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The Journal of Immunology, 2000, 164: 4307-4312.
Copyright © 2000 by The American Association of Immunologists

Calcitonin Gene-Related Peptide Induces IL-8 Synthesis in Human Corneal Epithelial Cells1

Mau T. Tran, Mary H. Ritchie, Robert N. Lausch and John E. Oakes2

Department of Microbiology/Immunology, College of Medicine, University of South Alabama, Mobile, AL 36688


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Calcitonin gene-related peptide (CGRP), a neuropeptide with proinflammatory activities, is released from termini of corneal sensory neurons in response to pain stimuli. Because neutrophil infiltration of the clear corneal surface is a hallmark of corneal inflammation in the human eye, we determined whether CGRP can bind to human corneal epithelial cells (HCEC) and induce expression of the neutrophil chemotactic protein IL-8. It was found that HCEC specifically bound CGRP in a saturable manner with a Kd of 2.0 x 10-9 M. Exposure of HCEC to CGRP induced a significant increase in intracellular cAMP levels and enhanced IL-8 synthesis nearly 4-fold. The capacity of CGRP to stimulate cAMP and IL-8 synthesis was abrogated in the presence of the CGRP receptor antagonist CGRP8–37. CGRP stimulation had no effect on the half-life of IL-8 mRNA while increasing IL-8 pre-mRNA synthesis >2-fold. In contrast to IL-8, CGRP did not induce monocyte chemotactic protein-1 or RANTES synthesis, nor did the neuropeptide enhance detectable increases in steady state levels of mRNA specific for these two ß-chemokines. The results suggest that HCEC possess CGRP receptors capable of initiating a signal transduction cascade that differentially activates expression of the IL-8 gene but not the genes for monocyte chemotactic protein-1 or RANTES. The capacity of CGRP to stimulate IL-8 synthesis in HCEC suggests that sensory neurons are involved in induction of acute inflammation at the eye surface.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cornea is a specialized transparent epidermal tissue that covers the eye surface. Although corneal clarity can be diminished by inflammatory reactions, inflammation characterized by an infiltration of neutrophils can be generated within diseased or damaged corneal tissues (1, 2, 3). The mechanisms by which neutrophils are recruited to the eye surface from blood vessels surrounding the clear cornea surface are not clear. It has been shown that IL-1{alpha} can induce corneal epithelia to synthesize neutrophil chemotactic chemokines such as IL-8 (4, 5). Because abundant quantities of IL-1{alpha} are stored within epithelial cells (6, 7, 8), release of IL-1{alpha} from these cells may be one important inducer of chemokine synthesis at the eye surface. However, certain pathogens infecting corneal surfaces initiate inflammatory responses and neutrophil infiltration even though they do not induce release of IL-1{alpha} (8). This suggests that there may be other mechanisms for inducing the synthesis of chemokines involved in corneal inflammation.

Pain sensations are transmitted from peripheral tissues to the CNS by sensory neurons. CGRP3 is a 37-aa neuropeptide released from C-type sensory nerve fibers by the same kinds of irritants that initiate sensory impulses (9, 10). In vascularized tissues, CGRP plays a role in inflammatory responses by dilating blood vessels and stimulating leukocyte extravasation (11, 12, 13). In addition to regulating vascular function, CGRP can also influence immune responses. For example, CGRP can induce synthesis of several cytokines as well as interact with lymphocytes, Langerhans cells, and macrophages to modulate their immunological functions (14, 15, 16, 17, 18). Within the skin epidermis, release of CGRP from sensory nerves can inhibit Ag presentation by Langerhans cells as well as impair contact hypersensitivity by triggering mast cell release of TNF-{alpha} (18, 19).

