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Inflammatory response to cold injury in remote organs is reduced by corticotropin-releasing factor
Regulatory Peptides 99 Ž2001. 131–139 www.elsevier.comrlocaterregrep
Inflammatory response to cold injury in remote organs is reduced by corticotropin-releasing factor ¨ c , Serap Arbak c , Ayhan Bozkurt a , Salah Ghandour a , Nesime Okboy b, Susanne Oner Tamer Cos¸kun a , Berrak C¸ . Yegen ˘ a,) a
˙ Department of Physiology, School of Medicine, Marmara UniÕersity, Haydarpas¸a, Istanbul, 81326, Turkey b ˙ Department of Forensic Medicine, Cerrahpas¸a Medical Faculty, Istanbul UniÕersity, Istanbul, Turkey c ˙ Department of Histology, School of Medicine, Marmara UniÕersity, Haydarpas¸a, Istanbul, 81326, Turkey Received 10 July 2000; received in revised form 1 February 2001; accepted 6 February 2001
Abstract Current experimental evidence concerning the potential activity of corticotropin releasing factor ŽCRF. in inflammatory processes still remains controversial. To determine whether CRF has protective effects on three remote organs Žliver, lung and stomach. affected by cold injury and to characterize the role of neutrophils in cold-induced inflammation, dorsums of anesthetized rats were exposed for 5 min to a 22% NaCl solution maintained at y20 " 0.58C and the rats were sacrificed at 24 h after the cold injury. The results indicate that cold-exposure-induced edema in the liver, lung and stomach was blocked by subcutaneous Žs.c.; 1.2 and 12 nmolrkg; 30 min before cold trauma. CRF pretreatment, while the central administration of CRF Žintracisternally Ži.c..; 0.30 and 1.5 nmolrrat; 15 min before cold . had the similar effect at the higher dose. Histological assessment and the tissue myeloperoxidase activities also revealed that CRF given peripherally has a protective role in damage generation. Moreover, CRF had a facilitatory effect in the recovery of the body temperature following cold exposure. In conclusion, CRF is likely to act on its peripheral receptors in the inflamed remote organs, suppressing the edematogenic effects of inflammatory mediators, some of which are neutrophil-derived. q 2001 Elsevier Science B.V. All rights reserved. Keywords: MPO; Edema; Inflammation; Neutrophils
1. Introduction Corticotropin releasing factor ŽCRF., a 41 amino acid neuropeptide, was originally isolated and sequenced from the ovine hypothalamus and identified by and named for its property to stimulate anterior pituitary secretion of adrenocorticotropic hormone ŽACTH., the systemic hormone that regulates production of glucocorticoids by the adrenal cortex w1,2x. Besides its hypophysiotropic effects, CRF acts within the central nervous system to initiate coordinated behavioral, autonomic and visceral responses to stress w3–5x. Peptides of the CRF family have been shown to have either pro- or anti-inflammatory activities w6,7x. By activating glucocorticoid and catecholamine secretion, CRF participates in the anti-inflammatory and immunosuppressive mechanisms at multiple levels w8x. Immunohistochemical and in situ hybridization studies have ) Corresponding author. Tel.: q90-216-414-4736; fax: q90-216-4144731. E-mail address: [email protected] ŽB.C¸ . Yegen ˘ ..
shown that immune challenge or cytokines activate CRF neurons and increase the expression of CRF mRNA in the paraventricular nucleus of the hypothalamus w9–11x. CRF and its receptors are found in the mammalian brain, as well as in peripheral tissues such as the skin, testes, pancreas, adrenal medulla, gut, placenta, and cells of the immune system w12x. The presence of CRF in cells of the immune system and in inflamed tissues has recently been reviewed w13x. CRF is found in inflamed synovial tissues at the same concentrations detected in hypophyseal portal venous blood w14x. Moreover, CRF produced in high amounts in inflammatory sites was shown to promote inflammation by potentiating the pro-inflammatory activities of cytokines and other mediators of inflammation w15x. In contrast to its systemic indirect immunosuppressive effects, the enhanced local expression of CRF and its receptors within inflamed tissue suggests that CRF acts as an autocrine or paracrine inflammatory cytokine w6x. Direct local administration of CRF-specific antiserum was reported to suppress the inflammatory response to carrageenin. Using intravital microscopy, it was shown that
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 3 9 - 7
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Table 1 Criteria for scoring the histopathological findings
tissue damage in cold injury is attractive since the period between freezing and thawing offers an opportunity for therapeutic intervention. In this context, Serda and Wei w27x have demonstrated that CRF inhibited the local inflammatory response to cold injury. The current experimental evidence concerning the potential activity of CRF in inflammatory processes in vivo still remains controversial. The first aim of this study was to assess the extent of distant organ damage following freezing cold injury and to assess the role of neutrophils in systemic hypothermia by determining tissue associated myeloperoxidase activity ŽMPO. —a reliable index of neutrophil infiltration w28x. The second aim of this study was to determine whether CRF has protective effects on remote organs affected by cold injury and to characterize the involved mechanisms to some extent.
Liver 1. pyknotic hepatocyte nuclei 2. hepatocyte degeneration 3. sinusoidal widening 4. Kupffer cell infiltration 5. parenchymal damage Lung 1. alveolar cell degeneration 2. cellular infiltration 3. intraalveolar hemorrhage 4. interstitial edema 5. congestion 6. septal irregularity Stomach 1. cellular infiltration 2. hemorrhage 3. glandular irregularity 4. epithelial damage Scores are given as 0: none, 1: mild, 2: moderate, 3: maximum for each criterion. At least five microscopic areas were examined to score each specimen.
