Hyperstimulation of leukocytes by plasma from cardiopulmonary bypass patients is diminished by α-MSH pretreatment

International

Journal

of

cardiology ELSEVIER

International Journal of Cardiology 53 Suppl.(1996) S47-S53

Hyperstimulation of leukocytes by plasma from cardiopulmonary bypass patients is diminished by a-MSH pretreatment Thomas V. Bilfinger *a*b-,Thomas K. Hughes ‘, Madeline Rodriguezb, Richard Glassb, Frederic0 Casaresb, George B. StefanoaTb “Depurtment

of Surgery,

University

Medical

Center, State University of New York at Stony Brook, Stony New York 11794-8191. USA bMultidisciplinary Center for the Study of Aging, Neuroscience Research Institute, State University of New YorklCollege at Old Westbury, New York, USA ‘Depurtment of Microbiology, University.of Texas Medical Branch, Galveston, Texas, USA

Brook,

Abstract

Cardiopulmonarybypass(CPB) resultsin a diffuseinflammatory responsecharacterizedin part by hyperstimulation of leukocytes.We have previously shownthat this hyperstimulationappearsto be due, in part, to an increase in the releaseof biological responsemodifiers(BRMs) such as cytokines. In the presentstudy, we evaluatedthe ability of a naturally occurring immunocyteinhibitory substance,a-melanocyte-stimulatinghormone(a-MSH), to prevent the hyperstimulationcausedby CPB. Monocytes and granulocyteswere pretreatedwith a-MSH (10m6M) before exposing the cells to plasma obtained from patients who had undergoneCPB, as CPB plasmawould stimulatenaive monocytesand granulocytesin a mannersimilar to that observedin CPB patients. Pretreatment of thesecellswith a-MSH significantly diminishedthe hyperstimulation induced by CPB plasmain a concentration-dependentmanner.In contrast, when the cells werefirst or simultaneouslyexposedto CPB plasmaand then to a-MSH, a-MSH had no effect. Furthermore, useof the specificneutral endopeptidaseinhibitor, phosphoramidon, significantly increased the efficacy of a-MSH in inhibiting CPB-induced immunocyte activation. The data demonstratethat pretreatment of monocyte/macrophagesand granulocytes with a-MSH effectively inhibits the immune hyperstimulation induced by CPB-plasmaexposure. In addition, the data strongly suggestthat preexposureto other naturally occurring immuneinhibitory substances may diminishthe hyperstimulationassociated with CPB. The study also further confirmsthat this hyperstimulationmay, in part, be due to BRMs releasedfrom immunocytes.

Keywords:

Alpha-MSH; Granulocytes; Monocytes; Cardiopulmonarybypass

*Corresponding author. 0167-5273/96/$15.00 0 1996ElsevierIrelandLtd. All rightsreserved PII SO167-5273(96)02571-S

1. Introduction

2. Materials

and methods

Cardiopulmonary bypass (CPB) has been implicated in causing a diffuse inflammatory response [1,2], and results in general immune dysfunction [3-71. This dysfunction may arise from the effects of surgical trauma, and also from the rapid movement of immune cells over artificial surfaces (e.g. CPB circuit), resulting in cellular destruction, complement activation, and functional, as well as morphological, changes in immunocytes indicative of activation [4,6,7- 131. More recently, it has been shown that many of the pathological sequelae related to CPB are the result of an increase in the level of endogenous biological response modifiers (BRMs) normally present in plasma which may arise from immunocytes. It appears that increased levels of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-cc are produced by monocytes/macrophages, granulocytes and other cells [14], which in turn further exacerbates the cellular trauma associated with CPB by further enhancing immunocyte activation to the point that the cells are hyperstimulated and thus rendered dysfunctional [15]. Our laboratory, using computer-assisted microscopic image analysis, has studied the behavior of leukocytes before, during and after CPB [6,7]. We found that leukocytes obtained from patients undergoing CPB and those obtained 38 h postCPB do not respond chemotaxically to naturally occurring signal molecules, regardless of whether they are excitatory or inhibitory, and that a large number exhibit chemokinesis [6,7,15]. Furthermore, plasma obtained from CPB patients induces similar effects on naive, non CPB-exposed cells [6]. The present study tested the hypothesis that naturally occurring immunoinhibitory signal molecules (e.g., cc-melanocyte-stimulating hormone (c(-MSH) [16-181) may be used preoperatively to inhibit leukocyte hyperstimulation associated with CPB by downregulating these cells before CPB exposure. We specifically sought to antagonize the stimulatory effect of CPB plasma on naive monocyte/macrophages and granulocytes, since these cells give rise to many BRMs found in CPB plasma [14,19].

