Effects of mechanical stress on cytokine production in mandible-derived osteoblasts.

OBJECTIVE: Mechanical stress is known to be an important factor in the regulation of bone remodeling, and mandibular bone is continuously exposed to mechanical stressors such as occlusal force. Therefore, in this study, we investigated the effects of mechanical stress approaching occlusal force, to which mandible-derived osteoblasts (MDOB) are exposed, on cytokine expression and production using an original hydrostatic pressure apparatus. MATERIALS AND METHODS: The levels of cytokine in MDOB were examined by real-time RT-PCR, ELISA, and western blotting. In addition, mitogen-activated protein kinase inhibitor for ERK1/2, JNK, and p-38 pathways was used to identify the signal transduction pathway. RESULTS: Hydrostatic pressure increased the expression of IL-6 and TNF-α mRNA in a magnitude- and time-dependent manner and also enhanced IL-6 and TNF-α protein production. Furthermore, hydrostatic pressure changed the RANKL/OPG ratio in favor of RANKL for both mRNA and protein levels. Specific inhibitor of p-38 pathway but not that of the ERK1/2 and JNK pathways suppressed the up-regulation of RANKL production induced by hydrostatic pressure loading. CONCLUSION: These results suggest that MDOB play a role in cytokine production in response to mechanical stress and that occlusal force may support the maintenance of mandible bone homeostasis by activating bone remodeling through osteoclastogenesis.

Eradication of the commensal intestinal microflora by oral antimicrobials interferes with the host response to lipopolysaccharide.

The host components and commensal microorganisms of the intestinal microenvironment play roles in the development and maintenance of the host defence. Recent observations have suggested that toll-like receptors (TLRs) are involved in the recognition of innate immunity against intestinal microbes. However, little is known regarding the role of TLR in the maintenance of systemic host defence by intestinal microorganisms. We studied the expression and function of TLR4 and TLR2 on alveolar and peritoneal macrophages in mice after 3 weeks of oral administration of streptomycin and cefotaxime. After active treatment, the intestinal microorganisms were nearly completely eradicated, and the surface expression of TLR4 and TLR2 on the peritoneal macrophages was prominently downregulated. When the actively treated mice were challenged with lipopolysaccharide (LPS), a TLR4 ligand, the host response was markedly impaired. Our results suggest that the oral administration of antimicrobials downregulates the expression of surface TLR on the peritoneal macrophages and modulates the host immune responses against LPS by modifying the intestinal environment.

Production and characterization of tumor-degenerating factor.

Degenerative changes of human tumor cells or continuous cell lines occur when they are cocultured with human embryonic fibroblasts. The present study confirmed that the degenerative changes of the target cells were due to a factor secreted from the human fibroblast culture, because the culture supernatant of human fibroblast caused the same degenerative changes as those induced by the coculture. This factor was termed "tumor-degenerating factor" (TDF). TDF was produced in the fibroblast culture as early as the 1st day and increased gradually up to the 8th day. TDF induced the degenerative changes in human KB cells, HeLa cells, FL cells, and PLC/PRF/5 cells but not in human WiDr cells or in fibroblasts. Also, it did not induce the degenerative changes in various murine cells, bovine cells, rabbit cells, or monkey cells, suggesting that TDF has species specificity. Furthermore, human leukocyte interferon enhanced the activity of TDF. TDF with the specific activity of 2.9 U/mg protein was purified by several chromatographies. At a final recovery rate of 14.6%, the specific activity was increased to 9,010 U/mg protein. Its molecular weight was estimated about 26,500-30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TDF was relatively stable to heat treatment.

Inhibitory effects of alpha-carotene on proliferation of the human neuroblastoma cell line GOTO.

alpha-Carotene inhibited the proliferation of the human neuroblastoma cell line GOTO in a dose- and time-dependent manner. In addition, it was about 10 times more inhibitory than beta-carotene. Northern blot analysis indicated that alpha-carotene caused maximum suppression of the level of the N-myc messenger RNA of GOTO cells. This suppression occurred within 18 hours of alpha-carotene treatment, after which the level of the N-myc messenger RNA gradually recovered to the basal level. Analysis by flow cytometry indicated that when GOTO cells were exposed to alpha-carotene, they were arrested in the G0-G1 phase of their cell cycle. However, as the level of the N-myc messenger RNA was recovering, these cells resumed normal cycling. These results indicate that the reduction in the level of the N-myc messenger RNA caused by alpha-carotene is closely linked with G0-G1 arrest.