The human cornea is densely innervated with CGRP containing C-type sensory neurons (20, 21). Because CGRP has known proinflammatory properties, this study was initiated to investigate the possibility that CGRP plays a role in corneal inflammation by specifically binding to epithelial cells and inducing expression of proinflammatory chemokines (22). It was found that HCEC have functional receptors for CGRP and that activation of these receptors by CGRP differentially induces expression of the neutrophil chemotactic chemokine IL-8, but not the monocyte chemotactic chemokine MCP-1 or the lymphocyte chemotactic chemokine RANTES. The role that sensory neurons within the human cornea may play in induction of acute inflammation after injury to the corneal epithelium is discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of HCEC

Human corneas were obtained from the National Disease Research Interchange (Philadelphia, PA). Pure cultures of HCEC were established within 48 h of enucleation as previously described (5, 23). IL-8 levels in cell cultures exposed to adrenomedullin, CGRP, or CGRP receptor antagonist (CGRP8–37, purity >97%) (Sigma, St. Louis, MO) were quantitated with ELISA kits purchased from R&D Systems (Minneapolis, MN). cAMP was quantitated with a cAMP enzyme immunoassay kit purchased from Stratagene (La Jolla, CA). The significance in chemokine or cAMP levels between unstimulated and stimulated cells was determined by using small sample paired t statistics. p < 0.05 was considered significant.

CGRP receptor binding assay

Confluent cultures of HCEC were exposed to 1 x 10-16 M to 1 x 10-8 M 125I-CGRP (Amersham, Arlington Heights, IL) with a specific activity of >2000 Ci/mmol in a final volume of 200 µl for 3 h on ice. A duplicate set of experiments was conducted in which 1 µM unlabeled CGRP was added to the binding buffer to determine nonspecific binding. Duplicate unlabeled wells were used to count cell numbers. After incubation with label, the supernatants were removed from the cultures, and the monolayers were washed twice with 200 µl PBS and then lysed using 200 µl 0.6% SDS in 10 mM Tris-EDTA buffer for 10 min. The lysates and supernatants were mixed with a scintillation cocktail and counted in a Beckman LX500CE scintillation counter (Beckman, Fullerton, CA) to determine levels of bound and unbound ligand. Nonspecific binding was found to be <15%. Scatchard analysis of the equilibrium saturation binding data was performed with the GraphPad Prizm program (GraphPad, Harvard University, Cambridge, MA).

Primer selection

cDNA sequences were obtained using the Wisconsin Genetics Computer Group program (Genetics Computer Group). PCR primers were then selected with the aid of OLIGO primer selection software (Eccles Institute for Human Genetics and Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT) run on the National Cancer Institute-Frederick Cancer Research Center Vax 6620 (Frederick, MD). Primers selected to amplify sequences complementary to mRNA molecules were chosen such that forward and reverse primers span at least one intron. Primers used to amplify pre-mRNA molecules were chosen such that forward primers were complementary to intron sequences whereas reverse primers were complementary to exon sequences as described previously (24). Each primer pair and the predicted size of its RT-PCR product are as follows: human IL-8 mRNA (253 bp), forward 5'-CTC TCT TGG CAG CCT TCC TGA TT -3', reverse 5'-AAC TTC TCC ACA ACC CTC TGC AC-3'; human IL-8 pre-mRNA (592 bp), forward 5'-CTC TCT TGG CAG CCT TCC TGA TT-3', reverse 5'-AAC TTC TCC ACA ACC CTC TGC AC-3; human CGRP-1 receptor mRNA (444 bp), forward 5'-AGA CAT CCA GCA AGC AAC AGA AC-3', reverse 5'-CCA GCC AAG AAA ATA ATA CCA CAT T-3'. Previously published primers were used to amplify human MCP-1, RANTES, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs, and GAPDH pre-mRNA molecules (24, 25). The identity of all RT-PCR products was verified by size and by sequencing on an Applied Biosystems 373XL Sequencer (Perkin-Elmer, Division PE/ABI, Foster City, CA). The sequences of the PCR products provided an exact match to the selected regions of the published sequences of these genes.