Human granulocytes and macrophages for cellular analysis were obtained from the Long lsland Blood Services (Melville, NY). Plasma was obtained from controls (n = 5 normal healthy individuals) and CPB patients (n = 10) at University Medical Center who had given their informed consent. Blood was obtained through a central venous access after 30 min of CPB. After centrifugation (500 x g for 25 min), plasma from controls and CPB patients (38 h after surgery) were immediately frozen at - 70°C and stored for up to one year .before use. Patient eligibility included admission for elective CPB only. Patients with chronic illnesses such as diabetes or cancer, as well as acute processes (e.g., known infections, trauma, etc.) were not eligible. In all patients undergoing CPB, the same anesthetic technique was used, which consisted of induction with fentanyl (fentanyl citrate adjusted for pH) or sufentanyl (N-[-Lt(methoxymethy1 O&l-[2-(thienyl) ethyl]-4-piperidinyl]-jV-phenylpropanamide 2 hydroxy-1,2,3 propanetricarboxylate) up to 15 pg/kg. Maintenance was achieved with the same agents. Standard CPB was instituted at moderate hypothermia (28’C). The circuit consisted of polyvinyl chloride tubing with roller pumps (Sarns Co., Ann Arbor, MI), a cardiotomy sucker and reservoir and a hollow fiber oxygenator (Baxter Univox, Irvine, CA). 2.1. Analysis of cellular activity The analysis of cell activation and inhibition was determined as noted extensively elsewhere [6,7,20,21]. Normal cells (granulocytes and macrophages not exposed to CPB) were obtained by Ficoll-Hypaque centrifugation. Briefly, 100 ~1 of RPM1 containing naive cells were placed in a ring of petroleum jelly on a glass slide. The cells were then treated as required with c(-MSH and/or phosphoramidon (100 PM) with 100 pl of CPB plasma or non-CPB plasma, and covered with a glass cover slip. The cells were observed for periods up to 1 h at 37 “C. Activation (change of cellular conformation and movement) was determined through the use of cell image analysis software (American Innovi-

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sion, Inc., San Diego, CA; [21-241). Changes in cellular conformation ranging from inactiverounded to active-amoeboid were determined by measurements of cellular area and perimeter and were mathematically expressed by use of the shape-factor formula of the American Innovision Analysis System. The following formula was used: AC/AT = (LC/LT)2, in which AT represents the area of a circle with the same perimeter as that of a given cell, and AC and LC represent the actual area and perimeter of the cell [22-253. The lower the number, the higher the perimeter and the more amoeboid the cellular shape. The proportion of activated cells was determined as noted elsewhere [25]. Activated cells not only changed their conformation in response to a pharmacological stimulus, they also became mobile and were capable of phagocytosis [25]. Cells (n = 39-52) were observed for each 400 pm viewing diameter, and four additional viewing diameters were observed per slide. This was treated ten times with CPB-exposed plasma from ten different patients and five times from controls. The resulting mean (k S.D.) was graphed. 2.2. Reagents a-M SH and phosphoramidon were obtained from Sigma, Inc. (St. Louis, MO), and human TNF-cr antibody was obtained from Repligen (Boston, MA).

cytes with a-MSH progressively reduced the level of spontaneous activation in a concentration-dependent manner over a 50-min observation period (Fig. 1). A concomitant incubation of the granulocytes with 10e9 M a-MSH, a concentration shown to be ineffective in inhibiting the spontaneous activation level of this cell, and 100 FM phosphoramidon, a specific neutral endopeptidase inhibitor which alone does not alter granulocyte activity [20], resulted in inhibiting the spontaneous activation level during the 50min observation period (Fig. 2). Control cells incubated with CPB plasma, obtained from patients 38 h postsurgery (diluted 1:l