Characterization of new human pancreatic cancer cell lines which propagate in a protein-free chemically defined medium.

Many human cancer cell lines which have been maintained in fetal bovine serum (FBS)-supplemented medium produce and secrete many substances such as transferrin, alpha 1-antitrypsin, alpha 2-macroglobulin, alkaline phosphatase, gamma-glutamyltranspeptidase, creatine kinase, carcinoembryonic antigen, alpha-fetoprotein, carbohydrate antigen 19/9, and cytokines including colony-stimulating factors and transforming growth factor, and further they may produce small amounts of unknown substances. Usually, small amounts of substances have to be concentrated as highly as possible for detection, but FBS interferes with this procedure. A protein-free culture system is an ideal method for detecting small quantities of substances which originate from cancer cells without interference by FBS. However, we were concerned that protein-free culture may interrupt the production of the substances which have been produced in FBS-supplemented medium. In this study, we investigated the productibility of 46 kinds of well-known substances in ten newly established cell lines derived from human pancreatic cancer. These cell lines were propagated in a protein-free non-FBS-supplemented medium. Of the ten cases, one cell line alone that was derived from acinal cell carcinoma propagated as a semisuspension; on the other hand, nine cell lines that were derived from ductal cell carcinoma propagated as monolayers without piling up. This method prolongs the doubling time, which is not affected by the addition of FBS. The spent media of these cell lines were collected aseptically after the removal of cell debris and concentrated by ultrafiltration using a Pericon cassette followed by lyophilization. Using 46 kinds of available antibodies, we investigated whether or not the substances which react to these antibodies could be detected in the spent media and in the cells by enzyme-linked immunosorbent assay, Western blot analysis, and immunocytochemistry. Among these cell lines, HPC-Y11 produced and secreted the most kinds of substances, and the production of those substances was lowest in HPC-Y0. In conclusion, our protein-free culture system can be available in every laboratory, since this is not only an economical method, but also an effective method for the saving of purification procedures. Moreover, this is a most suitable method for surveying unknown substances derived from cancer cell lines.

Characterization of a cathepsin L-like enzyme secreted from human pancreatic cancer cell line HPC-YP.

Spent culture medium from the human pancreatic carcinoma cell line HPC-YP, which can propagate in a protein-free, chemically defined medium without any other supplements, was analyzed for the presence of the cysteine protease, cathepsin L. The secreted form of cathepsin L was distinguished from the lysosomal form by its increased stability at alkaline pH, by its strong thermostability, and by its larger molecular size. HPC-YP cathepsin L was still stable at pH 7.4 and at 56 degrees C after 60-min preincubation. The molecular weight of this enzyme was estimated to be 68,000, compared with a molecular weight of 29,000 for normal liver cathepsin L. By Western blot analysis, HPC-YP enzyme was found to be composed of two components, one with a molecular weight of 37,000 and the other of 31,000. This result suggests that HPC-YP enzyme in the spent medium may be a complex of the proenzyme (in the case of liver proenzyme; Mr 39,000) and the mature enzyme (in the case of liver mature enzyme; Mr 29,000). Interestingly, an intrinsic inhibitor was also separated from the spent medium by gel filtration. The molecular weight of this inhibitor was estimated to be approximately 13,000. The cathepsin L of HPC-YP proved more resistant toward leupeptin than did liver cathepsin L. On the other hand, the former was more sensitive than the latter toward the diazomethane inhibitors, Z-Phe-Phe-CHN2 and Z-Phe-Ala-CHN2. These results indicate that cathepsin L secreted from cancer cell lines may play a role in the destruction of basal lamina, invasion of tissue, and formation of metastasis.

Tumor degeneration by human embryonic fibroblasts and its enhancement by interferon.

KB cells from a human nasopharyngeal tumor were cocultivated with human embryonic fibroblasts (HF 8101 cells); 7 to 14 days after incubation, "spongy degeneration"-like changes developed in the target cell-growing area. These changes developed in other target cells [HeLa cells from human cervical cancer, human hepatoma cells (PLC/PRF/5), and human amnion FL cells] cocultured with several kinds of human embryonic fibroblasts [HF 8101 cells, HF 8103 cells, and HEL cells]; however, HF 8101 cells did not cause degenerative changes in murine L929 cells. The degenerative changes were enhanced by treatment with human leukocyte interferon or human fibroblast interferon at a dose of 1,000 or 10,000 IU/ml, but there was no significant difference in the enhancing effect between human leukocyte and human fibroblast interferons. Mouse L929 interferon did not enhance the degenerative changes in KB cells caused by HF 8101 cells. It was concluded that human fibroblasts caused the degenerative changes in the human tumor cells and the continuous cell line and that the changes were enhanced by treatment with either human leukocyte interferon or human fibroblast interferon.

