Peptides,Vol. 13, pp. 429-434, 1992
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
CGRP Stimulates the Adhesion of Leukocytes to Vascular Endothelial Cells C H E N G - P O S U N G , l A N T H O N Y J. A R L E T H , N A M B I A I Y A R , P R A D I P K. B H A T N A G A R , P A U L G. L Y S K O A N D G I O R A F E U E R S T E I N
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Received 5 D e c e m b e r 1991 SUNG, C.-P., A. J. ARLETH, N. AIYAR, P. K. BHATNAGAR, P. G. LYSKO AND G. FEUERSTEIN. CGRPstimulates the adhesion ofleukocytes to vascularendothelialcells. PEPTIDES 13(3) 429-434, 1992.--Calcitonin gene-related peptide (CGRP) stimulates the adhesiveness of human umbilical vein endothelial cells for U937 cells and human neutrophils in a dose- and timedependent manner. The onset of CGRP-induced adhesives of HUVEC was rapid (30 min), independent of protein synthesis, and lasted over 24 h in the continuous presence of the peptide. The stimulatory effect of CGRP was completely blocked by the CGRP antagonist, CGRP(8-37). The present study provides evidence in support of the potential role of sensorynerve-derivedneuropeptides in the modulation of leukocyte adhesion to vascular endothelial cells. CGRP Neuropeptide Neuroimmunology
Leukocyte--endothelialcell adhesion
THE central nervous system has been implicated in modulation of immune and inflammatory reactions. Reciprocal interactions between the nervous system and immune and inflammatory cells have been demonstrated at both the cellular and humoral levels (1). The cellular mode of interaction is exhibited by the intense innervation of primary (thymus, bone marrow) and secondary (lymph nodes, spleen) immune organs by sensory and autonomic nerves (10). The humoral mode of interaction is supported by demonstrations that neural-derived factors, such as substance P (SP), VIP, opioid peptides, nerve growth factor (NGF), somatostatin, and other peptides, activate or suppress circulating immune/inflammatory cells (17). Moreover, specific neuropeptides, (e.g., a-MSH) possess antipyretic (18) and antiinflammatory activities, probably by modulation of inflammatory cell mediators such as tumor necrosis factor (cachectin, TNFa) or intefleukin-1 (23). While there is ample evidence in support of a role for neuronal-derived factors in the activation and regulation of immune and inflammatory cells, no evidence so far has implicated sensory nerve-derived factors in the regulation of a critical step in the initiation of inflammation, i.e., the adhesion of leukocytes to the endothelium. Since the initial events in inflammation involve adhesion of leukocytes to vascular endothelial cells, we hypothesized that neuronal-derived peptides might modulate leukocyte adhesion to vascular endothelial cell. In the only report available to date, we showed that NPY increased U937 cell adhesion to HUVEC, suggesting a role for a sympathetic nerve-derived neuromodulator in regulation of endothelium adhesiveness (26). The present study provides evidence supporting that the sensory
Adhesion molecules
Endothelium
nerve-derived peptide, CGRP, also promotes the adhesiveness of human vascular endothelium for leukocytes. METHOD
Reagents Human CGRP (98% purity, 69% peptide content), indomethacin, and nordihydroguaiaretic acid (NDGA) were purchased from Sigma Chemical Co. (St. Louis, MO). Lipopolysaccharide (LPS) from S. typhosa 0901 was purchased from Difco Laboratories (Detroit, MI). Cycloheximide was obtained from Calbiochem Corp. (San Diego, CA). Human recombinant tumor necrosis factor-alpha (TNFa) (specific activity = 2 X l07 units/ mg, LPS content = 0.04 ng/mg) was kindly supplied by the Departments of Molecular Genetics and Cell Sciences, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). All other agents were purchased from common commercial sources.