Analysis of mRNA and pre-mRNA levels by RT-PCR

RT-PCR products were generated from total cellular RNA with a GeneAmp RT-PCR kit (Perkin-Elmer, Norwalk, CT) according to the manufacturer’s instructions. In mRNA stability assays, 10 µg/ml actinomycin D were added to cell cultures 1 h after stimulation with 1 nM CGRP. At selected times post-actinomycin D treatment, total RNA was harvested and specific RNA molecules were amplified by RT-PCR. All RT-PCR amplifications used thermocycles of 30 s at 95°C, 30 s at 65°C and 2 min at 72°C. PCR products were analyzed on a 1.5% agarose gel stained with 1 µg/ml ethidium bromide and photographed with a Kodak Digital Science SP700 camera (Kodak Scientific Imaging Systems, New Haven, CT). The digitized images of the PCR products were quantitated using Kodak Digital Science Software (Kodak Scientific Imaging Systems, New Haven, CT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HCEC express receptors for CGRP

The CRLR gene product has been identified as the CGRP-binding component of the CGRP receptor (26). In preliminary experiments, it was found that a RT-PCR product complementary to the mRNA of the human CRLR protein could be amplified from HCEC RNA samples (Fig. 1Go, Inset A). These results suggest, therefore, that HCEC express a CGRP-binding protein. Equilibrium-binding experiments with 125I-CGRP were then performed to determine whether HCEC specifically bind this neuropeptide. Analysis of the binding data showed that HCEC bound CGRP in a specific and saturable manner (Fig. 1Go). A Scatchard plot revealed ~500 binding sites per cell with a Kd of 2.0 x 10-9 M (Fig. 1Go, inset B). This dissociation constant is consistent with Kd values for CGRP receptors found on other cell types (27, 28, 29).



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  		FIGURE 1. Steady state binding of 125I-CGRP to HCEC. Confluent monolayers of cells were incubated with increasing concentrations of 125I-CGRP. Specific binding was determined by subtraction of nonspecific binding measured in the presence of 1 µM unlabeled CGRP from total binding. Inset A, ethidium bromide-stained gel containing CRLR and GAPDH RT-PCR products amplified from RNA harvested from HCEC; Inset B, Scatchard plot generated from the radioligand binding data. The results are representative of three independent experiments done with three different donors.

 
CGRP stimulates IL-8 gene expression

To determine whether CGRP induces IL-8 gene expression in HCEC, a dose-response experiment was performed. HCEC from individual corneal donors were stimulated with 10-3–10 nM CGRP for 3 h. Media were then removed from stimulated cells and assayed for IL-8. As noted in earlier studies (5), unstimulated HCEC produced constitutive levels of IL-8 (Fig. 2GoA). However, exposure of HCEC to increasing concentrations of CGRP resulted in a dose-dependent increase in IL-8 synthesis resulting in significant IL-8 production in the presence of both 0.01 and 0.1 nM CGRP (p < 0.05). The C-terminal fragment of CGRP (CGRP8–37) is a specific CGRP receptor antagonist (27, 28, 29, 30). When corneal epithelial cell cultures were exposed to CGRP in the presence of 1 µM CGRP8–37, the amount of IL-8 secreted was not significantly enhanced (p > 0.05) (Fig. 2GoB). Thus, specific inhibition of the binding of CGRP to HCEC blocked induction of IL-8 synthesis.



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  		FIGURE 2. Effects of CGRP and its receptor antagonist on IL-8 synthesis. A, HCEC were cultured for 3 h with CGRP in the concentrations indicated. Culture supernatants were then collected and assayed for IL-8 by ELISA. The results are representative of the mean ± SEM from four corneal donors. B, Confluent monolayers of human epithelial cells were stimulated with either fresh medium, CGRP or CGRP plus 1 µM CGRP receptor antagonist CGRP8–37. At selected times poststimulation, supernatants were collected and assayed for IL-8 by ELISA. The results represent the mean ± SEM from three different donors.

 
In addition to its interaction with CGRP, the CRLR gene product can be modified by receptor-activity-modifying proteins to interact with adrenomedullin, a neuropeptide related to CGRP (26). To determine whether adrenomedullin can also function as a stimulator of IL-8 synthesis, we compared IL-8 synthesis in HCEC stimulated with adrenomedullin with its synthesis in cells stimulated with CGRP. In contrast to CGRP, it was found that adrenomedullin had no effect on IL-8 synthesis (Fig. 3Go). These results suggest, therefore, that adrenomedullin does not function as a physiological inducer of IL-8 synthesis in HCEC.



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  		FIGURE 3. Effect of adrenomedullin (Adm) on IL-8 synthesis in HCEC. Confluent cultures of HCEC were stimulated with either CGRP or adrenomedullin. At 3 h poststimulation, culture medium was harvested and assayed for IL-8. The results represent the mean ± SEM from four independent experiments.

