Perioperative Measurements of Interleukin-6 and ar-Melanocyte-Stimulating Hormone in Cardiac Transplant Patients

T. Sakai, MD,T.W. Latson, MD, C.W. Whit-ten, MD, W.S. Ring, MD, J.M. Lipton, PhD,A.H. Giesecke, MD, and

D.N. O’flaherty, MB

Interleukin-6 (IL-6) and amelanocyte-stimulating hormone (aMSH) are important modulators of the immunologic re- sponse to tissue injury and antigenic challenge. Serial changes in the plasma concentrations of these two peptides were measured in 12 patients undergoing heart transplantation. Tissue concentrations of IL-6 in atrial samples from both donor and recipient hearts were also compared. Plasma IL-6 concentration remained stable prior to cardiopulmonary bypass (CPB), initially decreased with the onset of CPB, and then increased significantly over control values at the end of CPB (160 f 40 v 53 f 60 pg/mL). Plasma IL-6 remained elevated for at least 60 minutes after CPB, and then it returned to control values by 24 hours postoperatively (67 + g pg/mL). Examination of IL-6 changes after CPB in 10 additional patients undergoing nontransplant cardiac sur- gery with CPB revealed a similar elevation in IL-6 at 60 minutes after CPB (290 -C 76 pg/mL). However, IL-6 in the nontransplant group remained significantly elevated at 24 hours (138 * 42 pg/mL). These combined results suggest

I NTERLEUKIN-6 (IL-6) is a pro-inflammatory cytokine that increases in the circulation during infection,’

injury,2x” or antigenic challenge.4 Due to its multiple ac- tions, this cytokine has been previously identified as inter- feron B2, B cell-stimulating factor 2, and hybridoma growth factor. IL-6 is one of the key mediators of the “acute phase response,“5 which is a coordinated systemic response to microbial invasion, inflammation, immune challenge, and tissue injury. In response to tissue injury, increases in IL-6 have been shown to precede changes in IL-l or other acute phase proteins. When IL-6 gains access to the circulation, it induces a broad spectrum of changes in immunologic, hematologic, and endocrinologic factors6 IL-6 produced within tissue (either by specific tissues or by local fibro- blasts, macrophages, and endothelial cells) may also be important in local modulation of the inflammatory re- sponse. Locally released IL-6 may have antiviral activity (similar to interferon), promote expansion of locally acti- vated T cells, and enhance production of antibody by activated B cells.6 Recent evidence suggests that IL-6 may play a role in the host response to organ transplantation. Transient elevations in plasma and urine IL-6 were ob- served 2 days after renal transplantation, with recurrent elevations preceding rejection.’ Two other recent reports have suggested that IL-6 may play a role in accelerated graft atherosclerosis following cardiac transplantation.7,8

a-Melanocyte-stimulating hormone (aMSH) is a trideca- peptide that shares the 1-13 amino acid sequence with ACTH. Similar to IL-6, this molecule may also be impor- tant in modulating the inflammatory response.9,‘0 When given either centrally or peripherally to conscious subjects, uMSH has potent antipyretic and anti-inflammatory ef- fects.” Prior studies suggest that aMSH can antagonize the actions of both IL-l and IL-6.9

As part of an ongoing study investigating the neurohu- moral changes accompanying organ transplantation, the

that CPB causes a marked increase in IL-6, and that implanta- tion of a new heart in transplant patients does not augment this increase. The return of IL-6 to control values by 24 hours in the patients who have had transplants suggests that immunosuppression has an appreciable effect on IL-6 at this time. In contrast to IL-6, plasma aMSH never increased above control values. The absence of a significant change in aMSH suggests that there is no interaction between these two immunologic modulators under the conditions of this study. Tissue IL-6 in hearts with idiopathic cardiomyopathy was significantly less than that in other hearts (ie, donor hearts and hearts with ischemic cardiomyopathy). Additional stud- ies are required to determine if this reduced tissue concentra- tion of IL-6 is related to the cardiomyopathic process. Copyright 0 1993 by W. B. Saunders Company

KEY WORDS: immune response, cardiac surgery, cardiomy- opathy, cardiopulmonary bypass

acute changes in plasma IL-6 and aMSH that occur during cardiac transplantation were measured. To determine if atria1 tissue is capable of producing IL-6, and to determine if its production is influenced by cardiomyopathy, the tissue concentration of IL-6 in both native and recipient atria1 tissue was also assayed. The results documented changes in plasma IL-6, which appear to be different from that reported with other types of surgery. To assess the influence of heart implantation and immunosuppression on these IL-6 changes, IL-6 levels were subsequently measured in plasma samples from 10 additional patients undergoing nontransplant cardiac surgery with cardiopulmonary bypass (CPB).