2. Materials and methods 2.1. Animals
CRF, given intravenously or intracisternally, increased the number of rolling and adhered leukocytes in postcapillary mesenteric venules w6,16x, indicating a local proinflammatory action of CRF. However, systemic as well as local administration of CRF reduces edema, swelling and protein extravasation in different experimental models w17–25x. How the peptide reduces the edema is not fully known, but several possibilities have been documented. It seems clear that CRF exerts its effects locally and these effects are not mediated by secondary release of ACTH, beta-endorphin or corticosteroids. In all of these experimental models, however, the anti-inflammatory role of CRF was investigated at the site of inflammation, while its potential effects on organs distant from the original injury remain to be studied. Systemic hypothermia occurs when the loss of body heat exceeds heat production. When living tissues are frozen and rewarmed, there is initially vasoconstriction, followed by hyperemia, and then a rapid development of edema w26x. The pathogenesis of freezing cold injuries is not yet entirely understood. The use of drugs to modify
Adult Wistar Albino rats of both sexes Ž250–300 g. were kept in a light- and temperature-controlled room on a 12:12-h light–dark cycle, where the temperature Ž22 " 0.58C. and relative humidity Ž65–70%. were kept constant and fed a standard diet and water ad libitum. Experiments were approved by the Marmara University, School of Medicine, Animal Care and Use Committee. 2.2. Experimental protocol Experiments were performed between 9:00 and 12:00 a.m. Rats were anesthetized by intraperitoneal Ži.p.. injection of a mixture of ketamine Ž100 mgrkg. and chlorpromazine Ž12.5 mgrkg.. Cold exposure was applied on the shaved dorsums Ž30% of the total body surface area. of rats for 5 min by laying the animals on the ice block Ž22% NaCl., which was kept at y20 " 0.58C in an insulated box. In anesthetized animals, rectal temperatures were recorded 30 min before, immediately, 30 and 60 min after cold application by an electronic thermometer. Following
Table 2 Changes in rectal temperatures Ž8C. in control rats and in saline or CRF-pretreated Ž12 nmolrkg, subcutaneously, s.c. and 1.5 nmolrrat, intracisternally, i.c.. rats following cold injury
Before cold trauma During cold application 30 min after cold trauma 60 min after cold trauma
Control
i.c. saline
s.c. saline
i.c. CRF
s.c. CRF
37.0 " 0.2 35.4 " 0.3 35.8 " 0.4 35.7 " 0.3
36.4 " 0.2 31.5 " 0.4 ) ) ) 31.2 " 0.5 ) ) ) 32.4 " 0.5 ) ) )
36.8 " 0.2 33.0 " 0.5 ) ) ) 32.7 " 0.5 ) ) ) 33.9 " 0.5 ) ) )
36.7 " 0.3 33.3 " 0.6 ) ) ) ,† 32.9 " 0.7 ) ) ) ,† 33.5 " 0.5 ) ) )
36.4 " 0.2 34.6 " 0.2 ) ) ) ,† 34.3 " 0.1) ) ) ,† 35.5 " 0.2 ) ,†
Values are means" SE. ) p - 0.05, compared to the measurement of the same group before cold application. ))) p - 0.001, compared to the measurement of the same group before cold application. † p - 0.05, compared to the respective saline-treated group.
A. Bozkurt et al.r Regulatory Peptides 99 (2001) 131–139
cold exposure, the rats were towel-dried and kept warm until they recovered from anesthesia. In order to rule out the effects of anesthesia, the same protocol was applied in the control rats, except that they were exposed to a 22% NaCl solution maintained at room temperature Ž22 " 0.58C.. The rats were returned to their home cages until they were sacrificed at 24 h following cold exposure. In all groups, the stomach, liver and right lung were rapidly removed, tissue wet weights were recorded and corrected
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for 100 g body weight. Tissue samples were stored at y708C to determine tissue-associated myeloperoxidase activity ŽMPO. and some samples were fixed in 10% formalin solution for light microscopic examination. 2.3. Administration of drugs CRF ŽSigma. was dissolved in saline and aliquots were stored at y208C until use. Freshly prepared dilutions were
Fig. 1. Effects of intracisternal Ži.c.. or subcutaneous Žs.c.. CRF pretreatment on wet weights of the liver Ža., lung Žb. and stomach Žc. following cold injury. Control rats received vehicle injections but were not exposed to cold injury. Results are expressed as means" SE. ) p - 0.05, ) ) p - 0.01, and ))) p - 0.001; compared to the control group, qp - 0.05; compared to the respective saline-treated group.
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A. Bozkurt et al.r Regulatory Peptides 99 (2001) 131–139
injected subcutaneously Žs.c.; 0.12, 1.2 and 12 nmolrkgf 0.57, 5.7 and 57 mgrkg. or intracisternally Ži.c.; 0.15, 0.30 and 1.5 nmolrrat. 30 or 15 min before the cold trauma, respectively. In the vehicle groups, saline was injected Ž1 mlrkg; s.c. and 5 mlrrat; i.c.. before the cold trauma at the same time points. Control rats also received saline Ži.c.
or s.c.. in the same time course. Each subgroup consisted of 8 to 10 animals. CRF or saline was injected intracisternally by puncture of the occipital membrane with the needle of a Hamilton syringe in rats fixed in ear bars of a stereotaxic device ŽStoelting Lab... The presence of cerebrospinal fluid in the
Fig. 2. Effects of intracisternal Ži.c.. or subcutaneous Žs.c.. CRF pretreatment on tissue MPO activities measured in the liver Ža., lung Žb. and stomach Žc. following cold injury. Control rats received vehicle injections but were not exposed to cold injury. Results are expressed as means" SE. ) p - 0.05, )) p - 0.01, and ) ) ) p - 0.001; compared to the control group, qp - 0.05; compared to the respective saline-treated group.
A. Bozkurt et al.r Regulatory Peptides 99 (2001) 131–139 Table 3 Histologic scores in control rats and in subcutaneously saline or CRF-pretreated Ž1.2 nmolrkg. rats following cold injury
Liver Lung Stomach
Control
Saline
CRF
0.33"0.33 3.0"0.58 0.67"0.33
8.67"0.33 ) ) ) 14.0"0.58 ) ) ) 6.0"0.01) ) )
6.0"0.58 ) ) ,a 7.67"1.20 ) ) ,a 4.0"0.01) )
In each group ns 3. At least five microscopic areas are examined to score each specimen. Values are means"SE. )) p- 0.01 compared to control group. ))) p- 0.001 compared to control group. a p- 0.01 compared to respective vehicle group.