 
CGRP induces cAMP synthesis

CGRP receptors are G-linked proteins that initiate signal transduction by activating adenylyl cyclase to catalyze formation of the intracellular second messenger cAMP (10). To test the hypothesis that increased IL-8 production in CGRP-stimulated cells is accompanied by increased intracellular cAMP levels, cell lysates were harvested at selected times after CGRP stimulation, and intracellular concentrations of cAMP were determined by an enzyme immunoassay. CGRP stimulation nearly doubled the concentration of intracellular cAMP (Fig. 4Go). This increase in cAMP levels was highly significant (p < 0.05). The dependence of cAMP formation on activation of the CGRP receptor was confirmed by demonstrating that CGRP did not stimulate cAMP synthesis in the presence of the receptor antagonist CGRP8–37 (Fig. 3Go). These results suggest, therefore, that CGRP receptors expressed on HCEC stimulate cAMP synthesis.



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  		FIGURE 4. Induction of intracellular cAMP levels in CGRP-stimulated cells. Confluent monolayers of HCEC were exposed to medium, CGRP, or CGRP plus 1 µM CGRP receptor antagonist CGRP8–37. At selected times postexposure, cell lysates were assayed for cAMP. Results are the mean concentration of cAMP generated from three different donors.

 
CGRP stimulates IL-8 gene expression by enhancing IL-8-specific RNA synthesis

IL-8 gene expression in CGRP-stimulated cells could potentially be induced at either the level of IL-8-specific RNA synthesis or at the level of IL-8 mRNA stability (31, 32, 33, 34). To determine whether increases in specific RNA synthesis contribute to the enhanced IL-8 production observed in CGRP-stimulated cells, IL-8 pre-mRNA levels were measured by RT-PCR. It was found that CGRP stimulation more than doubled the amounts of IL-8 pre-mRNA expressed as compared with levels expressed in unstimulated cells (Fig. 5Go). To determine whether IL-8 mRNA stability is increased in CGRP-stimulated cells, 10 µg/ml actinomycin D was added to cell cultures 1 h after the addition of CGRP. At selected times post-actinomycin D treatment, the levels of both IL-8 pre-mRNA and mRNA were measured by RT-PCR. After actinomycin D treatment, the half-life of IL-8 mRNA was <1.5 h in both CGRP-stimulated and unstimulated cells (Fig. 6Go). It was clear that actinomycin D inhibited IL-8 transcription in these experiments because IL-8-specific pre-mRNA synthesis was undetectable 1 h after actinomycin D treatment. These two experiments suggest, therefore, that CGRP stimulation increases levels of nuclear IL-8 pre-mRNA without significantly enhancing the stability of IL-8 mRNA.



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  		FIGURE 5. Effects of CGRP on steady state levels of intracellular IL-8 pre-mRNA. Confluent monolayers of HCEC were stimulated with either CGRP or medium alone. At selected times poststimulation, total RNA was isolated and IL-8 pre-mRNA molecules were amplified by RT-PCR. The RNA products were then quantitated by scanning the gels on an imager and normalizing with respect to the GAPDH (GAPD) pre-mRNA internal standard. The relative density represents the fold increase in the density of pre-mRNA amplified from stimulated cells over that of pre-mRNA amplified from unstimulated cells. RT-PCR products were not detected in parallel samples that did not contain either RNA or reverse transcriptase. Identical experiments were conducted with two additional donors and gave similar results.

 


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  		FIGURE 6. Stability of IL-8 mRNA in corneal epithelial cells after stimulation with CGRP. Confluent monolayers of HCEC were stimulated with either CGRP or with fresh medium. One hour poststimulation, 10 µg/ml actinomycin D were added to the cultures. At selected times after actinomycin D treatment, intracellular IL-8 pre-mRNA and mRNA molecules were amplified by RT-PCR. The levels of IL-8-specific mRNA were then quantitated by scanning gels on an imager and normalizing with respect to the GAPDH (GAPD) internal standard. The relative densities were then plotted against time. Arrows, Times post-actinomycin D treatment in which approximately one-half of the mRNA was degraded. A, Unstimulated cells; B, CGRP-stimulated cells. Insets, Agarose gels containing the IL-8 mRNA and pre-mRNA RT- PCR products. Identical experiments were done with two additional donors and gave similar results.