METHODS

Transplant Patient:?

After Institutional Review Board approval, 12 patients undergo- ing heart transplantation were studied; informed written consent was obtained from each of them. The patients ranged in age from 43 to 65 years. Upon arrival in the operating room, each patient was sedated with midazolam (0.03 to 0.05 mg/kg) IV. Intravenous and radial artery catheters were placed percutaneously under local anesthesia. Anesthesia was induced with sufentanil (0.15 to 0.85 pg/kg) and/or etomidate (0.1 to 0.25 mgikg), along with vecuro- nium (0.14 to 0.18 mg/kg) for muscle relaxation. After induction of anesthesia, a pulmonary artery catheter was inserted in each patient. Anesthesia was maintained with additional doses of sufentanil and/or supplemental isoflurane (0.2% to 1%). Ventila- tion was mechanically controlled at a tidal volume of approximately

From the Departments of Anesthesiology and Surgery, University of Texas Southwestern Medical Center at Dallas, Dallas, TX.

Address reprint requests to Teq W Larson, MD, Department of Anesthesiology, University of Texas Southwestern Medical Center at Dallas, 5323 Hany Hines Blvd, Dallas, TX 752358894.

Copyright 0 1993 by KB. Saunders Company 1053-0070/931070.1-0004$03.0010

Journalof Cardiothoracic and VascularAnesthesia, Vol7, No 1 (February), 1993: pp 17-22 17

18

10 mL/kg and a rate of X to 10 breaths/minute adjusted to maintain the PaC02 at 35 +- 5 mmHg.

Blood samples were withdrawn from the arterial catheter at the following times: before induction (Pl); after induction (P2); after incision (P3); immediately before CPB (P4); during CPB, immedi- ately before removal of the old heart (P5); during CPB, immedi- ately before reperfusion of the new heart (P6); during CPB, immediately before weaning from CPB (P7); 10 minutes after discontinuance of CPB (P8); 60 minutes after discontinuance of CPB (P9); and 24 hours postoperatively (PlO).

All samples of arterial blood (4 mL) were collected in chilled tubes containing EDTA and 100 pL of aprotinin (0.67 trypsin- inhibiting units/mL of blood). The samples were immediately placed in ice and centrifuged within 10 minutes (3,000 rpm, 4”C, 15 minutes). After transfer of the platelet-rich plasma to polypropy- lene Eppendorf tubes, the samples were centrifuged in a microfuge for 1 minute. The plasma was stored at -70°C until the assays were performed. Plasma samples were assayed for IL-6 using a radioim- munoassay kit (R&D Systems, Minneapolis, MN). For this assay, sensitivity is 3.5 pg/mL, intra-assay variation is 7OJ to 9%, and interassay variation 5% to 10%. This is a highly specific assay for IL-6 with no measurable cross-reactivity with IL-l, IL-2, IL-3, IL-4, or tumor necosis factor (TNF). A radioimmunoassay kit (Milab, Maemii Immun Laboratorium AB, Sweden) was used to measure the concentration of nMSH in unextracted plasma samples (n = 9 due to insufficient plasma sample for adequate assay in 3 patients). The sensitivity of this assay is 3 pmol/L; the coefficient of variation is 3.9% to 4.1%. Cross-reactivity with ACTH (l-24), ACTH (l-39), p and y uMSH is less than 0.002%. Cross-reactivity with des-acetyl- (r-MSH is 100%.