Hamilton syringe upon aspiration for injection insured the correctness of needle placement into the cisterna magna w29x. 2.4. Tissue MPO measurement Tissue-associated MPO activity was utilized as an index of tissue accumulation of neutrophils w28x. Briefly, the tissue samples Ž0.2–0.3 g. were homogenized in 10 vol. of ice-cold potassium phosphate buffer Ž20 mM K 2 HPO4 , pH 7.4.. The homogenate was centrifuged at 10,000 rpm for 20 min at 48C. The pellet was homogenized with an equivalent volume of 50 mM acetic acid ŽpH 6.0. containing 0.5% Žwtrvol. hexadecyltrimethylammonium bromide ŽHETAB.. MPO activity was assessed by measuring the H 2 O 2 -dependent oxidation of 3,3X ,5,5X-tetramethylbenzidine. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance of 1.0rmin at 655 nm and 378C w30x.
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2.5. Histologic examination The tissue samples of the liver, lung and stomach were fixed in 10% formalin solution. Following dehydration in ascending series of ethanol, tissue samples were cleared in xylene and embedded in paraffin. Tissue sections of 6–7 mm were stained with hematoxylin–eosin and investigated under a light microscope ŽOlympus BH-2, Tokyo, Japan. for characterization of histopathological changes by an observer unaware of the treatment condition of the animal. Histological score of the observed organ was calculated as the sum of the scores Ž0–3. given for each criterion, using the semiquantitative scale shown on Table 1. 2.6. Statistical analysis The results are expressed as mean " SEM. Analysis of variance ŽANOVA. and Tukey–Kramer multiple comparison tests were used for multiple comparisons and Student’s t-test was used to evaluate the level of statistical significance between two groups. Differences were considered statistically significant if p - 0.05.
3. Results The data following CRF administration are only given for the higher Ž12 nmolrkg, s.c. and 1.5 nmolrrat, i.c.. and lower Ž1.2 nmolrkg, s.c. and 0.3 nmolrrat, i.c.. doses, because the lowest doses Ž0.12 nmolrkg, s.c. and 0.15 nmolrrat, i.c.. had negligible effects on the inflammatory parameters.
Fig. 3. Micrographs demonstrate ŽA. normal liver lobular morphology in the control group, ŽB. vacuolated hepatocytes Ž™. and vascular congestion in dilated sinusoids Ž-. in the cold injury group, and ŽC. low degree of degeneration in lobular morphology with few vacuolated hepatocytes Ž≠. in CRF-treated Ž1.2 nmolrkg, s.c.. group. Magnification ŽA. and ŽB. 66 = , ŽC. 33 = , Haematoxylin and Eosin.
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Fig. 4. Micrographs demonstrate ŽA. normal lung parenchyme in the control group, ŽB. a prominent disturbance in alveolar architecture with wide-spread leukocyte infiltration Ž'., fused interalveolar septa Ž ) . in the cold injury group, and ŽC. thin interalveolar septa Ž ) ., low degree of leukocyte infiltration Ž'. in CRF-treated Ž1.2 nmolrkg, s.c.. group. Magnification ŽA. 33 = ; ŽB. and ŽC. 66 = , haematoxylin and eosin.
3.1. Effect of CRF on cold injury-induced change in body temperature The body temperature dropped significantly following cold exposure Ž p - 0.05–0.001; Table 2.. CRF administered at 12 nmolrkg Žsc. and 1.5 nmolrrat Ži.c.. doses before cold exposure attenuated the drop in body temperature when compared with rats which received saline Ž p 0.05., while the lower doses did not significantly affect the cold-induced drop in body temperature Ždata not shown.. 3.2. Effect of CRF on cold injury-induced change in tissue wet weights Following systemic cold injury, tissue wet weights of the liver, lung and stomach were increased significantly in both vehicle groups, where saline was given either i.c. or s.c. Ž p - 0.05–0.01, compared to control group; Fig. 1.. The central administration of CRF at the higher dose reduced the cold-induced edema significantly in the lung and stomach Ž p - 0.05; Fig. 1.. Peripheral administration of CRF at the lower dose Ž p - 0.05. abolished the cold-induced increases in the wet weights of the studied tissues, while the higher dose was not effective in the liver and stomach.
those of the i.c.-saline group and were increased significantly following cold injury Ž p - 0.05–0.001; Fig. 2.. These increases in MPO activities were not reduced by either doses of centrally administered CRF ŽFig. 2.. On the other hand, CRF given peripherally at both doses blocked the elevation of MPO activity in the liver, while this effect was observed only at the higher dose in the lung tissue. However, none of the CRF doses had a significant effect on the elevated MPO activity in the stomach. 3.4. Effect of CRF on cold injury-induced histopathological changes Systemic cold application significantly increased the histologic scores in the liver, lung and stomach Ž p - 0.001; Table 3., when compared with the control group. Peripheral administration of CRF at the lower effective dose Ž1.2 nmolrkg; s.c.. before cold injury reduced the histological scores Ž p - 0.05–0.001. in the liver and lung ŽTable 3; Figs. 3 and 4., but not in the stomach. On the other hand, centrally given CRF decreased the severity of cold-induced damage of the lung and liver, only at the higher dose Ž1.5 nmolrrat; data not shown..
