Immunological Flemming

Aspects of Cardiopulmonary Knudsen,

MD, PhD and Lars Willy Andersen,

E

XTRACORPOREAL circulation (ECC) using the heart-lung machine has been known to contribute to the morbidity and mortality of cardiac surgery since the first operations were performed.‘V2 Cardiopulmonary bypass (CPB) can never be truly physiological, primarily because of the artificial environment of the extracorporeal circuit. Interaction among the patient’s blood, the artificial surfaces, and the pump results in generation of various mediators in plasma and direct cellular injury, leading to a systemic inflammatory response.3 Although the immune system normally serves to protect the host from offenders, overreacting ongoing immunological responses may be detrimental and result in the postperfusion syndrome. In the most severe cases, pulmonary and renal dysfunction, a bleeding tendency, neurological disorders, and fevers of noninfectious origin may develop. Closed cardiac surgery without the use of CPB and other major procedures have fewer sequelae.4-7 The present review updates the most important theories concerning the systemic inflammatory response initiated by CPB in patients undergoing cardiac surgery, and discusses possible therapeutic interventions. COMPLEMENT

ACTIVATION

The Biology of the Complement System It is beyond the framework of the present review to give a detailed description of all the various aspects of complement biology. However, knowledge relevant to complement activation during ECC will be briefly reviewed. The complement system is known to consist of 20 plasma proteins and is a part of the normal host defense system participating in the elimination of various infectious agents. Although usually beneficial, activation may play an adverse role in a number of situations, including endotoxin shock, adult respiratory distress syndrome (ARDS), and various modalities of ECC.s Complement is traditionally activated through either the classical or the alternative pathway.g Known activators of the classical pathway include the lipid A moiety of endotoxin, certain carbohydrate groups, some gram-negative bacteria, enveloped virus particles, parasites,

Bypass

MD

mitochondrial membranes, and polynucleotides, which may be released from damaged cells in such situations as trauma and sepsis.* Activators of the alternative pathway include bacteria, yeast, viruses, virus-infected cells, tumor cells, certain parasites,* and artificial surfaces encountered during ECC.” A simplified schematic presentation of the various steps operating during complement activation is given in Fig 1. Activation of the complement system generates a variety of biologically active split products*-” that either operate through a direct effect or because of their stimulating effect on neutrophiles and mononuclear phagocytes (Table 1). Several studies have indicated that anaphylatoxin C5a (C5a) is the main offender generated during massive complement activation. Its amino acid sequence is now known, and the compound consists of 14 amino acid residues cross-linked by three intrachain disulfide bonds. In addition, it contains a complex oligosaccharide (molecular weight approximately 3,000) attached to the side chain of asparagine at the 64 position, and thus is a glycoprotein.i2 Through release of histamine from mast cells and basophil leukocytes, ‘I C5a increases vascular permeability. Binding of C5a to specific receptors on polymorphonuclear leukocytes13 stimulates chemotaxis, leukocyte aggregation, and release of toxic oxygen radicals and lysosomal enzymes.” C5a is eliminated from plasma in two ways. After binding to the specific receptor, C5a is internalized and degraded.14 A potent carboxypeptidase (ie, serum carboxypeptidase B, an anaphylatoxin inactivator) exists in plasma, and, upon generation of C5a by cleavage of native C5 by C5 convertase, C5a is largely, if not totally, converted by this enzyme to a degradation product, C5a des Arg. I5 C5a des Arg is inactive as From the Department of Anesthesia,Odense University Hospital, Odense. and the Department of Anesthesia, University Hospital, Copenhagen, Denmark. Address reprint requests to Flemming Knudsen, MD. PhD. Dept of Anesthesia, Odense University Hospital, DK5000 Odense C, Denmark. o 1990 by W.B. Saunders Company. 0888~6296/90/0402-0015$03.00/O

Journal of CardiorhoracicAnesrhesia, Vol 4, No 2 (April), 1990: pp 245-258

245

KNUDSEN AND ANDERSEN

246

Alternative

Classical piGq Ca++ 1 m

L

]pqEq Mg++

Cl -Esterase inhibitor

Surfacebound C3b lela Ba Wt++

+

C3bBb

C4a+C2a C4b2b -m

1tF.l C3bBpP Bab

_C3a

Lc4BpI

loop

Mg+’