Differential susceptibility of HLA class II antigens induced by gamma-interferon in human neuroblastoma cell lines.

Five neuroblastoma cell lines have been examined for the induction of HLA class II antigens by a recombinant gamma-interferon. The expression of HLA-DR and -DP was induced on up to 80% of cells in two of five neuroblastoma cell lines examined (KP-N-SI and KP-N-RT). Low expression of HLA-DR and -DP was induced on the SK-N-DZ line in the presence of recombinant gamma-interferon for 10 days. In contrast HLA-DQ was not induced on any of five neuroblastoma cell lines studied. HLA-DR and -DP antigen induction was reversible, falling to nondetectable levels when interferon was removed from the culture medium. The reinduction of interferon to the culture medium again induced HLA-DR and -DP antigen expression in a fashion similar to that originally observed. These results were confirmed by Northern blot analysis using a probe to HLA-DR alpha mRNA. Recombinant interferon appears to induce HLA class II expression at the level of gene transcription or posttranscription. The results also indicate that HLA-DR, -DP, and -DQ antigens are independently regulated. Treatment of neuroblastoma cell lines with gamma-interferon results in the induction of a differentiated phenotype. Although the cytokine gamma-interferon induces neurofilament expression in some of the cell lines, this was not the case for all lines studied. Thus no correlation could be established between the morphological differentiation and either HLA class II or neurofilament expression. In addition, no correlation between response to recombinant interferon and N-myc amplification was noted. The biological significance of HLA class II expression on neuroblastoma cell lines by gamma-interferon may be related to the differentiation stage of neuroblastoma cells or may enable gamma-interferon-treated neuroblastoma cells to be recognized by cytotoxic T-cells.

Growth factors: importance in wound healing and maintenance of transparency of the cornea.

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.

Successful transfer of ADA gene in vitro into human peripheral blood CD34+ cells by transfecting EBV-based episomal vectors.

We report a novel non-viral system for transfecting human immature hematopoietic cells in vitro. Epstein-Barr virus (EBV)-based episomal vectors carrying human adenosine deaminase (ADA) gene cDNA were transfected by electroporation into human peripheral blood (PB) CD34+ cells. The transgene-specific mRNA were detected from 37 to 100% of CFU-c (colony forming unit in culture) colonies derived from the transfected cells. A two-fold increase in enzyme activity was also found. These results indicate the successful transfer and expression of genes in human immature hematopoietic cells using the EBV-based episomal vector system.

Expression of IFN-gamma in cerebrovascular endothelial cells from aged mice.

Recently, it has become clear that interferon-gamma (IFN-gamma) plays a role in the central nervous system (CNS) as well as in the immune system. However, the reason for the alteration in IFN-gamma production in the brain with aging remains unknown. In this study, we investigated the expression of IFN-gamma in the brain in terms of both mRNA and protein and compared the expression in young adult brain with that in aged mice. The cerebrum and cerebellum were collected from young adult (8-10 weeks old) and aged (24-26 months old) BALB/c mice, and the expressions of IFN-gamma and IFN-gamma receptor-1 (IFNGR-1) mRNA were examined by RT-PCR. Expression of IFN-gamma mRNA was detected in the brains from aged mice but not in those from young adult mice. However, IFNGR-1 mRNA was expressed in the brains from both young adult and aged mice. Moreover, IFN-gamma levels in the cerebrum and cerebellum from aged mice were detectable by ELISA, but IFN-gamma was undetectable in these tissues from young adult mice. To identify the cellular source of IFN-gamma in the brain of aged mice, immunostaining using antimouse IFN-gamma monoclonal antibody (mAb) was done. Immunoreactivity of IFN-gamma appeared to be located in cerebrovascular endothelial cells, including the choroid plexus of the cerebellum from aged mice. Expression of IFN-gamma and IFNGR-1 was also identified in isolated microvessels from brains. These results suggest that IFN-gamma plays a role in age-associated changes.

Cytokine expression and induction of acinar cell apoptosis after pancreatic duct ligation in mice.