O-O .-.

a-MSH a-MSH

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2.3. Statistical analysis Analysis was performed by using Student’s t-test to compare controls with either drug or CPB-exposed cells as noted in the text. A Pvalue < 0.05 was considered significant. 3. Results The level of spontaneous activation (conformational changes, mobility) of control granulocytes and macrophages as determined by shape factor analysis was the same as previously reported [22, 23, 251. Spontaneous activation of granulocytes or macrophages increased to 8.3% + 2.7% (S.D.) and 7.8% + 2.4%, respectively, during the 60-min incubation period. Incubation of control granulocytes and mono-

Fig. 1. r-MSH downregulation of spontaneously activated (top) granulocytes and (bottom) monocytes. The number of cells observed for each time period varied from 67 to 94. Activation is defined as cells exhibiting a form factor (4 x H x area/per”) of <0.6, whereas inactivation form factors are >0.8. Each value graphed represents the mean of the mean ( f SD.) of three replicates. Statistical significance was obtained from a one-tailed Student’s r-test.

s50

.-: r;

T. V. BirJsnger et al. I InternationaI

Journal of Cardiology 53 Suppl. (1996) S47-S53

60 I ”

/ 0

0

a-MSH

(1O-g

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100 &

.-6 50 z .1. 4 40 al x 0" 5

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60

(mln)

Fig. 2. a-MSH inhibition of spontaneously active human granulocytes in the absenceor presenceof the specific neutral endopeptidase inhibitor, phosphoramidon (100 PM). The 10m9 M concentration of wMSH without phosphoramidon is without effect on altering granulocyte activation (see Fig. 1). Measurements were made as described in the text. Each value represents 20 to 50 readings. The percentage of both nontreated spontaneously active (9.5+ 1.2% (S.D.)) and phosphoramidon treated cells were significant at the PcO.05 level (30 min) when compared to control and or-MSH plus phosphoramidon treated cells alone. Data was analysed by Student’s t-test.

with RPMI), became activated (changes in confirmation and motility) within 30 min (45.6% and 49.9% for granulocytes and monocytes, respectively; Fig. 3). a-MSH (lO-‘j M) pre-exposure (50-min incubation) resulted in a reduction in the number of cells activated when later exposed to CPB plasma for 30 min. a-MSH reduced the activation level to 15% and 13.1% (P < 0.01 compared to CPB-exposed plasma alone) for granulocytes and monocytes, respectively. In contrast, when cells were pretreated or concomitantly exposed to CPB plasma and then to aMSH, immunocyte inhibition did not occur (Fig. 3). Analysis of the CPB plasma revealed the following TNF-a levels: control, < 10 pg/ml; preCPB, 12.2 + 3.4 pg/ml; on CPB (30 min), 23.8 + 4.9 pg/ml; 38 h post-CPB, 114.0 + 37.2 pg/ml. Since TNF-a has been shown to activate granulocytes, we attempted to inhibit this excitatory action by pretreating the cells with TNF-a antibody in order to inhibit the immunocyteactivation by CPB plasma containing this signal

7

8 a-MSH

-__5

6 (lo-”

M)

Fig. 3. The effect of cl-MSH on the activation rate after preor post-exposure of immunocytes to CPB plasma. Cells exposed to CPB alone (cont. mark on y-axis) exhibit an activation level of 48% + 5.2% (combined; S.E.M.) within 30 min. Triangles represent cells that were exposed to CPB plasma first and then to a-MSH, whereas circles represent cells pre-incubated with a-MSH (45 min) and then with CPB plasma for 30 min. The number of cells per a-MSH concentration varied between 70 and 79. Each value graphed represents the mean of the mean (+S.D.) of three replicates. Statistical significance was determined by Student’s f-test.

molecule. The antibody partially antagonized (P < 0.05) the immunocyte activation of the CPB plasma4 indicating that TNF was in part contributing t’o activation. This experiment further sug60

6--6Mon

+ MSH

w-Cant.

t PC0 05 100

10 TNF-a

10

0 I

(U/ml)

Fig. 4. TNF-antibody partially inhibits the activation of human granulocytes exposed to CPB-plasma. Cells were first incubated in anti-TNF-a (10 U/ml for 45 min) and then exposed to CPB-plasma for 30 min before cellular determination began. Each value represents 37 to 42 readings replicated three times and the mean of each value was combined and the resultant mean (+S.D.) graphed. The percentage of nontreated spontaneously active cells was 8.3% f 1.1% (SD.). Data was analysed by Student’s t-test.