4. Discussion 3.3. Effect of CRF on cold injury-induced change in tissue MPO actiÕity Tissue MPO activities measured in the liver, lung and stomach of the s.c.-saline group were not different from
The results of the present study indicate that cold-exposure-induced edema in the liver, lung and stomach was blocked by subcutaneous CRF treatment. Central administration of CRF had the similar edema-preventive effect
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only at the higher doses, implicating the presence of a peripheral action. Histological assessment also revealed that peripheral CRF pretreatment has a protective role in damage generation. In accordance with the histological findings and edema formation, tissue associated MPO activity—known as the index of neutrophil infiltration— increased by cold trauma in the liver, lung and stomach. Peripherally administered CRF reduced the elevations in MPO in the liver and lung. In addition, CRF had a facilitatory effect in the recovery of the body temperature following cold exposure, which may have a contribution to its protective effect in distant organ injury. Amelioration of cold-induced remote organ injury by the subcutaneous administration of CRF in all the studied tissues suggests that CRF has a peripheral anti-inflammatory action. The most common cause of accidental hypothermia is exposure to environmental low temperature, which can occur during hiking and skiing expeditions, in shipwrecks, or in connection with insufficient heating w31x. The pathogenesis of freezing cold injuries is not entirely understood. If hypothermia is severe, hemoconcentration, vascular stasis, and microcirculatory insufficiency may occur leading to tissue necrosis in the frozen part w32x. Various pharmacological agents, including antihistamines, inhibitors of the arachidonic acid cascade, anticoagulants, vasodilators and sympatholytic drugs, have been tried to improve the survival of frozen tissues. In an attempt to search an opportunity for therapeutic intervention, CRF was shown to have potent inhibitory effects on the acute local inflammatory response to thermal injury w19x. It was shown that exposure of tissues to exogenous CRF produces a reduction in the detrimental effects of several injuries. Systemic as well as local administration of CRF decreases protein extravasation, edema and swelling elicited by antidromic stimulation of peripheral nerves, application of heat or extreme cold stimuli, injection of inflammatory mediators w7,24x. CRF also reduces edema and swelling in the rat carrageenan model of inflammation w17x. It is noteworthy that CRF induces a thermogenic effect and stimulates energy expenditure in laboratory animals w33x, with a reduction in food intake, by increasing the activity of the sympathetic nervous system w34x. In the present study, the thermogenic effect of CRF, as observed with its facilitatory effect on the recovery of the body temperature after cold application, may be participating by reducing the microcirculatory problems and the formation of tissue edema. In addition to its role and distribution throughout the brain and spinal cord, it is also shown that CRF is expressed in various peripheral tissues including stomach and duodenum w35x, lung and adrenal glands w36x and leukocytes and lymphocytes w37,38x. Recently, mRNA coding for the CRF1 receptor gene was found to be present in neutrophils w39x suggesting that local tissue modulation of CRF levels and its receptor activities can affect the course of inflammation with a paracrinerautocrine mechanism
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w6,40,41x. CRF could produce its anti-inflammatory effects by inhibiting the release of inflammatory mediators or by interfering with the actions of these mediators on the nerve or on the endothelium w42x. It is possible that some of the inflammatory mediators are neutrophil-derived. Since the present results show that cold injury-induced elevation in MPO activity is reversed by peripheral CRF, it is likely that CRF has an anti-inflammatory effect by suppressing the accumulation of tissue neutrophils. However, it seems likely that the anti-inflammatory effect of CRF on the stomach is not neutrophil-dependent. The observed variability of the tissue responses to CRF pretreatment may be attributed to dose-dependent anti- or proinflammatory effects of the peptide. Moreover, it may be suggested that the anti-inflammatory effects of CRF are peripherally mediated, since the lower dose of CRF applied subcutaneously Ž1.2 nmolrkg. reduced the inflammatory parameters, while the dose Ž0.3 nmolrrat. given centrally Žwhich is f 1.2 nmolrkg when corrected for the body weight. was not effective. Similarly, Kiang and Wei w19x have suggested that intravenous CRF may be a powerful inhibitor of the inflammatory response of the skin to thermal injury and its effects are not mediated by secondary release of ACTH and beta-endorphin, products that may be derived from the CRF-proopiomelanocortin axis. Dexamethasone, a potent synthetic corticosteroid was also found to be inactive in reducing the tissue response to thermal injury. Moreover, the anti-inflammatory effects of CRF are apparently not mediated via activation of the hypothalamo-pituitary adrenal axis and subsequent steroid action, because similar effects were observed after hypophysectomy or adrenalectomy w17,18,22,23x. The effect of CRF was not antagonized by nalaxone, suggesting that it was not mediated via activation of opioid receptors w22x. Indirect, systemic cardiovascular effects of CRF would also not account for its effect because CRF did not produce hypotension at doses that inhibited vascular leakage w22x. Moreover, topical application of CRF still inhibited vascular leakage w43x. On the other hand, CRF produced in high amounts in inflammatory sites would promote inflammation by potentiating the pro-inflammatory activities of cytokines and other mediators of inflammation w15x. CRF is found in inflamed tissues at the same concentration as in pituitary portal venous blood w14x. In two recent studies, CRF given intravenously increased the number of rolling and adhered leukocytes in postcapillary venules w16x and carrageenin injection into a subcutaneous air-pouch of rats increased local CRF immunoreactivity in parallel to the monoclonal cell infiltration w6x. Local as well as systemic application of a CRF-specific antiserum decreased both the volume and the cellularity of the inflammatory exudate w6x. These findings suggest that CRF has predominantly proinflammatory effects in peripheral inflammatory sites. However, the present data suggest that the local effects of CRF in the freezing cold injury are anti-inflammatory.
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In conclusion, freezing cold injury results in edema formation and histopathological damage in the liver, lung and stomach, accompanied by neutrophil infiltration, suggesting that infiltrated neutrophils are responsible for the injurious effect of freezing cold insult on the liver, lung and stomach. CRF is likely to act on its peripheral receptors in the remote organs to ameliorate the extent of injury, by facilitating the recovery of body temperature and suppressing the edematogenic effects of inflammatory mediators, some of which are neutrophil-derived. Since the consequences of freezing cold injury to tissues are severe, further studies on the use of CRF in this condition merit consideration and future studies are necessary to elucidate the potent inhibitory actions of CRF on inflammatory processes.