To clarify the role of cytokines and acinar cell apoptosis in the pathogenesis of acute pancreatitis, we investigated the expression of intrapancreatic cytokines and apoptosis-related molecules in mice after pancreatic duct ligation (PDL). From day 1 or 3 after PDL, the expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-1 receptor antagonist, IL-6, IL-10, and tumor necrosis factor (TNF-alpha) mRNA were up-regulated in the pancreas, suggesting that these cytokines may be involved in the development of pancreatitis after PDL. Acinar cell apoptosis was observed in the pancreas at rates of 0.13 +/- 0.03, 1.32 +/- 0.38, and 0.86 +/- 0.23% on days 1, 3, and 7 after PDL, respectively. Significant increases in intrapancreatic mRNA levels of TNF-alpha, Fas ligand (FasL), and IL-1beta-converting enzyme (ICE) were observed from day 3 after PDL with the appearance of acinar cell apoptosis. The serum amylase activity peaked on day 1 after PDL and gradually decreased on days 3 and 7 after PDL. These results suggest that acinar cell apoptosis induced after PDL may modulate the progression of acute pancreatitis by reducing the release of digestive enzymes and may therefore be a host defense mechanism, and that acinar cell apoptosis after PDL may be mediated by the TNF-alpha and/or Fas/FasL and ICE system.

Determination of interferon-alpha2 allele composition in the genomic DNA from healthy volunteers and leukemic patients in Japan.

The three interferon-alpha2 (IFN-alpha2) sequences identified to date differ from each other in just two nucleotide positions, both of which result in changes in amino acids. Thus, the mature IFN-alpha2a protein product is characterized by a lysine residue at position 23 (AAA) and a histidine at position 34 (CAA), IFN-alpha2b has an arginine at position 23 (AGA) and histidine at position 34 (CAT), and IFN-alpha2c has arginine residues at both positions 23 (AGA) and 34 (CGT). These nucleotide variations in the DNA sequence can be distinguished by selective restriction enzyme analysis. We studied the distributions of the three IFN-alpha2 variants by analyzing chromosomal DNA from 103 Japanese volunteers and 33 patients with hematologic disorders. Fragments of 238 bp and 617 bp of the IFN-alpha2 gene containing codons 23 and 34 were amplified by PCR using specific primers, and the PCR products were analyzed with specific restriction nucleases to identify the IFN-alpha2 variant sequences. Only IFN-alpha2b gene was detected in normal volunteers, and no IFN-alpha2a gene was detected in Japanese subjects. However, IFN-alpha2c was detected in 4 of 33 (12.1%) patients with leukemia.

Kinetics of cytokine production in the cornea and trigeminal ganglion of C57BL/6 mice after corneal HSV-1 infection.

To investigate the role of cytokines in the pathogenesis of acute herpetic keratitis (HK), we examined the kinetics of cytokine expression in the corneas and the trigeminal ganglia (TG) of C57BL/6Cr (B6) mice after herpes simplex virus type 1 (HSV-1) infection and observed the influence of the targeted disruption of interferon-gamma (IFN-gamma) gene on the clinical course of HK and/or viral clearance. Following corneal infection with HSV-1 Amakata strain, all corneas developed a typical dendritic keratitis. Quantitative analysis using enzyme-linked immunosorbent assay (ELISA) revealed that the expression of interleukin-1alpha (IL-1alpha), IL-5, IL-6, and IFN-gamma in corneas and TGs significantly elevated immediately after infection, peaked between days 2 and 7 postinfection (p.i.), and then diminished. One exception was IFN-gamma, whose expression significantly persisted in the TGs until day 30 p.i. An additional experiment using IFN-gamma-/- (gko) mice revealed that there was no significant difference in the peak level of viral replication in corneas and TGs between gko and B6 mice, although gko mice showed a significant delay of virus clearance in both corneas and TGs (p < 0.005) and higher mortality rate than B6 mice after HSV-1 infection (p < 0.01). These data suggest that the production of proinflammatory cytokines closely correlates with the pathogenesis of HK, and that IFN-gamma plays an important role in enhancing viral clearance from the cornea and TG.

Highly efficient suicide gene expression in hepatocellular carcinoma cells by epstein-barr virus-based plasmid vectors combined with polyamidoamine dendrimer.

The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the beta-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, < or =50-fold higher beta-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100- to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 microM), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC.

Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter.

The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.

Extremely efficient gene transfection into lympho-hematopoietic cell lines by Epstein-Barr virus-based vectors.