For measurement of IL-6 concentrations in atria1 tissue, atrial tissue samples were obtained from the nonauricular free wall of the right atrium for both native (n = 12) and donor (n = 8) hearts. In donor hearts, this sample was taken when the preserved heart was brought into the “recipient” operating room (donor hearts were perfused with cardioplegia before excision, and then immediately placed in iced saline). For the native heart, this sample was taken immediately after the native heart was excised. Tissue samples were immediately placed in iced saline, and subsequently frozen in liquid nitrogen until assays were performed. For measurements of IL-6 tissue concentration, the tissue samples were placed in an ice-cold solution of 0.1 M acetic acid with 0.02 N HCI and homogenized with a polytron (VirTishear, Virtis Co, Gardiner, NY) for six 30-second intervals. Tissue homogenants were then sonicated for three 20-second intervals using a Virsonic 50 (Virtis Company). IL-6 in tissue homogenates was then measured using the same assay used for plasma IL-6. IL-6 concentrations are reported as pg of IL-6 per milligram of total protein. Total protein was assessed by comparing the optical density of dyed tissue homogenates with the density of known standards (Biorad Labora- tories, Richmond, CA).

The significance of changes in plasma IL-6 and aMSH measured at different sample times was assessed with the Wilcoxon signed ranks test. The significance of differences in tissue concentrations of IL-6 in native and donor atria was assessed using the Mann- Whitney U test and the Fisher exact test. For all statistical comparisons, P values <0.05 were considered significant. All values are reported as mean 2 standard error of the mean.

Nontransplant Cardiac Surgical Patients

To assess the possible influence of heart implantation and immunosuppression on IL-6 changes measured in transplant pa- tients, IL-6 was subsequently measured in plasma samples from 10 patients undergoing nontransplant cardiac surgery with CPB. To

SAKAI ET AL

control for possible effects of CPB duration, all patients used for comparison had bypass times of 100 minutes or greater; mean bypass times were similar in both groups (transplant group: 140 t 9 minutes; nontransplant group: 138 ? 9 minutes). These patients were enrolled in a separate study examining immuno- endocrine responses to CPB, and informed consent was obtained from all patients. Anesthetic technique (etomidate, sufentanil, isoflurane, and vecuronium) and CPB management were similar to that used with patients undergoing transplant surgery. Sampling times equivalent to those evaluated in transplant patients included (1) prior to induction; (2) 60 minutes after CPB; (3) 24 hours postoperatively.

RESULTS

Transplant Patients

Anesthesia and surgical stress before the start of CPB had no appreciable effect on plasma IL-6 (Pl-P4, Fig 1). A small decrease in plasma IL-6 was observed during hypother- mic CPB (P6). With rewarming and reperfusion of the donor heart (P7), there was a consistent increase in plasma IL-6 in all patients (Pl: 53 + 6; P7: 180 -C 40 pg/mL). This increase in IL-6 persisted for at least 60 minutes (P8: 205 5 28; P9: 206 + 34 pg/mL). Between sample point PY and PlO (24 hours postoperatively), plasma IL-6 decreased in every patient, such that IL-6 levels at PlO were not significantly different from control values (PlO: 67 2 9

pg/mL). Similar to IL-6, plasma aMSH showed an initial decrease

with onset of CPB and hypothermia, followed by an increase during rewarming and reperfusion of the donor heart (P7, Fig 2). However, in contrast to IL-6, plasma aMSH at the end of CPB was not significantly elevated above control measurements. All subsequent posttrans- plant measurements of plasma clMSH were also not signifi- cantly different from control measurements.

Significant quantities of IL-6 were detected in all atrial tissue samples. When considered as a uniform group, the tissue concentration of IL-6 in native atria (298 2 90 pgimg) was not significantly different from the concentra- tion in donor atria (271 + 72 &mg). However, subdivision

200 Plasma II-6

(P&a 100

L~_______I 01 j j ’ ’ c Pl P2 P3 P4 P5 P6 P7 P8 P9 PlO

Sample Point

Fig 1. Perioperative changes in plasma IL-6 concentration. See text for explanation of sample points. Brackets depict standard error of the mean. ??P c .05; #P c .Ol; Sample vs control (Pl).