 
CGRP does not stimulate MCP-1 or RANTES gene expression

Even though potent chemokine inducers such as IL-1{alpha} and TNF-{alpha} stimulate IL-8 synthesis in HCEC, they do not enhance synthesis of chemokines such as MCP-1 or RANTES (25). Therefore, we were curious to know whether CGRP-stimulated HCEC produce a similar pattern of chemokine expression. To test this possibility, medium was removed from CGRP-stimulated cultures and assayed for MCP-1 and RANTES (Fig. 7Go). It was found that exposure of HCEC to CGRP did not stimulate significant increases in either MCP-1 or RANTES production. In addition, CGRP stimulation did not result in detectable increases in steady state levels of either RANTES or MCP-1 transcripts (data not shown). The cells were metabolically active in these experiments because CGRP induced the synthesis of significant levels of IL-8. These results suggest, therefore, that in contrast to the IL-8 gene, the genes for RANTES and MCP-1 are not up-regulated in HCEC in response to CGRP.



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  		FIGURE 7. Effects of CGRP on production of MCP-1 and RANTES. HCEC were stimulated with CGRP. At selected times poststimulation, culture media were assayed for IL-8, MCP-1, and RANTES by ELISA. Results represent the mean ± SEM from three different donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Even though the CRLR gene product has been identified as the CGRP receptor (26), little is known about the precise signal transduction pathways initiated by this protein. However, it is likely that the receptor stimulates IL-8 synthesis in HCEC by enhancing transcription of the IL-8 gene. This conclusion is supported by the fact that exposure of HCEC to CGRP had no effect on the half-life of IL-8 mRNA while increasing levels of nuclear IL-8 pre-RNA by >2-fold. These results would be expected if CGRP up-regulated the IL-8 gene by enhancing its transcription. It was also found that CGRP readily enhanced intracellular cAMP levels in stimulated cells. The transcriptional factor AP-2 has been reported to mediate signal transduction in response to cAMP (35). Because the IL-8 promoter possesses an AP-2-binding site, this factor could be involved in linking CGRP receptors to IL-8 gene expression (36). The IL-8 promoter also possesses a consensus binding site for the transcriptional factor AP-1 (36). In some cells, AP-1 is activated by stimulation of G-linked cell surface receptors through a mitogen-activated protein kinase cascade (37). Thus, it is possible that IL-8 gene expression was activated in CGRP-treated cells through the capacity of CGRP receptors to initiate a G-linked biochemical cascade that resulted in AP-1 activation.

In our study, it was found that the concentration of CGRP responsible for stimulating maximum levels of IL-8 synthesis was ~20-fold less than the Kd of its receptor. A similar situation has been found in lymphocytes where CGRP concentrations involved in stimulating these cells is also significantly less than the Kd (16). These results suggest that only a fraction of the cell surface CGRP receptors need to be occupied for the neuropeptide to produce significant biological responses. This finding is not unique to CGRP receptors given that it has been reported that the peptide hormone insulin can also produce maximal responses in its target tissues when only a fraction of cell surface receptors are occupied (38, 39). A contributing factor in the capacity of small numbers of CGRP receptors to activate IL-8 gene expression may be that these receptors are linked to the IL-8 gene promoter through more than one signal transduction pathway, providing opportunities to dramatically amplify signals initiated by receptor occupancy (35, 37).

It was interesting that exposure of HCEC to CGRP did not enhance either MCP-1 or RANTES synthesis because it was found in earlier experiments that potent chemokine inducers such as IL-1{alpha} and TNF-{alpha} also failed to stimulate production of these two chemokines (25). The assemblage of transcriptional factor consensus binding elements within the IL-8 promoter is considerably different from those found within promoters of the MCP-1 and RANTES genes (33, 40, 41). Therefore, we can speculate that these two chemokine genes are unresponsive to stimulation in these experiments either because one or more of the specific transcriptional factors needed to induce MCP-1 and RANTES genes are missing in HCEC or because transcriptional factors needed to up-regulate the two genes are not activated by the same stimuli responsible for IL-8 gene activation.