III

C3h -

C4c+C4d+’ . 1

C4b2b3b

&+C3d

C5a C5b 1

~lc_rllcsllcs]

C5b6789 Membrane Attack Complex (MAC) an anaphylatoxin, but retains its ability to cause leukocyte chemotaxis and release of lysosomal enzymes.“~i5 Conversion of C5 by C5 convertase generates not only C5a but also C5b. The latter constitutes, together with C6, C7, C8, and C9, the terminal complement complex (TCC) designated C5b-9. This major complex exists in two forms: the membrane-bound complex (C5b-9m), which is the eytolytic mediator of complement (membrane attack complex), and the fluid-phase Table 1. Some Biplogically Significant Effects of the Various Complement-Split

BiologicalEffect

products Complement-Split Product

Mast-cell degranulation, contraction of smooth muscle. increased vascular permeability

C3a. C5a

Chemotaxis of neutrophils Neutrophil aggregation

C5a. C5a des Arg

Lysosomal enzyme release

C5a. C3b

C5a. C5a des Arg

Leukocytosis

C3e

Immune adherencelopsonization

C3b. C4b

Membrane lysis

C5b-g (membrane attack complex)

C3bBb3b

Simplified scheFig 1. matic presentation of the current concepts of the complement system. B, D. and P, factors B. D. and P. resoectively; H and I denote ‘the negative regulatory proteins C3b-inactivator and @,-H. respectively.

TCC, which is noncytolytic and bound to the S protein (SC5b-9). Complement and Hemodialysis

Transitory profound leukopenia during the initial phase of hemodialysis was noticed as early as 1968.16 The reports by Craddock et a1,‘0”7in 1977, of a linkage among complement activation, leukopenia, and pulmonary dysfunction during hemodialysis were a potent stimulus for a new area of extensive research dealing with ECC and the immune system. In a series of elegant studies of animals and humans, these investigators showed that interaction between plasma and the artificial surfaces within the dialyzer leads to complement activation. Generation of split products with significant biological effects causes increased adherences of leukocytes, pulmonary leukoembolization, transitory leukopenia, and acute deterioration of pulmonary function. Subsequent studies further elaborated these findings and stressed the role of the material used for the production of the dialyzer membrane. The authors were able to show a dialyzer membrane-

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AND CPB

247

dependent generation of anaphylatoxin CSa, leukopenia, release of granulocyte elastase, and decrease in arterial oxygen tension during hemodialysis.‘8M20Similar findings have been reported by other groups of investigators.21-24 Complement and Cardiopulmonary

Bypass

Changes in complement after CPB were first reported in 1972 by Parker et a1.25Using hemolytic assays, they reported a decrease in total complement after the procedure. Because of the methodology, they were not able to elucidate whether the depletion was a result of the activation of complement or simple denaturation of proteins. Subsequent studies confirm that complement is affected by CPB, and depletion of native C3, C4, and factor B26*27 and conversion of C328 are documented. Hammerschmidt et a129linked the transitory neutropenia observed during CPB to complement activation. They observed a decrease in neutrophil count during bypass. Neutrophils were not, however, corrected for hemodilution. Simultaneously, a depletion of total hemolytic complement and hemolytic C3 were observed, interpreted as a sign of complement activation. With the isolation of the anaphylatoxins C3a and C5a, determination of the amino acid sequence, and subsequent synthesis performed by Hugli and Chenoweth,30 specific assays for these and other complement-split products became available. The question of complement and CPB could thus be further addressed. Direct evidence of complement activation with generation of biologically active split products came in 198 1 with the report by Chenoweth et a131of an increase in plasma C3a during the procedure. C3a concentration increased steadily during ECC, and maximal levels reached more than five times preoperative values. The presence of C5a, however, was not demonstrable. This finding has since been confirmed by others32 despite documented complement activation, and is most probably caused by rapid binding, internalization, and degradation of C5a by polymorphonuclear leukocytes.33 Complement activation during CPB has been confirmed in several studies either as an increase in plasma C3a394*35or in plasma C3d.32,36*37 Complement activation during ECC does not stop with the formation of anaphylatoxins