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receptors in rats with painful hindlimb inflammation. Eur J Pharmacol 1996;311:221–31. w41x Schafer M, Mousa SA, Zhang Q, Carter L, Stein C. Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia. Proc Natl Acad Sci 1996;93:6096– 100. w42x Kiang JG, Poree L, Wei ET. Anti-inflammatory activity of corticotropin-releasing factor. II. Mechanisms of action. Proc West Pharmacol Soc 1987;30:63–5. w43x Joyner WL, Wei ET. Mechanism for the anti-inflammatory effect of corticotropin-releasing factor ŽCRF.. FASEB J 1989;3:272. Žabstract..
Inflammatory response to cold injury in remote organs is reduced by corticotropin-releasing factor ¨ c , Serap Arbak c , Ayhan Bozkurt a , Salah Ghandour a , Nesime Okboy b, Susanne Oner Tamer Cos¸kun a , Berrak C¸ . Yegen ˘ a,) a
˙ Department of Physiology, School of Medicine, Marmara UniÕersity, Haydarpas¸a, Istanbul, 81326, Turkey b ˙ Department of Forensic Medicine, Cerrahpas¸a Medical Faculty, Istanbul UniÕersity, Istanbul, Turkey c ˙ Department of Histology, School of Medicine, Marmara UniÕersity, Haydarpas¸a, Istanbul, 81326, Turkey Received 10 July 2000; received in revised form 1 February 2001; accepted 6 February 2001
Abstract Current experimental evidence concerning the potential activity of corticotropin releasing factor ŽCRF. in inflammatory processes still remains controversial. To determine whether CRF has protective effects on three remote organs Žliver, lung and stomach. affected by cold injury and to characterize the role of neutrophils in cold-induced inflammation, dorsums of anesthetized rats were exposed for 5 min to a 22% NaCl solution maintained at y20 " 0.58C and the rats were sacrificed at 24 h after the cold injury. The results indicate that cold-exposure-induced edema in the liver, lung and stomach was blocked by subcutaneous Žs.c.; 1.2 and 12 nmolrkg; 30 min before cold trauma. CRF pretreatment, while the central administration of CRF Žintracisternally Ži.c..; 0.30 and 1.5 nmolrrat; 15 min before cold . had the similar effect at the higher dose. Histological assessment and the tissue myeloperoxidase activities also revealed that CRF given peripherally has a protective role in damage generation. Moreover, CRF had a facilitatory effect in the recovery of the body temperature following cold exposure. In conclusion, CRF is likely to act on its peripheral receptors in the inflamed remote organs, suppressing the edematogenic effects of inflammatory mediators, some of which are neutrophil-derived. q 2001 Elsevier Science B.V. All rights reserved. Keywords: MPO; Edema; Inflammation; Neutrophils
1. Introduction Corticotropin releasing factor ŽCRF., a 41 amino acid neuropeptide, was originally isolated and sequenced from the ovine hypothalamus and identified by and named for its property to stimulate anterior pituitary secretion of adrenocorticotropic hormone ŽACTH., the systemic hormone that regulates production of glucocorticoids by the adrenal cortex w1,2x. Besides its hypophysiotropic effects, CRF acts within the central nervous system to initiate coordinated behavioral, autonomic and visceral responses to stress w3–5x. Peptides of the CRF family have been shown to have either pro- or anti-inflammatory activities w6,7x. By activating glucocorticoid and catecholamine secretion, CRF participates in the anti-inflammatory and immunosuppressive mechanisms at multiple levels w8x. Immunohistochemical and in situ hybridization studies have ) Corresponding author. Tel.: q90-216-414-4736; fax: q90-216-4144731. E-mail address: [email protected] ŽB.C¸ . Yegen ˘ ..
shown that immune challenge or cytokines activate CRF neurons and increase the expression of CRF mRNA in the paraventricular nucleus of the hypothalamus w9–11x. CRF and its receptors are found in the mammalian brain, as well as in peripheral tissues such as the skin, testes, pancreas, adrenal medulla, gut, placenta, and cells of the immune system w12x. The presence of CRF in cells of the immune system and in inflamed tissues has recently been reviewed w13x. CRF is found in inflamed synovial tissues at the same concentrations detected in hypophyseal portal venous blood w14x. Moreover, CRF produced in high amounts in inflammatory sites was shown to promote inflammation by potentiating the pro-inflammatory activities of cytokines and other mediators of inflammation w15x. In contrast to its systemic indirect immunosuppressive effects, the enhanced local expression of CRF and its receptors within inflamed tissue suggests that CRF acts as an autocrine or paracrine inflammatory cytokine w6x. Direct local administration of CRF-specific antiserum was reported to suppress the inflammatory response to carrageenin. Using intravital microscopy, it was shown that
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 2 3 9 - 7
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Table 1 Criteria for scoring the histopathological findings
tissue damage in cold injury is attractive since the period between freezing and thawing offers an opportunity for therapeutic intervention. In this context, Serda and Wei w27x have demonstrated that CRF inhibited the local inflammatory response to cold injury. The current experimental evidence concerning the potential activity of CRF in inflammatory processes in vivo still remains controversial. The first aim of this study was to assess the extent of distant organ damage following freezing cold injury and to assess the role of neutrophils in systemic hypothermia by determining tissue associated myeloperoxidase activity ŽMPO. —a reliable index of neutrophil infiltration w28x. The second aim of this study was to determine whether CRF has protective effects on remote organs affected by cold injury and to characterize the involved mechanisms to some extent.
Liver 1. pyknotic hepatocyte nuclei 2. hepatocyte degeneration 3. sinusoidal widening 4. Kupffer cell infiltration 5. parenchymal damage Lung 1. alveolar cell degeneration 2. cellular infiltration 3. intraalveolar hemorrhage 4. interstitial edema 5. congestion 6. septal irregularity Stomach 1. cellular infiltration 2. hemorrhage 3. glandular irregularity 4. epithelial damage Scores are given as 0: none, 1: mild, 2: moderate, 3: maximum for each criterion. At least five microscopic areas were examined to score each specimen.