Recruitment of neutrophils to the eye surface is an important host defense mechanism against infectious agents that impinge on the corneal epithelium (3). IL-8 plays a crucial role in this process because it is the major chemotactic protein made by corneal epithelial cells (4, 5). Previous studies have identified IL-1{alpha} and TNF-{alpha} as major inducers of IL-8 synthesis in inflamed corneal tissues (4, 7). Data in this report suggest that release of CGRP from sensory neurons represents a third mechanism for induction of IL-8 synthesis at the eye surface. An explanation for this finding may be that HCEC have evolved the capacity to express active CGRP receptors to stimulate IL-8 synthesis in response to stimuli too weak to elicit IL-1{alpha} and TNF-{alpha} production. For example, IL-1{alpha} can be released from epithelial cells only by insults that disrupt membrane integrity whereas TNF-{alpha} is produced primarily by macrophages and lymphocytes that migrate into inflamed corneas during cell-mediated responses (42, 43). In contrast, CGRP is released from sensory nerves by insults that stimulate pain (9, 10). Since the cornea is exquisitely sensitive to pain stimuli, CGRP may be the sole inducer of IL-8 synthesis in response to minor insults or infections that are not severe enough to disrupt epithelial cell membranes or to stimulate cell-mediated immune responses. In addition, some pathogens can initiate acute inflammation without inducing detectable IL-1{alpha} release (8). Under these circumstances, CGRP released from sensory neurons may be an important mechanism whereby IL-8 expression is stimulated.

In summary, the accumulation of neutrophils within the cornea can alter the structure of this sensitive tissue, leading to vision loss. IL-8 plays an important role in inducing neutrophil infiltrates onto the eye surface because it is the major chemotactic protein made by corneal epithelium. In this study, CGRP receptors were found on HCEC with the capacity to stimulate IL-8 synthesis. These findings represent a novel mechanism for stimulating IL-8 gene expression within this ocular tissue. Future studies need to be directed at the identification of signal transduction pathways by which CGRP activates IL-8 gene expression and to determine whether antagonists of CGRP can be used as therapeutic agents to dampen corneal inflammatory responses.


   Acknowledgments
 
We thank Dr. David R. Poyner and Dr. Jonathan Scammel for their many helpful discussions and Patricia Couling for the careful preparation of the manuscript.


   Footnotes
 
1 This work was supported by in part by National Eye Institute Grant EY 03621. Back

2 Address correspondence and reprints requests to Dr. John E. Oakes, Department of Microbiology, University of South Alabama, MSB 2096, Mobile, AL 36688. Back

3 Abbreviations used in this paper: CGRP, calcitonin gene-related peptide; CRLR, calcitonin receptor-like receptor; HCEC, human corneal epithelial cells; MCP, monocyte chemotactic protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Back