C3a and CSa, but proceeds to involve the terminal complement complex C5b-9.37 In an elegant study, Salama et a13’recently documented that erythrocyte ghosts carrying C5b-9 appeared during ECC. Deposition of the terminal-membrane attack complex on erythrocytes may be a previously unknown mechanism causing hemolysis during these procedures. Granulocytes were also shown to carry C5b-9.38 Attachment of C5b-9 to granulocytes elicits leukotriene B, generation by stimulation of arachidonate metabolism in these cells.39 Therefore, it is likely that granulocytes are stimulated not only by C5a but also by prostaglandin metabolites during ECC in cardiac surgery.38 The kinetics of complement activation during CPB have not been totally established, but maximal generation of C3a apparently occurs at normothermia during institution of ECC and during rewarming.40 Final elucidation of the various factors that cause complement activation during CPB requires further study, but several mechanisms seem to operate. Incubation of blood with a nylon-mesh liner from a bubble oxygenator, as well as vigorous oxygenation of whole blood, promote conversion of complement.31 At the termination of bypass, complement may be further activated by the heparin-protamine complex. 41,42This activation seems to progress along the classical pathway and is associated with an acute increase in plasma C4a leve1.43 Circulating endotoxins, known activators of both the classical and alternative complement pathway,’ have recently been described by this group and may be an additional factor causing complement activation during CPB. Hypothermia, hemodilution, and heparinization, conditions customarily used during CPB, attenuate complement activation in vitro43 and may at least theoretically protect the patient from excessive complement activation during perfusion. The clinical significance of complement activation during ECC remains to be elucidated fully. Although many studies26-29.31Y32J4,35-38 incriminate complement activation as a major contributing factor to the postperfusion syndrome, postoperative morbidity, or even mortality, there are only a few well-conducted studies relating the degree of complement activation to outcome.

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Kirklin et al3 found that plasma C3a, measured 3 hours after the end of CPB, predicted the occurrence of postoperative pulmonary dysfunction, renal dysfunction, cardiac dysfunction, abnormal bleeding, and overall morbidity. These results were obtained in a prospective series of 116 consecutive patients. In a series of patients reported by Moore et al,44plasma C3a concentration was greater in 13 patients requiring mechanical ventilation for longer than 1 day than among 67 patients without respiratory complications. The cardiovascular effects of protamine injection after termination of bypass can be explained by generation of anaphylatoxins C3a and C5a. Although some complement activation occurs in all patients during CPB, a significant secondary increase may result from the activation of the classical pathway by the protamineheparin complex. The variable hemodynamic response to protamine administration by patients receiving heparin could be a result of variations in the degree of complement activation or differences in vasomotor responsiveness.43 Although one study shows that membrane oxygenators activate complement to a lesser extent than do bubble oxygenators,35 another did not report this difference.34 No studies have been done to date to assess the influence of the type of oxygenator on patient outcome and degree of complement activation.

KNUDSEN AND ANDERSEN

differential counts before and after cardiac surgery involving CPB has shown that the procedure is associated with postoperative leukocytosis with increases in band forms and lymphopenia.47~48 The changes in leukocyte and differential counts that follow cardiac surgery are not unique to patients undergoing CPB. Apparently, the elevation in band count and the decrease in lymphocyte count correlate primarily with the extent of the trauma caused by the operation.48 In hemodialysis, complement activation during ECC elicits neutropenia, leukopenia,‘8120 and pulmonary sequestration of leukocytes.49 The apparent lack of neutropenia and leukopenia during CPB, despite evidence of ongoing complement activation, is not in conflict with findings during hemodialysis. After weaning from bypass and reestablishment of the pulmonary circulation, neutrophils are trapped in the lung vessels.31950The extent of pulmonary leukocyte sequestration seems to be related to the extent of complement activation evoked by the ECC.35 Neutrophils and Toxic Oxygen Radicals