IL-6 IN HEART TRANSPLANT PATIENTS 19

, ’ CPB : I I

(Pg/mL) 15

10

5

0 Pl P2 P3 P4 P5 P6 P7 PB P9 PlO

Sample Point

Fig 2. Perioperative changes in plasma aMSH concentration. See text for explanation of sample points. Brackets depict standard error of the mean. ‘P c .02; Sample vs control (Pl).

of the native atria1 tissue samples into two sets based on the etiology of heart failure revealed relatively low atria1 IL-6 concentrations in a high percentage of tissue from patients with idiopathic cardiomyopathy. Four of five patients with idiopathic cardiomyopathy had atria1 tissue IL-6 concentra- tions less than 70 ugimg of protein, as compared to only two of seven patients with ischemic cardiomyopathy and one of eight donor patients (P < .05).

Nontransplant Patients

Similar to observations in patients who had undergone transplants, analysis of plasma samples in nontransplant patients revealed significant increases in IL-6 over control measurements at 60 minutes after CPB (290 ? 76 vs 7 ? 2 pg/mL, P < .Ol). In contrast to what was observed in transplant patients, IL-6 remained significantly elevated over control measurements at 24 hours postoperatively (138 ? 42 pg/mL, P < .Ol).

DISCUSSION

The “acute phase response” is a term that is used to describe a coordinated systemic response to significant tissue injury and/or microbial invasion.12-*5 This response is currently believed to have more than 15 components including fever, leucocytosis, tachycardia, net catabolism, and altered concentrations of various circulating proteins known as “acute phase proteins.” Recent investigations suggest that a “network” of various cytokines may be instrumental in orchestrating this acute phase response.6 Of particular importance are IL-l, TNF and IL-6. IL-6 appears to play a pivotal role in production of the acute phase proteins.2 IL-6 is identical to hepatocyte-stimulating factor, which has been shown to be the major inducer of acute phase proteins in rat hepatoma cell cultures.16

Previous studies by Cruickshank et al2 have documented an increase in IL-6 accompanying various types of surgical procedures (hip replacement, vascular surgery, colorectal surgery, cholecystectomy, and minor peripheral surgery).

An initial increase in IL-6 occurred in all patients within 2 to 4 hours of incision, with concentrations reaching a peak at 6 to 12 hours, postincision. The magnitude and time course of IL-6 elevations appeared to correlate with the extent and duration of surgery, and it was not influenced by the anesthetic technique. Changes in IL-6 preceded changes in C-reactive protein, and were a more sensitive indicator of the magnitude of surgical trauma.

In the present study, no significant change was found in IL-6 after anesthesia induction or surgical stimulation in the prebypass period. This absence of an initial response may be due to the limited time interval between incision and initiation of CPB in this study. This time interval ranged from 28 to 145 minutes (mean 58 t 9 minutes), but was less than 70 minutes in 11 of the 12 patients. Although one previous study documented increases in IL-6 as early as 60 minutesi others suggest a more delayed response (1.5 to 4 hours).2~1s-2’1 As pointed out by Cruickshank et aL2 such a delayed response may reflect the time required for IL-6 transcription. In stimulated monocytes, an increase in IL-6 mRNA was detectable at 1 hour, with peak expression at 3 hours2i

With initiation of CPB, there was a small decrease in IL-6. Possible etiologies for this decrease include the effects of hypothermia, hemodilution, and alterations in periph- eral blood lymphocytes induced by CPB.22-24 After rewarm- ing and reperfusion of the newly implanted heart, there was a significant increase in IL-6. This increase in IL-6 persisted for at least 60 minutes. Considering the half-life of IL-6 (initial 3-minute distribution half-life, followed by a slow elimination half-life of 55 minutes),25 this persistent eleva- tion suggests that augmented release of IL-6 continued into the postbypass period. Plasma IL-6 then returned to control levels by 24 hours.

Similar to the results of Cruickshank et al2 in patients having noncardiac surgery, the increase in IL-6 in the present study may be a delayed response to tissue injury. The magnitude of IL-6 increase in these patients is similar to that reported in their previous study in the subgroups of patients having hip surgery, vascular surgery, or colorectal surgery.2 However, the elevation in plasma IL-6 in the present study appeared to be more acutely related to CPB and/or events in the latter part of CPB rather than a gradual response to continuing surgical trauma. Although studies in patients having smaller operations (eg, cholecys- tectomy) have shown peaks in IL-6 as early as 1.5 hours after incision,is the studies of Cruickshank et al2 in patients having major surgery documented peak IL-6 concentrations between 6 and 12 hours after incision. In contrast, mean IL-6 in the present !itudy reached a plateau 10 minutes after CPB; this corresponds to a mean time from skin incision of only 201 ? 12 minutes.