Received for publication May 20, 1999. Accepted for publication February 7, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. III Waring, G. O., M. M. Rodrigues. 1987. Patterns of pathologic response in the cornea. Surv. Ophthalmol. 31:262.[Medline]
  2. Mondino, B. J.. 1988. Inflammatory diseases of the peripheral cornea. Ophthalmology 95:463.[Medline]
  3. Friedlaender, M. H.. 1993. The ocular immune response. Allergy and Immunology of the Eye 53. Raven Press, New York.
  4. Elner, V. M., R. M. Strieter, M. A. Pavilack, S. G. Elner, D. G. Remick, J. M. Danforth, S. L. Kunkel. 1991. Human corneal interleukin-8. IL-1 and TNF-induced gene expression and secretion. Am. J. Pathol. 139:977.[Abstract]
  5. Cubitt, C. L., Q. Tang, C. A. Monteiro, R. N. Lausch, J. E. Oakes. 1993. IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes. Invest. Ophthalmol. Vis. Sci. 34:3199.[Abstract/Free Full Text]
  6. Wilson, S. E., Y. G. He, S. A. Lloyd. 1992. EGF, EGF receptor, basic FGF, TGF ß-1, and IL-1 {alpha} mRNA in human corneal epithelial cells and stromal fibroblasts. Invest. Ophthalmol. Vis. Sci. 33:1756.[Abstract/Free Full Text]
  7. Rosenbaum, J. T., S. T. Planck, X. N. Huang, L. Rich, J. C. Ansel. 1995. Detection of mRNA for the cytokines, interleukin-1 {alpha} and interleukin-8, in corneas from patients with pseudophakic bullous keratopathy. Invest. Ophthalmol. Vis. Sci. 36:2151.[Abstract/Free Full Text]
  8. Tran, M. T., D. A. Dean, R. N. Lausch, J. E. Oakes. 1998. Membranes of herpes simplex virus type-1-infected human corneal epithelial cells are not permeabilized to macromolecules and therefore do not release IL-1{alpha}. Virology 244:74.[Medline]
  9. Wimalawansa, S. J.. 1997. Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily. Crit. Rev. Neurobiol. 11:167.[Medline]
  10. Brain, S. D.. 1997. Sensory neuropeptides: their role in inflammation and wound healing. Immunopharmacology 37:133.[Medline]
  11. Smith, C. H., J. N. Barker, R. W. Morris, D. M. MacDonald, T. H. Lee. 1993. Neuropeptides induce rapid expression of endothelial cell adhesion molecules and elicit granulocytic infiltration in human skin. J. Immunol. 151:3274.[Abstract]
  12. Sung, C. P., A. J. Arleth, N. Aiyar, P. K. Bhatnagar, P. G. Lysko, G. Feuerstein. 1992. CGRP stimulates the adhesion of leukocytes to vascular endothelial cells. Peptides 13:429.[Medline]
  13. Brain, S. D., J. R. Tippins, H. R. Morris, I. MacIntyre, T. J. Williams. 1986. Potent vasodilator activity of calcitonin gene-related peptide in human skin. J. Invest. Dermatol. 87:533.[Medline]
  14. Fox, F. E., M. Kubin, M. Cassin, Z. Niu, J. Hosoi, H. Torii, R. D. Granstein, G. Trinchieri, A. H. Rook. 1997. Calcitonin gene-related peptide inhibits proliferation and antigen presentation by human peripheral blood mononuclear cells: effects on B7, interleukin 10, and interleukin 12. J. Invest. Dermatol. 108:43.[Medline]
  15. Sakuta, H., K. Inaba, S. Muramatsu. 1995. Calcitonin gene-related peptide enhances cytokine-induced IL-6 production by fibroblasts. Cell. Immunol. 165:20.[Medline]
  16. McGillis, J. P., S. Humphreys, S. Reid. 1991. Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes. J. Immunol. 147:3482.[Abstract]
  17. Vignery, A., F. Wang, M. B. Ganz. 1991. Macrophages express functional receptors for calcitonin-gene-related peptide. J. Cell. Physiol. 149:301.[Medline]
  18. Hosoi, J., G. F. Murphy, C. L. Egan, E. A. Lerner, S. Grabbe, A. Asahina, R. D. Granstein. 1993. Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363:159.[Medline]
  19. Niizeki, H., P. Alard, J. W. Streilein. 1997. Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity. J. Immunol. 159:5183.[Abstract]
  20. Udea, S., M. del Cerro, LoCascio, and J. Aquavella. 1989. Peptidergic and catecholaminergic fibers in the human corneal epithelium. Acta Ophthalmol. 67:80.
  21. Muller, L. J., L. Pels, G. F. Vrensen. 1996. Ultrastructural organization of human corneal nerves. Invest. Ophthalmol. Vis. Sci. 37:476.[Abstract/Free Full Text]
  22. Baggiolini, M., B. Dewald, B. Moser. 1994. Interleukin-8 and related chemotactic cytokines: CXC and CC chemokines. Adv. Immunol. 55:97.[Medline]