Reactive oxidant free radicals, including superoxide anion, hydroxy radical, and hydrogen peroxide, have recently received considerable attention as important mediators of tissue damage during inflammation and ischemia and subsequent reperfusion.5’-54 Toxic oxygen radicals apparently exert their major effects by lipid LEUKOCYTES AND CARDIOPULMONARY peroxidation of phospholipids in cell membranes, BYPASS although several intracellular and extracellular Leukocyte and Diferential Counts During macromolecules may serve as targets.5’S54 Free Cardiopulmonary Bypass radicals of biological interest have a life span of A decrease in leukocyte count during CPB microseconds; therefore, evidence for their existhas been observed for years,45 and has been ence is frequently indirect and based on the confirmed by several subsequent studies.29*31*34*36 appearance of peroxidation products.” GeneraThe decrease in absolute number of leukocytes tion of free radicals seems to progress along two reported in some studies29*45can probably be major pathways: stimulation of neutrophils by explained by lack of correction for hemodilution. CSa, arachidonic acid, and leukotriene D4; and Others have observed significant neutropenia ingestion of bacteria that induces a respiratory after institution of bypass36 when correction for burst, generating both superoxide radicals and hemodilution was based on changes in packed hydrogen peroxide. The simultaneous release of cell volume. This is inappropriate because equatmyeloperoxidase enhances the oxidant activity.55 ing changes in hematocrit directly with changes Reperfusion after ischemia also generates free in plasma volume is mathematically incorrect.46 radicals, but via the xanthine dehydrogenaseIf correction for hemodilution is performed, leuoxidase mechanism.s’“4 kocyte and neutrophil counts remain unchanged Royston et a15’report that cardiac operaduring CPB, whereas the lymphocyte count detions using CPB are associated with generation creases slightly. 32 Comparison of leukocyte and of free radicals. Using a thiobarbituric acid

IMMUNOLOGY

AND CPB

reaction, they documented an increase in plasma concentrations of peroxidation products during CPB. The peroxidation products appeared after removal of the aortic crossclamp, and, at the same time, paired samples from central venous and left atria1 blood showed pulmonary sequestration of neutrophils. The close temporal relationship between lung neutrophil ingress and peroxidation-product egress from the lung indicates that the peroxidation products mainly are produced by complement-stimulated neutrophils, although reperfusion of the lungs after resumption of cardiac activity also may contribute. van Oeveren et a134used a chemoluminescence luminol-dependent photometric assay for detection of reactive oxygen species from polymorphonuclear leukocytes. They found that leukocytes isolated from patients perfused with bubble oxygenators showed evidence of enhanced generation of reactive oxygen species both in the resting state and when stimulated by the chemotaxin N-formyl-methionyl-leucyl-phenylalanine, compared with cells isolated from patients perfused with membrane oxygenators. Maximal enhancement of chemoluminescence did not coincide with maximal complement activation. These results may indicate that factors other than complement split products contribute to generation of reactive oxygen species during CPB. Using a cytochrome c reduction assay, Kharazmi et a156confirmed that CPB is followed by an increase in neutrophil release of superoxide when the cells are stimulated by a synthetic chemotactic peptide. Cells isolated 24 hours after the operation exhibited enhanced generation of toxic oxygen radicals. Plasma endotoxin levels were elevated 5 minutes after the removal of the aortic crossclamp and decreased over the following 24 hours. In vitro incubation of neutrophils with lipopolysaccharides enhanced generation of reactive oxygen species by neutrophils. Taken together, these results suggest that neutrophils are filled with endotoxins during CPB resulting in enhanced generation of superoxide. The contribution of reactive oxygen species to morbidity after cardiac surgery involving CPB remains to be established, although they have been suggested to play a role in the occurrence of postoperative pulmonary problems.34’50V56 The elevated levels of hydrogen peroxide in breath

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condensate from patients with ARDS could favor a biologically significant effect.” Neutrophils and Lysosomal Enzymes

Neutrophil granulocytes contain an array of lysosomal enzymes that are primarily involved in the defense against invading micoorganisms. These include elastase, collagenase, and cathepsins B, D, and G. Elastase and cathepsin G belong to the family of neutral proteinases and are stored in the azurophil granules. Lysosomal enzymes are released extracellularly during cell death, exposure to antigen-antibody complexes, activated complement, and toxic substances such as endotoxin. Granulocyte elastase is a cationic protein consisting of three isoenzymes with a molar weight of 29,000 to 31,000 d. Elastase is probably largely responsible for tissue injury, because of its broad substrate specificity, digesting elastin, protoglycan subunits, native collagen, and its abundance in neutrophil leukocytes.58 Upon release, granulocyte elastase is immediately inactivated in plasma by complex formation with proteinase inhibitors. Approximately 92% is bound to Lu,-proteinase inhibitor (cuiantitrypsin), and 8% is inactivated by ay2-macroglobulin.59 Both complexes are eliminated by the reticuloendothelial system, and a,-proteinase inhibitor is assumed to function as both a carrier protein for the transfer of the enzyme to CQmacroglobulin and as a “suicidal” protein.58 In plasma, the in vivo half-life of elastase bound to a,-proteinase inhibitor is 1 hour, whereas elastase bound to Lu,-macroglobulin has an elimination half-life of approximately 12 minutes.60 Release of lysosomal enzymes during CPB was documented years ago. Starling et a16i reported as early as 1975 that plasma concentrations of cathepsin D were elevated 10 minutes after beginning CPB compared with preoperative values. Using a priming solution containing whole blood, they found the highest levels in the pump priming solution. Degranulation of neutrophils in the pump priming solution before institution of CPB could therefore account for some of the elevation observed in patient plasma during the subsequent bypass. A later study confirms the release of lysosomal enzymes during CPB in humans@ and animals63 by reporting an increase in the plasma concentration of P-glucuronidase and N-acetyl ,&glucosaminidase. However, both