We have estimated the efficiency of Epstein-Barr virus (EBV)-based vectors in transfecting genes into cell lines of lympho-hematopoietic lineages. The transfection efficiency was estimated both at transient and stable phases, in terms of expression of a marker gene and acquisition of drug resistance, respectively. Plasmid vectors carrying EBV oriP (replication origin of plasmid), EBNA (EBV nuclear antigen)-1 and as the marker genes, murine CD8 alpha cDNA and neoR (neomycin resistant) genes were transfected into various cell lines by electroporation. When cell lines constitutively expressing EBNA-1 were transduced, virtually all the cells expressed CD8 alpha on day 3 and acquired G418 resistance thereafter. In the case of K562 cells, which do not express EBNA-1, approximately 40% of cells expressed the marker gene product on day 3 posttransfection, and 30% of cells became stable transfectants. These data suggest a broader application of the EBV vector system in basic immunology and medicine.

Postexposure prophylaxis of varicella in family contact by oral acyclovir.

OBJECTIVE: To determine whether varicella can be prevented by administration of oral acyclovir (ACV) during the incubation period of the disease. SUBJECTS AND METHODS: ACV (40 or 80 mg/kg daily in four divided doses) was given orally to 25 exposed infants and children for 7 days, starting 7 to 9 days after exposure from the index case in their families. Their clinical features were compared with those of 25 age-matched control subjects who had been exposed in their families but did not receive ACV. A fluorescent antibody to membrane antigen assay was used for determination of the antibody to varicella-zoster virus, and a nested polymerase chain reaction method was used for detection of viremia. RESULTS: Among the 25 who received ACV, 4 (16%) developed the disease and 1 (4%) had a fever. On the other hand, all of 25 control subjects developed the disease and 17 (68%) had a fever. The incidence of fever and the severity of skin rashes were significantly lower (P < .01) in the subjects who received oral ACV than in the control group. Seroconversion was observed in 84% of subjects who received ACV. In some cases, varicella-zoster virus DNA was detected by polymerase chain reaction amplification in peripheral blood mononuclear cells from blood drawn approximately 14 days after exposure. CONCLUSIONS: Varicella can be prevented or modified by administration of oral ACV late in the incubation period.

Distribution of epidermal growth factor receptors in rabbit lens epithelial cells.

PURPOSE: This study examined the growth-promoting effect of epidermal growth factor (EGF) on rabbit lens epithelial cells and the distribution of EGF receptors in these cells. METHODS: Rabbit lens epithelial cells were cultured in TC-199 medium containing 0.5% fetal bovine serum and EGF for 1 to 5 days, and the growth-promoting effects of EGF were calculated with absorbance of methylene blue staining method. EGF receptors in the cells were analyzed by Scatchard plots using 125I-EGF in serum-free culture. RESULTS: EGF, which enhanced the cell growth in a dose-dependent manner, was stimulatory at 0.1-100 ng/ml and maximal (328.2%) with 10 ng/ml on day 5. The cells had two different affinity receptors. The number and the dissociation constant of the low affinity receptors were 5.81 x 10(4)/cell and 1.488 nM, respectively, and those of the high affinity receptors were 1.53 x 10(4)/cell and 0.159 nM, respectively. CONCLUSION: EGF may contribute to the development of living lenses.

Enhancement of fibronectin-induced migration of corneal epithelial cells by cytokines.

PURPOSE: To examine the enhancing effect of cytokines on the corneal epithelial cell migration induced by fibronectin (FN). METHODS: A modified Boyden chamber method was used to detect chemotactic cell migration. Cells plated in the inner chamber were incubated with FN, cytokines, or both in the outer chamber at 37 degrees C for 24 hours. Cells that had migrated were stained and counted under a microscope. Checkerboard analysis was used to distinguish chemotaxis from chemokinesis. RESULTS: FN induced epithelial cell migration, but interleukin (IL)-1 alpha, IL-6, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) alone did not. These cytokines, even at very low concentrations (0.1 to 100 pg/ml), enhanced FN-induced migration to levels about twofold those observed with FN alone. Checkerboard analysis demonstrated that EGF, but not IL-1 alpha, IL-6, or TNF-alpha, stimulated the chemokinesis of corneal epithelial cells in the presence of FN. CONCLUSION: EGF enhanced corneal epithelial cell migration by increasing chemokinesis, whereas IL-1 alpha, IL-6, and TNF-alpha enhanced this migration by increasing the FN-induced chemotactic activity, although these cytokines themselves do not have chemokinetic and chemotactic activity.