2. Materials and methods 2.1. Animals
CRF, given intravenously or intracisternally, increased the number of rolling and adhered leukocytes in postcapillary mesenteric venules w6,16x, indicating a local proinflammatory action of CRF. However, systemic as well as local administration of CRF reduces edema, swelling and protein extravasation in different experimental models w17–25x. How the peptide reduces the edema is not fully known, but several possibilities have been documented. It seems clear that CRF exerts its effects locally and these effects are not mediated by secondary release of ACTH, beta-endorphin or corticosteroids. In all of these experimental models, however, the anti-inflammatory role of CRF was investigated at the site of inflammation, while its potential effects on organs distant from the original injury remain to be studied. Systemic hypothermia occurs when the loss of body heat exceeds heat production. When living tissues are frozen and rewarmed, there is initially vasoconstriction, followed by hyperemia, and then a rapid development of edema w26x. The pathogenesis of freezing cold injuries is not yet entirely understood. The use of drugs to modify
Adult Wistar Albino rats of both sexes Ž250–300 g. were kept in a light- and temperature-controlled room on a 12:12-h light–dark cycle, where the temperature Ž22 " 0.58C. and relative humidity Ž65–70%. were kept constant and fed a standard diet and water ad libitum. Experiments were approved by the Marmara University, School of Medicine, Animal Care and Use Committee. 2.2. Experimental protocol Experiments were performed between 9:00 and 12:00 a.m. Rats were anesthetized by intraperitoneal Ži.p.. injection of a mixture of ketamine Ž100 mgrkg. and chlorpromazine Ž12.5 mgrkg.. Cold exposure was applied on the shaved dorsums Ž30% of the total body surface area. of rats for 5 min by laying the animals on the ice block Ž22% NaCl., which was kept at y20 " 0.58C in an insulated box. In anesthetized animals, rectal temperatures were recorded 30 min before, immediately, 30 and 60 min after cold application by an electronic thermometer. Following
Table 2 Changes in rectal temperatures Ž8C. in control rats and in saline or CRF-pretreated Ž12 nmolrkg, subcutaneously, s.c. and 1.5 nmolrrat, intracisternally, i.c.. rats following cold injury
Before cold trauma During cold application 30 min after cold trauma 60 min after cold trauma
Control
i.c. saline
s.c. saline
i.c. CRF
s.c. CRF
37.0 " 0.2 35.4 " 0.3 35.8 " 0.4 35.7 " 0.3
36.4 " 0.2 31.5 " 0.4 ) ) ) 31.2 " 0.5 ) ) ) 32.4 " 0.5 ) ) )
36.8 " 0.2 33.0 " 0.5 ) ) ) 32.7 " 0.5 ) ) ) 33.9 " 0.5 ) ) )
36.7 " 0.3 33.3 " 0.6 ) ) ) ,† 32.9 " 0.7 ) ) ) ,† 33.5 " 0.5 ) ) )
36.4 " 0.2 34.6 " 0.2 ) ) ) ,† 34.3 " 0.1) ) ) ,† 35.5 " 0.2 ) ,†
Values are means" SE. ) p - 0.05, compared to the measurement of the same group before cold application. ))) p - 0.001, compared to the measurement of the same group before cold application. † p - 0.05, compared to the respective saline-treated group.
A. Bozkurt et al.r Regulatory Peptides 99 (2001) 131–139
cold exposure, the rats were towel-dried and kept warm until they recovered from anesthesia. In order to rule out the effects of anesthesia, the same protocol was applied in the control rats, except that they were exposed to a 22% NaCl solution maintained at room temperature Ž22 " 0.58C.. The rats were returned to their home cages until they were sacrificed at 24 h following cold exposure. In all groups, the stomach, liver and right lung were rapidly removed, tissue wet weights were recorded and corrected
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for 100 g body weight. Tissue samples were stored at y708C to determine tissue-associated myeloperoxidase activity ŽMPO. and some samples were fixed in 10% formalin solution for light microscopic examination. 2.3. Administration of drugs CRF ŽSigma. was dissolved in saline and aliquots were stored at y208C until use. Freshly prepared dilutions were
Fig. 1. Effects of intracisternal Ži.c.. or subcutaneous Žs.c.. CRF pretreatment on wet weights of the liver Ža., lung Žb. and stomach Žc. following cold injury. Control rats received vehicle injections but were not exposed to cold injury. Results are expressed as means" SE. ) p - 0.05, ) ) p - 0.01, and ))) p - 0.001; compared to the control group, qp - 0.05; compared to the respective saline-treated group.
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injected subcutaneously Žs.c.; 0.12, 1.2 and 12 nmolrkgf 0.57, 5.7 and 57 mgrkg. or intracisternally Ži.c.; 0.15, 0.30 and 1.5 nmolrrat. 30 or 15 min before the cold trauma, respectively. In the vehicle groups, saline was injected Ž1 mlrkg; s.c. and 5 mlrrat; i.c.. before the cold trauma at the same time points. Control rats also received saline Ži.c.
or s.c.. in the same time course. Each subgroup consisted of 8 to 10 animals. CRF or saline was injected intracisternally by puncture of the occipital membrane with the needle of a Hamilton syringe in rats fixed in ear bars of a stereotaxic device ŽStoelting Lab... The presence of cerebrospinal fluid in the
Fig. 2. Effects of intracisternal Ži.c.. or subcutaneous Žs.c.. CRF pretreatment on tissue MPO activities measured in the liver Ža., lung Žb. and stomach Žc. following cold injury. Control rats received vehicle injections but were not exposed to cold injury. Results are expressed as means" SE. ) p - 0.05, )) p - 0.01, and ) ) ) p - 0.001; compared to the control group, qp - 0.05; compared to the respective saline-treated group.
A. Bozkurt et al.r Regulatory Peptides 99 (2001) 131–139 Table 3 Histologic scores in control rats and in subcutaneously saline or CRF-pretreated Ž1.2 nmolrkg. rats following cold injury
Liver Lung Stomach
Control
Saline
CRF
0.33"0.33 3.0"0.58 0.67"0.33
8.67"0.33 ) ) ) 14.0"0.58 ) ) ) 6.0"0.01) ) )
6.0"0.58 ) ) ,a 7.67"1.20 ) ) ,a 4.0"0.01) )
In each group ns 3. At least five microscopic areas are examined to score each specimen. Values are means"SE. )) p- 0.01 compared to control group. ))) p- 0.001 compared to control group. a p- 0.01 compared to respective vehicle group.