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enzymes are widely distributed in the body, including the liver, spleen, kidney, cardiac muscle, and white blood cells.62 Evidence of a specific release of granulocyte proteinases during CPB was found in an acute increase in plasma concentrations of granulocyte elastase complexed with a,-proteinase inhibitor.32*64*65 Using a nonblood pump priming solution, it was found that CPB elicited an acute increase in plasma concentrations of elastase complexed with cr,-proteinase inhibitor.32 These changes were observed in both absolute values and values corrected for hemodilution (Fig 2). Hind et a165 further reported that peak concentrations of elastase correlated closely with the duration of bypass. The finding of an increase in plasma elastase complexed with cur-proteinase inhibitor does not prove the existence of free elastase in tissues and organs, but merely reflects a “spillover” of elastase released somewhere in the body. Recent studies have suggested, however, that the

combination of local release of elastase and reactive oxygen species generated by either activated neutrophils66 or via the xanthine oxidase pathway6’ may act in concert and cause tissue injury. a,-Proteinase inhibitor apparently loses its ability to bind and inactivate granulocyte elastase when exposed to the myeloperoxidasehydrogen peroxide-halide system released from stimulated leukocytes.66 Trapping of activated neutrophils, for instance, in the lungs, simultaneous with release of toxic oxygen radicals and elastase, could be one of the mechanisms responsible for organ failure after CPB. Neutrophils and Chemotaxis

Granulocyte chemotaxis, the directed motion of granulocytes towards an area of inflammation, is an important aspect of the host defense system. Using a modified Boyden technique with cassein as chemoattractant, Bubenik and Meakins68 observed impaired active migration during CPB and for the following 3 days. How-

E-a,Pl

CPB

I t induction

I

I

I

0,5 h

t T Sternotomy

Prior

to CPB

I lh

Fig 2. Plama levels of granulocyte elastase complexed with n,-proteinase inhibitor (ng/mL) before, during, and after CPB. (- -_) Values that are corrected for hemodilution during CPB. Data from Knudsen et al.”

IMMUNOLOGY

AND CPB

ever, random migration was unchanged. With a similar technique, but using chemoattractant derived from Eschericia coli, Mayer et a169found an acute decrease in chemotaxis immediately after CPB. Granulocyte migration in patients with perfusion times greater than 90 minutes was more functionally defective than that in patients with shorter perfusions. The assays used in these investigations did not discriminate between changes in the capability of plasma to generate chemoattractants and impairment of cellular function. The authors have investigated chemotaxis during CPB using an assay that allows a separate study of active migration towards chemotactic factors generated in serum and towards the synthetic chemotactic polypeptide N-formylmethionyl-leucyl-phenylalanine (N-fMLP). Decreases in migration towards N-fMLP reflect the occurrence of a pure cellular defect. CPB was associated with an acute reduction in granulocyte chemotaxis towards both chemotactic factors in serum and towards N-fMLP.32 The decrease in active migration towards chemotactic factors generated in serum could be a result of hemodilution during CPB. The observation of a simultaneous decrease in migration to’wards IvfMLP, however, documents that CPB elicits an acute cellular defect. Granulocyte chemotaxis was normalized 18 hours after CPB. The cause of this functional impairment is unknown, but aircell interfaces and peristaltic pumping during CPB could possibly be of significance. The contribution of the transitory decrease in granulocyte chemotaxis to the increased incidence of infections among patients with cardiac diseases undergoing surgery with CPB remains to be established. The observation that granulocyte migration is more compromised in patients who develop sepsis after CPB than in uncomplicated cases indicates that this transitory defect may have clinical significance.” Neutrophils and Phagocytosis

