, vaccinated or hypervaccinated, carried significantly more liver-stage parasites than non-transgenic animals. These results suggest that although eosinophils from IL-5 transgenic mice are functional for a number of key parameters, large numbers of eosinophils and/or high levels of IL-5 may in some way impair clearance of S. mansoni. A re-evaluation of the roles of eosinophils and IL-5 in infections with this and other parasites may therefore be warranted.

Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblasts cultured within collagen matrices.

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.5-1.0 kVcm-1/960 microF in DMEM/F12/20 mM Hepes, pH 7.2) with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one direction. The lack of directed movement is due to depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (< or = 10 microM Ca2+) maintain their cytoskeleton and continue to migrate, when returned to culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accumulates near the base of pseudopodia and at the rear of the cell where it is subsequently left behind. We conclude that electroporation is a valuable technique for loading fibroblasts to study migration within ECM, provided that the conditions used support stability of the tubulin cytoskeleton.

Chemotaxis of polymorphonuclear leukocytes towards human pre-ovulatory follicular fluid and serum using a 'sparse-pore' polycarbonate filtration membrane.

The chemotactic responses of polymorphonuclear leukocytes (PMN) towards pre-ovulatory follicular fluid and serum from patients participating in an in vitro fertilization program were tested in a modified Boyden chamber using a 'sparse-pore' polycarbonate filtration membrane method. The chemotactic activities of follicular fluids were generally low, but were significantly higher in conceptual cycles than in non-conceptual cycles. The chemotactic activities of sera were generally high, but were significantly lower in conceptual cycles. The chemotactic activity of few follicular fluids ever exceeded that of serum, regardless of the occurrence of conception. These findings demonstrate for the first time using an appropriate technique, that pre-ovulatory follicular fluids can be chemotactic for PMN, but the relationship between this activity, serum chemotactic activity and successful pregnancy is not clear.

Neutrophil polarisation in plasma differs to that induced by endogenous chemoattractants with regard to frequency of uropod formation and requirement for divalent cations.

Human neutrophils suspended in Hanks' balanced salt solution (37 degrees C, 20 mM Hepes, pH 7.2) produced extensions, elongated and developed a polarised morphology with both a pseudopod and uropod when exposed to C5a (10 nM), leukotriene B4 (10 nM), platelet activating factor (40 nM) or interleukin-8 (12.5 nM). Responses to each mediator were generally enhanced or unaffected by chelators of extracellular Ca2+ and Mg2+. Neutrophils suspended in heparinised plasma (90-10% v/v in Hanks' balanced salt solution) produced extensions, elongated and developed a pseudopod, but rarely developed a uropod unless additional Mg2+ ions (0.5-5 mM) were added. These findings demonstrate that the polarisation of neutrophils in plasma is significantly different to that induced by endogenous chemoattractants with regard to the frequency of uropod formation and requirement for extracellular divalent cations.

Comparison of techniques for the assessment of polymorphonuclear leukocyte polarisation in suspension.

Polarisation of polymorphonuclear leukocytes (PMN) in suspension was assessed using three techniques: 1) visual classification; 2) computerised morphometry; and 3) flow cytometry. While visual classification detected the formation, polarisation and type of cytoplasmic extensions produced by PMN, morphometry and flow cytometry detected only the formation of extensions. The area, perimeter and ellipticity were, in general, statistically different for each subtype of PMN-shape identified by visual classification. Furthermore, the magnitude and direction of changes detected by flow cytometry were affected by the use of erythrocyte lysis (during isolation of the cells) and the fixative used prior to analysis. The findings of this study demonstrate that visual classification is a more sensitive, reliable and appropriate assay of PMN polarisation than current morphometric and flow cytometric methods.

Interleukin-8 and neutrophil leucocytes: adhesion, spreading, polarisation, random motility, chemotaxis and deactivation in assays using 'sparse-pore' polycarbonate (nuclepore) membranes in the Boyden chamber.

Adhesion, spreading, chemotaxis and deactivation of chemotactic responses of separated human peripheral blood neutrophil leucocytes under the influence of interleukin-8 (IL-8) were tested using a previously described Boyden chamber-type method involving 'sparse-pore' polycarbonate (Nuclepore) filtration membrane. The random motilities of neutrophils in similar concentrations of IL-8 were tested using corresponding chambers with polycarbonate membranes of standard pore densities. In addition, polarisation of neutrophils in suspension in various concentrations of IL-8, and the possibility of deactivation of this polarisation response by IL-8 itself or by N-formyl-methionyl-leucyl-phenylalanine (FMLP) were examined. Neutrophils exhibited maximum chemotaxis to IL-8 in a concentration of 100 ng/ml, and to a degree which was similar to the maximum response to FMLP (10(-7) M). This chemotactic response to IL-8 was markedly reduced by pretreatment of the cells with either 100 ng/ml IL-8 (deactivation) or 10(-7) M FMLP (cross-deactivation). On the other hand, the chemotactic response of the neutrophils to FMLP was reduced by pretreatment with FMLP but was not deactivated by pretreatment with IL-8 (i.e. deactivation but not cross-deactivation). Neutrophils in suspension were maximally polarised by 100 ng/ml IL-8, and to lesser degrees by 1, 10 and 1,000 ng/ml IL-8. The detailed morphology of the polar cells was not distinguishable from that induced by 10(-8) M FMLP. Pretreatment of the cells with either IL-8 or FMLP resulted in no reduction of polarisation in response to subsequent exposure to either agent (i.e. neither deactivation nor cross-deactivation).(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of extracellular divalent cations to polarisation of polymorphonuclear leukocytes induced by plasma.