Cell Culture Human umbilical vein endothelial cells (HUVEC) obtained as a primary culture (Cell Systems Corp., Kirklan, WA) or cell line (American Type Culture Collection, Rockville, MD) were maintained in F l2 media supplemented with 20% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 10/zg/l ECGF (Boehringer Mannheim, Indianapolis, IN), 20,000 U/l heparin (Elkins-Sinn Inc., Cherry Hill, NJ), and 50 mg/l ofgentamycin (Gibco, Grand Island, NY) at 37°C under constant humidity
Requests for reprints should be addressed to Cheng-Po Sung, Ph.D., Department of Pharmacology,UW2510, SmithKfine Beecham Pharmaceuticals, King of Prussia, PA 19406, 429
430
SUNG ET AL.
200•
A 250 U937, n=5
A
i
200
PMN, n=3
150
150
.o
g
E
IO0
o - -
50
100-1
*
C
4
.o
8
12
16
20
24
Q J¢ "O
95% as assessed by Wright's stained cytocentrifnge preparations and >99% viable as assessed by trypan blue exclusion. Human monocyfic line (25), U937 cells (American Type Culture Collections, Rockville, MD), were grown in spinner flasks (Bellco Bioteehnology) containing Dulbecco's modified Eagle's medium (Gibeo, Grand Island, NY) supplemented with 20% FIBS (MEM20% FBS). The 12937 cells were subeultured every 3-4 days and maintained at cell densities between 0.5-1.0 × 10~ cells/ml. PMN or U937 cells were incubated in 1 ml of DMEM-20% FBS with 300 ¢Ci of Na25~CrO4 for 90 rain. Excess chromium
ee
¢J 50 Time (hre) FIG. 2. (A) Time course of CGRP and TNFa for the stimulation of U937 cell adhesion to HUVEC. HUVEC cells were pcctreat~ with 2 /~M of CGRP (solid circle) or 100 U/rot of TNFa (open circle) for 1-24 h as indicated. A_Plerthe removal of medium oontaining OGRP or TNFa, cell monolayers were washed once prior to the addition of S~Cr-labcled U937 cells for 30-rain adhesion. Results arc expressed as the mean + SEM of values obtained from three separate experiments, each done i n triplicate.(B) StabilityofC G R P duringtheincubation,Incubationmedia from (A) were assayed for CGRP by PAA as described in the Method section. Results arc the mean of a duphcat¢ observation of a typical experiment.
Adhesion Assay HUVEC monolayers were washed once with 1 ml F12 medium and preincubated with the test compound, CGRP or TNFa, in F12-20% FBS for the time indicated in the figure legends. The incubation medium was removed and the monolayer was washed once with 1 ml ofF-12 media. Fresh medium was added prior to the addition of ra_diolabcled U937 cells (5 × 10~/weU) or PMN (2.5-5 × 10S/well). After a 30-rain incubation at 37°C, nonadherent leukocytes were aspirated, and monolayers were washed three times with F I 2 media. The number o f leukocytes that adhered to HUVEC monolaycrs was determined by either rapid filtration through GF/B glass fiber filters under reduced pressure (three washings with 2 ml each of cold P]BS) using a cell harvester, model M-24S ( ~ , Ga~qhersburg, MD), or by solublization with 1 N NaOH (overnight) followed by the determination of radioactivity associated with the leu-
kocytes.
was removed by three cyclesof centrifngationand washing with 50 ml of Dulbecco's p h o s p h a t e - b ~ saline(DPBS) solution.
Measurement of cAMP
Cells were resuspended in F I 2 media supplemented with 20% FBS (F12-20% FBS) and used within 20 min.
HUVEC were seeded into six-well Linbro plates and grown until 80-90% confluency (3--4 days). The protocols ofactivation
CGRP ON LEUKOCYTE-EC ADHESION
431 were assayed for CGRP using an RIA kit (Peninsula Laboratory Inc., Belmont, CA). The antisera provided in the kit cross-reacts 100% with a- or #-rat CGRP and human CGRP. The crossreactivity with rat calcitonin C-terminal adjacent peptide was less than 0.001%. Following the procedures recommended by the assay kit, we could reliably detect 1 tag (or 2.6 fmol) of CGRP with 95% confidence.
10000[]
T r e a t m e n t of H U V E C
[]
T r e a t m e n t of u g 3 7 c e l l s
8000' E
6000'
-T-
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-
.l= "O
0196-9781/92 $5.00 + .00 Copyright© 1992PergamonPress Ltd.