To assess the possible influence of cardiac implantation on this increase in IL-6 after CPB, IL-6 concentrations were subsequently measured in plasma samples from patients undergoing nontransplant cardiac surgery with CPB. Dura- tion of exposure to CPB was equivalent to that in the transplant patients. These nontransplant patients exhibited a similar increase in IL-6 at 60 minutes after CPB (290 2 76

20 SAKAI ET AL

pg/mL). This result suggests that the increase in IL-6 observed in the transplant patients was a nonspecific response to CPB and surgery rather than a specific response to heart implantation.

Prior studies have shown that CPB is associated with an array of changes in the immune system involving both cellular and humoral factors.2” Changes suggesting both depression and activation of the immune system have been described. The increase in IL-6 that was observed may be another important change in the immune system induced by CPB. This change may have particular importance in that increases in IL-6 appear to be one of the earliest cytokine responses to tissue injury and inflammation, and may play a pivotal role in modulating other cytokine responses.h

All patients in the present study did receive immunosup- pression with preoperative oral cyclosporine (4 to 6 mg/kg), oral azathioprine (2 mg/kg), and intraoperative methylpred- nisolone (500 mg IV administered at initiation of CPB). This immunosuppressive regimen may be responsible for the early return of IL-6 to control values in the transplant patients. In the nontransplant patients, IL-6 remained significantly elevated at 24 hours. This result in the nontrans- plant patients is consistent with studies of IL-6 changes in patients undergoing other types of major surgery. In these studies, plasma IL-6 did not return to control levels for well over 48 hours?J7J9

Cyclosporine A is known to inhibit the production and secretion of IL-2, IL-3, IL-4, IL-5, and interferon.lxJhx27 The observation that IL-6 returned to baseline concentrations within 24 hours in these patients indirectly suggests that this drug may similarly inhibit secretion of IL-6 in the postoper- ative period. However, possible effects of other immunosup- pressant drugs (methylprednisolone, azathioprine) cannot be ruled out.

Despite immunosuppressive therapy, patients in the present study still had significant intraoperative elevations of IL-6. Although this inability of preoperative immunosup- pression to ablate the intraoperative IL-6 response may be an inherent “limitation” of drug effects, it might also relate to poor absorption of orally administered drugs when given in the immediate preoperative period. Most patients in the present study received oral cyclosporine within 90 minutes of the start of surgery. Brown et a128 have shown that oral cyclosporine administered less than 4 hours prior to surgery may not produce therapeutic levels during the operative period. Gelb et a12Y have shown that the subtherapeutic intraoperative levels may relate to altered cyclosporine absorption during isoflurane anesthesia. Whether earlier oral administration (or IV administration) of preoperative immunosuppressant drugs might limit the observed in- crease in IL-6 will require additional study.

These observed changes in IL-6 should be contrasted to prior reports of changes in IL-l. One prior study in patients undergoing nontransplant cardiac surgery found no signifi- cant increase in IL-1 either during or 2 hours after CPB, but a subsequent twofold increase 24 hours later.30 In a subset of four patients in the present study, only a minimal increase in IL-1 was found at the end of CPB, followed by a twofold increase over control levels at 24 hours. Additional

studies in other types of surgeries have also documented early changes in IL-6, with delayed or absent responses in IL-1.i7,1’ These two interleukins thus appear to behave differently in response to tissue injury, with IL-6 consis- tently showing a more immediate response. These changes in IL-6 have also been shown to precede changes in acute phase proteins.2,‘y.2’j