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geststhat other immunocyte excitnrory molecules were present in the CPB plasma at sufficient levels to initiate activation. 4. Discussion Numerous studies have demonstrated that depression of cell-mediated immunity occurs following surgical procedures [7,26-351. Concerning CPB in particular, the activation of complement and the contact-activating system with cleavage of pre-kallikrein into kallikrein, in addition to direct cell damage, result in the activation of leukocytes [36,37]. These cells, in turn, play a major role in the release of a variety of chemotaxic, chemokinetic and vasoactive substances cascading into the whole body diffuse inflammatory response. The release of cytokines and other signal molecules as a consequence of surgery, especially CPB, is now well documented [19]. Since these cytokines act in an autocrine fashion, they may further induce their own release, compounding the resultant immunocyte dysfunction and hyperstim$ation. Much attention has been given to these proinflammatory molecules and their action, as well as to methods for blocking their effects. Very little attention, however, has been paid to modulators of these proinflammatory cytokines or to the cellular conditions leading to release of these BRMs. The use of naturally occurring signal molecules to regulate physiological activities is quite appealing, since problems (e.g. degradation) associated with pharmacological compounds may be avoided. In this regard, a-MSH appears to offer great potential for its ability to downregulate various types of immune activities, especially those associated with immunocyte functions (see [l&38,39]). a-MSH inhibits both the clinical and histological signs of experimental arthritis [40]. a-MSH has been shown to inhibit the acute inflammation induced in mice by IL-l, IL-6 and TNF-a [41]. Recent studies also demonstrate that this peptide derived from adrenocorticotrophic hormone (ACTH) can also inhibit the synthesis of interferon-gamma [42]. Thus, a-MSH may be used to limit the extent of damage due to sepsisand other

S51

conditions causing inflammation (see [39]). Furthermore, a-MSH has been shown to inhibit the migration of neutrophils as well as other types of immunocytes across diverse animal phyla [1618,431. In this regard, a-MSH has been implicated in the mode of immunosuppressive action of the human immunodefiency virus (HIV) L-181.In H9 cells, HIV causesthe production of ACTH which is converted by the “hosts” neutral endopeptidase to a-MSH, which in turn inhibits the migration (chemotaxis) of potential immuno-active immunocytes. In quite a different pathology, the human schistosoma worm, Schistosomamansoni, actually releasesACTH-like molecules which, as noted above, form a-MSH [17]. Thus, it would appear that a-MSH’s immunosuppressive activities have been used by many divergent organisms throughout evolution. The following conclusions can be drawn from the present study: (a) Plasma obtained from CPB patients contains granulocyte and monocyte stimulatory substance(s), i.e. TNF-a, which confirms our previous observations [6]; (b) a-MSH can reduce spontaneous activation; (c) a-MSH can similarly reduce stimulation induced by CPB plasma; (d) In contrast, pre-exposure of the cells to CPB plasma negated the a-MSH effect. In summary, active signal molecules, mainly IL-6 and in our study TNF-a, capable of stimulating cells are released into the plasma of patients undergoing CPB (see [6,44]). This stimulation can be prevented by treatment with a-MSH prior to CPB, but not by treatment during or after CPB. The stress that the body sustains with CPB is apparently more than it was “designed” to cope with and regulate. As a result, the body’s response may be regarded as inappropriate in magnitude as well as focus of the antigenic action, since the magnitude of the response appears to be detrimental. The induction of immunocyte downregulation prior to surgery may thus prevent or limit the over-production of cytokines to the point where they become cytotoxic [15,21]. aMSH is known to inhibit or limit the production of cytokines [45]. In the context of the present study, a-MSH may provide a pharmacological

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treatment whereby immunocytes made hyperactive, and thus dysfunctional, by CPB may be downregulated upon exposure to this peptide so as to limit immune responsiveness.

lowing cardiopulmonary

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