Printed in the USA.
CGRP Stimulates the Adhesion of Leukocytes to Vascular Endothelial Cells C H E N G - P O S U N G , l A N T H O N Y J. A R L E T H , N A M B I A I Y A R , P R A D I P K. B H A T N A G A R , P A U L G. L Y S K O A N D G I O R A F E U E R S T E I N
Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Received 5 D e c e m b e r 1991 SUNG, C.-P., A. J. ARLETH, N. AIYAR, P. K. BHATNAGAR, P. G. LYSKO AND G. FEUERSTEIN. CGRPstimulates the adhesion ofleukocytes to vascularendothelialcells. PEPTIDES 13(3) 429-434, 1992.--Calcitonin gene-related peptide (CGRP) stimulates the adhesiveness of human umbilical vein endothelial cells for U937 cells and human neutrophils in a dose- and timedependent manner. The onset of CGRP-induced adhesives of HUVEC was rapid (30 min), independent of protein synthesis, and lasted over 24 h in the continuous presence of the peptide. The stimulatory effect of CGRP was completely blocked by the CGRP antagonist, CGRP(8-37). The present study provides evidence in support of the potential role of sensorynerve-derivedneuropeptides in the modulation of leukocyte adhesion to vascular endothelial cells. CGRP Neuropeptide Neuroimmunology
Leukocyte--endothelialcell adhesion
THE central nervous system has been implicated in modulation of immune and inflammatory reactions. Reciprocal interactions between the nervous system and immune and inflammatory cells have been demonstrated at both the cellular and humoral levels (1). The cellular mode of interaction is exhibited by the intense innervation of primary (thymus, bone marrow) and secondary (lymph nodes, spleen) immune organs by sensory and autonomic nerves (10). The humoral mode of interaction is supported by demonstrations that neural-derived factors, such as substance P (SP), VIP, opioid peptides, nerve growth factor (NGF), somatostatin, and other peptides, activate or suppress circulating immune/inflammatory cells (17). Moreover, specific neuropeptides, (e.g., a-MSH) possess antipyretic (18) and antiinflammatory activities, probably by modulation of inflammatory cell mediators such as tumor necrosis factor (cachectin, TNFa) or intefleukin-1 (23). While there is ample evidence in support of a role for neuronal-derived factors in the activation and regulation of immune and inflammatory cells, no evidence so far has implicated sensory nerve-derived factors in the regulation of a critical step in the initiation of inflammation, i.e., the adhesion of leukocytes to the endothelium. Since the initial events in inflammation involve adhesion of leukocytes to vascular endothelial cells, we hypothesized that neuronal-derived peptides might modulate leukocyte adhesion to vascular endothelial cell. In the only report available to date, we showed that NPY increased U937 cell adhesion to HUVEC, suggesting a role for a sympathetic nerve-derived neuromodulator in regulation of endothelium adhesiveness (26). The present study provides evidence supporting that the sensory
Adhesion molecules
Endothelium
nerve-derived peptide, CGRP, also promotes the adhesiveness of human vascular endothelium for leukocytes. METHOD
Reagents Human CGRP (98% purity, 69% peptide content), indomethacin, and nordihydroguaiaretic acid (NDGA) were purchased from Sigma Chemical Co. (St. Louis, MO). Lipopolysaccharide (LPS) from S. typhosa 0901 was purchased from Difco Laboratories (Detroit, MI). Cycloheximide was obtained from Calbiochem Corp. (San Diego, CA). Human recombinant tumor necrosis factor-alpha (TNFa) (specific activity = 2 X l07 units/ mg, LPS content = 0.04 ng/mg) was kindly supplied by the Departments of Molecular Genetics and Cell Sciences, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). All other agents were purchased from common commercial sources.