  23. Cubitt, C. L., R. N. Lausch, J. E. Oakes. 1994. Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by pro-inflammatory cytokines. J. Immunol. 153:232.[Abstract]
  24. Cubitt, C. L., R. N. Lausch, J. E. Oakes. 1997. Differential induction of GRO{alpha} gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines. Invest. Ophthalmol. Vis. Sci. 38:1149.[Abstract/Free Full Text]
  25. Tran, M. T., M. Tellaetxe-Isusi, V. Elner, R. M. Strieter, R. N. Lausch, J. E. Oakes. 1996. Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells: ß-chemokine synthesis in corneal cells. Invest. Ophthalmol. Vis. Sci. 37:987.[Abstract/Free Full Text]
  26. McLatchie, L. M., N. J. Fraser, M. J. Main, A. Wise, J. Brown, N. Thompson, R. Solari, M. G. Lee, S. M. Foord. 1998. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393:333.[Medline]
  27. Aiyart, N., K. Rand, N. A. Elshourbagy, Z. Zeng, J. E. Adamou, D. J. Bergsma, Y. Li. 1996. A cDNA encoding the calcitonin gene-related peptide type 1 receptor. J. Biol. Chem. 271:11325.[Abstract/Free Full Text]
  28. Wimalawansa, S. J., R. D. Gunasekera, F. Zhang. 1993. Isolation, purification, and characterization of calcitonin gene-related peptide receptor. Peptides 14:691.[Medline]
  29. Sigrist, S., A. Franco-Cereceda, R. Muff, H. Henke, J. M. Lundberg, J. A. Fischer. 1986. Specific receptor and cardiovascular effects of calcitonin gene-related peptide. Endocrinology 119:381.[Abstract/Free Full Text]
  30. McMurdo, L., J. C. Lockhart, W. R. Ferrell. 1997. Modulation of synovial blood flow by the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8–37). Br. J. Pharmacol. 121:1075.[Medline]
  31. Caput, D., B. Beutler, K. Hartog, R. Thayer, S. Brown-Shimer, A. Cerami. 1986. Identification of a common nucleotide sequence in the 3'-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci. USA 83:1670.[Abstract/Free Full Text]
  32. Matsushima, K., K. Morishita, T. Yoshimura, S. Lavu, Y. Kobayashi, W. Lew, E. Appella, H. F. Kung, E. J. Leonard, J. J. Oppenheim. 1988. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167:1883.[Abstract/Free Full Text]
  33. Stein, B., Jr A. S. Baldwin. 1993. Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-{kappa} B. Mol. Cell. Biol. 13:7191.[Abstract/Free Full Text]
  34. Kunsch, C., C. A. Rosen. 1993. NF-{kappa}B subunit-specific regulation of the interleukin-8 promoter. Mol. Cell Biol. 13:6137.[Abstract/Free Full Text]
  35. Imagawa, M., R. Chiu, M. Karin. 1987. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251.[Medline]
  36. Mukaida, N., M. Shiroo, K. Matsushima. 1989. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J. Immunol. 143:1366.[Abstract]
  37. Gutkind, J. S.. 1998. The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J. Biol. Chem. 273:1839.[Free Full Text]
  38. Kono, T., F. Barham. 1971. The relationship between the insulin binding capacity of fat cells and the cellular response to insulin. J. Biol. Chem. 246:6210.[Abstract/Free Full Text]
  39. Gliemann, J., S. Gammeltoft, J. Vinten. 1975. Time course of insulin binding receptor binding and insulin-induced lipogenesis in isolated rat fat cells. J. Biol. Chem. 250:3368.[Abstract/Free Full Text]
  40. Udea, A., K. Okuda, S. Ohno, A. Shirai, T. Igarashi, K. Matsunaga, J. Fukushima, S. Kawamoto, Y. Ishigatsubo, T. Okubo. 1994. NF-{kappa}B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. J. Immunol. 153:2052.[Abstract]
  41. Nelson, P. J., B. D. Ortiz, J. M. Pattison, A. M. Krensky. 1996. Identification of a novel regulatory region critical for expression of the RANTES chemokine in activated T lymphocytes. J. Immunol. 157:1139.[Abstract]
  42. Stevenson, F. T., F. Torrano, R. M. Locksley, D. H. Lovett. 1992. Interleukin 1: the patterns of translation and intracellular distribution support alternative secretory mechanisms. J. Cell. Physiol. 152:223.[Medline]
  43. Beutler, B., A. Cerami. 1988. Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annu. Rev. Biochem. 57:505.[Medline]



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