The widespread distribution of otMSH receptors in the body suggests that this neurohormone may have important physiologic actions. Such actions may include: (1) a neuro- transmitter in the central nervous system; (2) an “endocrine stimulant”: (3) a pigmenting agent; and (4) a modulator of the immune-inflammatory response.“‘.” Some investigators have suggested that uMSH be renamed “intermodulin,” because it appears to modulate such a variety of biologic actions.“’ With respect to systemic inflammatory responses. (YMSH is a potent antagonist of the pyrogenic activity of IL-l and IL-6.” Other studies in mice have shown that (wMSH also inhibits neutrophilia. synthesis of serum amy- loid, and increases in plasma corticosterone induced by IL-1 and TNF.” The plasma concentrations of clMSH increase in rabbits given endotoxin or endogenous pyrogen, and in normal humans given bacterial lipopolysaccharides.q Recent unpublished observations suggest that these in- creases in aMSH tend to be associated with early, acute challenges to the host, and that they may not be sustained. The factors responsible for the increases in circulating aMSH are unknown. Because increases in circulating IL-6 also appear early after immunologic challenge and/or tissue trauma, a “feedback” interaction between IL-6 and aMSH has been suggested. The results from the present study suggest that elevations in circulating IL-6 arc not an essential factor in the “immunologic” release of aMSH. because a fourfold increase in plasma IL-6 was not associ- ated with any significant changes in plasma (uMSH levels. Possible explanations for this negative result include (1) a true dissociation between the actions of these two immuno- logic modulators; (2) anesthetic suppression of normal pituitary function: or (3) “pulsatile” secretion of CXMSH by the pituitary, such that increased (uMSH secretion may not be evident with only intermittent plasma sampling.

Although lymphocytes are felt to be the predominant source of circulating IL-h, other cell types known to produce IL-6 include fibroblasts, keratinocytes, endothelial cells, hepatocytes, and vascular smooth muscle cells.“J? Recent immunohistologic studies suggest that ventricular myocytes are also capable of IL-6 production.7 IL-6 produc- tion by ventricular myocytes and vascular smooth muscle may play a role in accelerated graft atherosclerosis.‘J Possible mechanisms by which IL-6 may influence graft atherosclerosis include enhancement of both smooth mus- cle libronectin synthesiss and smooth muscle cell prolifera- tion.“4--7h The present results document that human right atria1 tissue also contains significant concentrations of IL-6. Although some contribution of IL-6 from other cell types (eg, fibroblasts, macrophages) cannot be excluded, these results suggest that human atria1 myocytes may also bc capable of elaborating this cytokine. Production of IL-6 by these other tissues suggests a potential role for IL-6 in

IL-6 IN HEART TRANSPLANT PATIENTS 21

modulating the inflammatory response at a local level. In this regard, the finding of lower atria1 tissue concentrations of IL-6 in patients with idiopathic cardiomyopathy (relative to that measured in donor hearts and hearts with ischemic cardiomyopathy) may have important implications. These lower tissue levels might represent (1) a marker of an immunologic deficiency in patients prone to develop idio- pathic cardiomyopathy; (2) altered production of IL-6 resulting from the cardiomyopathic process; or (3) part of an ongoing immunologic response to the cardiomyopathic process. Alternatively, these lower tissue levels of IL-6 could be an indirect result caused by altered concentrations of total tissue proteins (which includes extracellular pro- teins) rather than altered cellular functions. Additional studies specifically comparing tissue concentrations of IL-6 with concentrations of more specific cellular proteins (in contrast to total tissue protein) are warranted.

In summary, these results document a significant in- crease in the plasma concentration of IL-6 during cardiac transplantation. This increase in IL-6 was temporally asso-

ciated with CPB rewarming and with reperfusion of the newly implanted heart. Subsequent measurement of IL-6 in plasma samples from patients undergoing noncardiac trans- plant surgery documented similar increases in IL-6 at 60 minutes after CPE, suggesting that the observed increase in IL-6 in transplant patients was a nonspecific reaction to CPB rather than a unique response to heart implantation. In contrast to prior surgical studies, and to the results in nontransplant cardiac surgical patients, IL-6 in transplant patients returned to control levels by 24 hours after operation. This early normalization of IL-6 suggests that perioperative immunosuppression had a significant influ- ence on IL-6 at this time. In contrast to plasma IL-6, no significant increase in plasma (uMSH over control values at any time during the study was found. Assays of tissue concentrations of IL-6 documented that most patients with idiopathic cardiomyopathy had relatively low atria1 IL-6 concentrations (pg/mg of total protein) compared to that measured in atria from other patients (ie, donors and patients with ischemic cardiomyopathy).

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