Cell Culture Human umbilical vein endothelial cells (HUVEC) obtained as a primary culture (Cell Systems Corp., Kirklan, WA) or cell line (American Type Culture Collection, Rockville, MD) were maintained in F l2 media supplemented with 20% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 10/zg/l ECGF (Boehringer Mannheim, Indianapolis, IN), 20,000 U/l heparin (Elkins-Sinn Inc., Cherry Hill, NJ), and 50 mg/l ofgentamycin (Gibco, Grand Island, NY) at 37°C under constant humidity
Requests for reprints should be addressed to Cheng-Po Sung, Ph.D., Department of Pharmacology,UW2510, SmithKfine Beecham Pharmaceuticals, King of Prussia, PA 19406, 429
430
SUNG ET AL.
200•
A 250 U937, n=5
A
i
200
PMN, n=3
150
150
.o
g
E
IO0
o - -
50
100-1
*
C
4
.o
8
12
16
20
24
Q J¢ "O
95% as assessed by Wright's stained cytocentrifnge preparations and >99% viable as assessed by trypan blue exclusion. Human monocyfic line (25), U937 cells (American Type Culture Collections, Rockville, MD), were grown in spinner flasks (Bellco Bioteehnology) containing Dulbecco's modified Eagle's medium (Gibeo, Grand Island, NY) supplemented with 20% FIBS (MEM20% FBS). The 12937 cells were subeultured every 3-4 days and maintained at cell densities between 0.5-1.0 × 10~ cells/ml. PMN or U937 cells were incubated in 1 ml of DMEM-20% FBS with 300 ¢Ci of Na25~CrO4 for 90 rain. Excess chromium
ee
¢J 50 Time (hre) FIG. 2. (A) Time course of CGRP and TNFa for the stimulation of U937 cell adhesion to HUVEC. HUVEC cells were pcctreat~ with 2 /~M of CGRP (solid circle) or 100 U/rot of TNFa (open circle) for 1-24 h as indicated. A_Plerthe removal of medium oontaining OGRP or TNFa, cell monolayers were washed once prior to the addition of S~Cr-labcled U937 cells for 30-rain adhesion. Results arc expressed as the mean + SEM of values obtained from three separate experiments, each done i n triplicate.(B) StabilityofC G R P duringtheincubation,Incubationmedia from (A) were assayed for CGRP by PAA as described in the Method section. Results arc the mean of a duphcat¢ observation of a typical experiment.
Adhesion Assay HUVEC monolayers were washed once with 1 ml F12 medium and preincubated with the test compound, CGRP or TNFa, in F12-20% FBS for the time indicated in the figure legends. The incubation medium was removed and the monolayer was washed once with 1 ml ofF-12 media. Fresh medium was added prior to the addition of ra_diolabcled U937 cells (5 × 10~/weU) or PMN (2.5-5 × 10S/well). After a 30-rain incubation at 37°C, nonadherent leukocytes were aspirated, and monolayers were washed three times with F I 2 media. The number o f leukocytes that adhered to HUVEC monolaycrs was determined by either rapid filtration through GF/B glass fiber filters under reduced pressure (three washings with 2 ml each of cold P]BS) using a cell harvester, model M-24S ( ~ , Ga~qhersburg, MD), or by solublization with 1 N NaOH (overnight) followed by the determination of radioactivity associated with the leu-
kocytes.
was removed by three cyclesof centrifngationand washing with 50 ml of Dulbecco's p h o s p h a t e - b ~ saline(DPBS) solution.
Measurement of cAMP
Cells were resuspended in F I 2 media supplemented with 20% FBS (F12-20% FBS) and used within 20 min.
HUVEC were seeded into six-well Linbro plates and grown until 80-90% confluency (3--4 days). The protocols ofactivation
CGRP ON LEUKOCYTE-EC ADHESION
431 were assayed for CGRP using an RIA kit (Peninsula Laboratory Inc., Belmont, CA). The antisera provided in the kit cross-reacts 100% with a- or #-rat CGRP and human CGRP. The crossreactivity with rat calcitonin C-terminal adjacent peptide was less than 0.001%. Following the procedures recommended by the assay kit, we could reliably detect 1 tag (or 2.6 fmol) of CGRP with 95% confidence.
10000[]
T r e a t m e n t of H U V E C
[]
T r e a t m e n t of u g 3 7 c e l l s
8000' E
6000'
-T-
O ¢n 4000
-
.l= "O