Hamilton syringe upon aspiration for injection insured the correctness of needle placement into the cisterna magna w29x. 2.4. Tissue MPO measurement Tissue-associated MPO activity was utilized as an index of tissue accumulation of neutrophils w28x. Briefly, the tissue samples Ž0.2–0.3 g. were homogenized in 10 vol. of ice-cold potassium phosphate buffer Ž20 mM K 2 HPO4 , pH 7.4.. The homogenate was centrifuged at 10,000 rpm for 20 min at 48C. The pellet was homogenized with an equivalent volume of 50 mM acetic acid ŽpH 6.0. containing 0.5% Žwtrvol. hexadecyltrimethylammonium bromide ŽHETAB.. MPO activity was assessed by measuring the H 2 O 2 -dependent oxidation of 3,3X ,5,5X-tetramethylbenzidine. One unit of enzyme activity was defined as the amount of the MPO present that caused a change in absorbance of 1.0rmin at 655 nm and 378C w30x.
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2.5. Histologic examination The tissue samples of the liver, lung and stomach were fixed in 10% formalin solution. Following dehydration in ascending series of ethanol, tissue samples were cleared in xylene and embedded in paraffin. Tissue sections of 6–7 mm were stained with hematoxylin–eosin and investigated under a light microscope ŽOlympus BH-2, Tokyo, Japan. for characterization of histopathological changes by an observer unaware of the treatment condition of the animal. Histological score of the observed organ was calculated as the sum of the scores Ž0–3. given for each criterion, using the semiquantitative scale shown on Table 1. 2.6. Statistical analysis The results are expressed as mean " SEM. Analysis of variance ŽANOVA. and Tukey–Kramer multiple comparison tests were used for multiple comparisons and Student’s t-test was used to evaluate the level of statistical significance between two groups. Differences were considered statistically significant if p - 0.05.
3. Results The data following CRF administration are only given for the higher Ž12 nmolrkg, s.c. and 1.5 nmolrrat, i.c.. and lower Ž1.2 nmolrkg, s.c. and 0.3 nmolrrat, i.c.. doses, because the lowest doses Ž0.12 nmolrkg, s.c. and 0.15 nmolrrat, i.c.. had negligible effects on the inflammatory parameters.
Fig. 3. Micrographs demonstrate ŽA. normal liver lobular morphology in the control group, ŽB. vacuolated hepatocytes Ž™. and vascular congestion in dilated sinusoids Ž-. in the cold injury group, and ŽC. low degree of degeneration in lobular morphology with few vacuolated hepatocytes Ž≠. in CRF-treated Ž1.2 nmolrkg, s.c.. group. Magnification ŽA. and ŽB. 66 = , ŽC. 33 = , Haematoxylin and Eosin.
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Fig. 4. Micrographs demonstrate ŽA. normal lung parenchyme in the control group, ŽB. a prominent disturbance in alveolar architecture with wide-spread leukocyte infiltration Ž'., fused interalveolar septa Ž ) . in the cold injury group, and ŽC. thin interalveolar septa Ž ) ., low degree of leukocyte infiltration Ž'. in CRF-treated Ž1.2 nmolrkg, s.c.. group. Magnification ŽA. 33 = ; ŽB. and ŽC. 66 = , haematoxylin and eosin.
3.1. Effect of CRF on cold injury-induced change in body temperature The body temperature dropped significantly following cold exposure Ž p - 0.05–0.001; Table 2.. CRF administered at 12 nmolrkg Žsc. and 1.5 nmolrrat Ži.c.. doses before cold exposure attenuated the drop in body temperature when compared with rats which received saline Ž p 0.05., while the lower doses did not significantly affect the cold-induced drop in body temperature Ždata not shown.. 3.2. Effect of CRF on cold injury-induced change in tissue wet weights Following systemic cold injury, tissue wet weights of the liver, lung and stomach were increased significantly in both vehicle groups, where saline was given either i.c. or s.c. Ž p - 0.05–0.01, compared to control group; Fig. 1.. The central administration of CRF at the higher dose reduced the cold-induced edema significantly in the lung and stomach Ž p - 0.05; Fig. 1.. Peripheral administration of CRF at the lower dose Ž p - 0.05. abolished the cold-induced increases in the wet weights of the studied tissues, while the higher dose was not effective in the liver and stomach.
those of the i.c.-saline group and were increased significantly following cold injury Ž p - 0.05–0.001; Fig. 2.. These increases in MPO activities were not reduced by either doses of centrally administered CRF ŽFig. 2.. On the other hand, CRF given peripherally at both doses blocked the elevation of MPO activity in the liver, while this effect was observed only at the higher dose in the lung tissue. However, none of the CRF doses had a significant effect on the elevated MPO activity in the stomach. 3.4. Effect of CRF on cold injury-induced histopathological changes Systemic cold application significantly increased the histologic scores in the liver, lung and stomach Ž p - 0.001; Table 3., when compared with the control group. Peripheral administration of CRF at the lower effective dose Ž1.2 nmolrkg; s.c.. before cold injury reduced the histological scores Ž p - 0.05–0.001. in the liver and lung ŽTable 3; Figs. 3 and 4., but not in the stomach. On the other hand, centrally given CRF decreased the severity of cold-induced damage of the lung and liver, only at the higher dose Ž1.5 nmolrrat; data not shown..