The roles of extracellular calcium and magnesium ions in the polarisation of human peripheral blood polymorphonuclear leukocytes (PMN) induced by autologous fresh heparinised plasma were investigated by studying the effects of 5 mM chelators of divalent cations [ethylenediamine tetra-acetic acid (EDTA), ethylenebis-(oxyethylene-nitrilo)-tetra-acetic acid (EGTA) or disodium hydrogen citrate]. In addition, the effects of a blocker of membrane calcium channels (verapamil) were studied. Polarisation of PMN suspended in plasma (84.1 +/- 11.9%) was reduced by each chelating agent over 30 min (to 20.0 +/- 15.6% by EDTA, to 42.5 +/- 19.3% by EGTA and to 29.4 +/- 22.9% by citrate). Polarisation of PMN suspended in plasma treated with EDTA or EGTA was restored by inclusion of equimolar additional Ca2+ ions, and in plasma treated with EDTA, EGTA or citrate, by equimolar additional Mg2+ ions. Additional Mg2+ had no effect on the spherical shape of PMN in Hanks' solution and additional cations had no effects on the polarisation of PMN induced by fMLP. Cells rendered spherical by each chelating agent in plasma for 30 min retained their ability to polarise on addition of fMLP to the plasma-chelator medium. Verapamil (10(-4) M) markedly reduced polarisation in plasma (to 52 +/- 11.3%) but the same drug (10(-5) M) had no such effect. In contrast to the polarisation of cells in plasma, the polarisation response of PMN to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) in buffered Hanks' solution was not affected by any of the chelating agents or by verapamil, even in high concentration. These results indicate that extracellular divalent cations are necessary for the polarisation of PMN suspended in autologous plasma and that the mechanism of polarisation of PMN in plasma may be different to that of polarisation induced by fMLP.

Effects of plasma proteins on the adhesion, spreading, polarization in suspension, random motility and chemotaxis of neutrophil leukocytes on polycarbonate (Nuclepore) filtration membrane.

The effects on adhesion, spreading, polarization in suspension, random motility and chemotaxis to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) of neutrophil leukocytes on polycarbonate (Nuclepore) membrane in the Boyden chamber of purified serum albumin, gamma globulin and fibrinogen, as well as of the Hanks'-soluble components of Cohn fractions III (beta and gamma globulins) and IV (albumin, alpha globulins and transferrin) of plasma proteins were tested in comparison to the corresponding effects of dilute fresh heparinized plasma. Serum albumin, fibrinogen and fibronectin inhibited adhesion of neutrophils, so that the spreading, random motility and chemotaxis of these cells could not be assessed in the presence of these proteins alone. These plasma proteins induced little or no polarization of the cells in suspension. Of the remaining preparations, gamma globulin promoted adhesion and excessive spreading and caused corresponding reduction of random motility and chemotaxis to FMLP of these cells. This plasma protein, even in low concentration, induced marked polarization of neutrophils in suspension. Both Cohn fractions III and IV supported adhesion and permitted random motility and chemotaxis to FMLP approximating that which occurs in dilute plasma. These fractions induced moderate polarization of cells in suspension. Addition of serum albumin or fibronection to the preparations of gamma globulin, or Cohn fractions III or IV did not alter the adhesion, random motility or chemotaxis of the neutrophils in these preparations. These results suggest that alpha and beta globulins, as well as gamma globulins are factors in plasma affecting adhesiveness and polarization of neutrophils and which provide for optimal motility and chemotaxis of these cells on solid substrata.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteoglycans synthesized by human polymorphonuclear leucocytes in vitro.

Polymorphonuclear leucocytes (PMN) were assessed in vitro for their ability to synthesize and secrete proteoglycans. The PMN were isolated from human peripheral blood and were found to contain less than 5% mononuclear cells. Following 24 h incubation in the presence of (35S)-sulfate, significant quantities of 35S-labelled macromolecules were detected both within the culture medium and cells. Although the PMN preparations contained some platelets (approximately five platelets:one PMN), culture of platelets alone did not result in the detection of any 35S-labelled macromolecules in either the medium or platelets. 35S/3H-labelled macromolecules from the PMN cultures were identified as proteoglycans on the basis of their degradation by papain, alkaline sodium borohydride, chondroitinase ACII, chondroitinase ABC and nitrous acid. The labelled proteoglycans isolated from the medium and cells eluted from Sepharose CL-4B with a Kav of 0.63; this indicated a small size compared with many other proteoglycans. The glycosaminoglycans associated with the proteoglycans were identified as heparan sulfate, chondroitin sulfate and dermatan sulfate, with chondroitin sulfate being the principal component. The average molecular weight of the glycosaminoglycans was determined to be 16,000. Therefore, the data from this study demonstrate the ability of human PMN to synthesize and secrete proteoglycans in vitro which appear to differ from those synthesized by mesenchymal cells with respect to molecular size and glycosaminoglycan composition.