4. Discussion 3.3. Effect of CRF on cold injury-induced change in tissue MPO actiÕity Tissue MPO activities measured in the liver, lung and stomach of the s.c.-saline group were not different from
The results of the present study indicate that cold-exposure-induced edema in the liver, lung and stomach was blocked by subcutaneous CRF treatment. Central administration of CRF had the similar edema-preventive effect
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only at the higher doses, implicating the presence of a peripheral action. Histological assessment also revealed that peripheral CRF pretreatment has a protective role in damage generation. In accordance with the histological findings and edema formation, tissue associated MPO activity—known as the index of neutrophil infiltration— increased by cold trauma in the liver, lung and stomach. Peripherally administered CRF reduced the elevations in MPO in the liver and lung. In addition, CRF had a facilitatory effect in the recovery of the body temperature following cold exposure, which may have a contribution to its protective effect in distant organ injury. Amelioration of cold-induced remote organ injury by the subcutaneous administration of CRF in all the studied tissues suggests that CRF has a peripheral anti-inflammatory action. The most common cause of accidental hypothermia is exposure to environmental low temperature, which can occur during hiking and skiing expeditions, in shipwrecks, or in connection with insufficient heating w31x. The pathogenesis of freezing cold injuries is not entirely understood. If hypothermia is severe, hemoconcentration, vascular stasis, and microcirculatory insufficiency may occur leading to tissue necrosis in the frozen part w32x. Various pharmacological agents, including antihistamines, inhibitors of the arachidonic acid cascade, anticoagulants, vasodilators and sympatholytic drugs, have been tried to improve the survival of frozen tissues. In an attempt to search an opportunity for therapeutic intervention, CRF was shown to have potent inhibitory effects on the acute local inflammatory response to thermal injury w19x. It was shown that exposure of tissues to exogenous CRF produces a reduction in the detrimental effects of several injuries. Systemic as well as local administration of CRF decreases protein extravasation, edema and swelling elicited by antidromic stimulation of peripheral nerves, application of heat or extreme cold stimuli, injection of inflammatory mediators w7,24x. CRF also reduces edema and swelling in the rat carrageenan model of inflammation w17x. It is noteworthy that CRF induces a thermogenic effect and stimulates energy expenditure in laboratory animals w33x, with a reduction in food intake, by increasing the activity of the sympathetic nervous system w34x. In the present study, the thermogenic effect of CRF, as observed with its facilitatory effect on the recovery of the body temperature after cold application, may be participating by reducing the microcirculatory problems and the formation of tissue edema. In addition to its role and distribution throughout the brain and spinal cord, it is also shown that CRF is expressed in various peripheral tissues including stomach and duodenum w35x, lung and adrenal glands w36x and leukocytes and lymphocytes w37,38x. Recently, mRNA coding for the CRF1 receptor gene was found to be present in neutrophils w39x suggesting that local tissue modulation of CRF levels and its receptor activities can affect the course of inflammation with a paracrinerautocrine mechanism
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w6,40,41x. CRF could produce its anti-inflammatory effects by inhibiting the release of inflammatory mediators or by interfering with the actions of these mediators on the nerve or on the endothelium w42x. It is possible that some of the inflammatory mediators are neutrophil-derived. Since the present results show that cold injury-induced elevation in MPO activity is reversed by peripheral CRF, it is likely that CRF has an anti-inflammatory effect by suppressing the accumulation of tissue neutrophils. However, it seems likely that the anti-inflammatory effect of CRF on the stomach is not neutrophil-dependent. The observed variability of the tissue responses to CRF pretreatment may be attributed to dose-dependent anti- or proinflammatory effects of the peptide. Moreover, it may be suggested that the anti-inflammatory effects of CRF are peripherally mediated, since the lower dose of CRF applied subcutaneously Ž1.2 nmolrkg. reduced the inflammatory parameters, while the dose Ž0.3 nmolrrat. given centrally Žwhich is f 1.2 nmolrkg when corrected for the body weight. was not effective. Similarly, Kiang and Wei w19x have suggested that intravenous CRF may be a powerful inhibitor of the inflammatory response of the skin to thermal injury and its effects are not mediated by secondary release of ACTH and beta-endorphin, products that may be derived from the CRF-proopiomelanocortin axis. Dexamethasone, a potent synthetic corticosteroid was also found to be inactive in reducing the tissue response to thermal injury. Moreover, the anti-inflammatory effects of CRF are apparently not mediated via activation of the hypothalamo-pituitary adrenal axis and subsequent steroid action, because similar effects were observed after hypophysectomy or adrenalectomy w17,18,22,23x. The effect of CRF was not antagonized by nalaxone, suggesting that it was not mediated via activation of opioid receptors w22x. Indirect, systemic cardiovascular effects of CRF would also not account for its effect because CRF did not produce hypotension at doses that inhibited vascular leakage w22x. Moreover, topical application of CRF still inhibited vascular leakage w43x. On the other hand, CRF produced in high amounts in inflammatory sites would promote inflammation by potentiating the pro-inflammatory activities of cytokines and other mediators of inflammation w15x. CRF is found in inflamed tissues at the same concentration as in pituitary portal venous blood w14x. In two recent studies, CRF given intravenously increased the number of rolling and adhered leukocytes in postcapillary venules w16x and carrageenin injection into a subcutaneous air-pouch of rats increased local CRF immunoreactivity in parallel to the monoclonal cell infiltration w6x. Local as well as systemic application of a CRF-specific antiserum decreased both the volume and the cellularity of the inflammatory exudate w6x. These findings suggest that CRF has predominantly proinflammatory effects in peripheral inflammatory sites. However, the present data suggest that the local effects of CRF in the freezing cold injury are anti-inflammatory.
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In conclusion, freezing cold injury results in edema formation and histopathological damage in the liver, lung and stomach, accompanied by neutrophil infiltration, suggesting that infiltrated neutrophils are responsible for the injurious effect of freezing cold insult on the liver, lung and stomach. CRF is likely to act on its peripheral receptors in the remote organs to ameliorate the extent of injury, by facilitating the recovery of body temperature and suppressing the edematogenic effects of inflammatory mediators, some of which are neutrophil-derived. Since the consequences of freezing cold injury to tissues are severe, further studies on the use of CRF in this condition merit consideration and future studies are necessary to elucidate the potent inhibitory actions of CRF on inflammatory processes.
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