Extracorporeal circulation during CPB affects not only the ability of neutrophils to orient themselves and move towards an area of infection/inflammation (chemotaxis), but also their ability to ingest and kill bacteria. As early as 1964, it was recognized that the phagocytic activity against Staphylococcus albus was acutely

251

depressed shortly after cardiac surgery with CPB. This depression lasted at least 1 week after surgery and was thought to account for some of the infectious complications in these patients.‘] In vitro simulation of ECC, using plastic pump tubing, showed that leukocytes from both pumped and passively rotated blood samples showed a varying but distinct decrease in phagocytic index. These studies suggested that perfusion- and surface-induced leukocyte trauma, not sufficient to cause outright cell destruction, bring about a severe functional impairment.72 Subsequent studies examined in more detail the decrease in phagocytic function during CPB. Silva et a17*used the nitroblue tetrazolium test, which indirectly measures the oxidative metabolism in phagocytes, to evaluate changes in phagocytosis during CPB. Anesthesia and surgery had only minor effects on the oxidative metabolism of granulocytes. During the first and second hours of CPB, both resting and latexengulfing phagocytes showed decreased metabolism. Preoperative granulocyte function was restored as early as the first postoperative day. These data suggest that CPB provokes a transient abnormality in the phagocytic capacity of granulocytes. Examination of granulocytes later in the postoperative period has suggested that their capability of phagocytosis and killing of bacteria may even be enhanced at that time. Schildt et al74reported increased oxygen metabolism in isolated blood granulocytes on the second postoperative day together with an increased capacity for killing of bacteria. Similar findings are reported by Palmblad, ” who found an enhanced capacity of isolated granulocytes to kill S. aureus 2 days after cardiac surgery involving CPB. The mechanisms accounting for the transient reduction in phagocytosis by polymorphonuclear leukocytes during and immediately after CPB have not been elucidated, but reduction in the opsonic capacity of post-CPB plasma76 caused by hemodilution may contribute, as may the deleterious effects of peristaltic pumping and air-cell interfaces. The enhancement, noted later during the postoperative course, may be a result of phagocytosis of degradation products such as blood-cell fragments and denatured proteins.74 Although it has been suggested that the transient reduction in phagocytosis and capacity for killing of bacteria

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may play a role in the occurrence of postoperative infections,71-73*76this issue needs further clarification. Lymphocytes

Numerous studies have reported that depression of cell-mediated immunity occurs after major surgical procedures. Studies relevant to cardiac surgery using CPB will be reviewed here. Salo7’ sequentially followed the mitogenic response of lymphocytes stimulated with phytohemagglutinin (PHA) and concanavalin A (Con A) during cardiac surgery. Anesthesia for 1.5 to 2 hours did not affect the cellular immunity before the beginning of surgery. The responses declined after the start of surgery, and a further decrease was observed after institution of CPB. Foreign lymphocytes isolated from the prime of the extracorporeal circuit had well-preserved responses, and this may tend to blur a deleterious effect of the ECC. Cell-mediated immunity remained depressed for days after the procedure; this was thought to contribute to the occurrence of postoperative viral, fungal, and certain bacterial infections. Using the transformation response to PHA and pokeweed mitogen (PWM), Ryhaenen et a147found depressed transformation 2 days after surgery, and normalization occurred at day 5. No differences were reported between bubble and membrane oxygenators, and parenteral nutrition did not protect against the immunosuppression. Roth et a17*further elaborated the findings of impairment of cellular immunity after CPB by investigating changes in transformation responses after stimulation with PHA, Con A, and PWM. The lymphocyte response to stimulation with mitomycin-treated, pooled allogenic lymphocytes (MLC) and circulating levels of T-, B-, and Fc-receptor-bearing lymphocytes were also determined. The T-cell-dependent lymphocyte responses (PHA, Con A, and MCL) decreased significantly 24 hours after operation, and this was accompanied by a 60% decrease in circulating T-cells. The responses towards PWM and the numbers of B- and Fc-receptor-bearing lymphocytes were unchanged. This indicated that B lymphocytes were unaffected by the procedure. Normalization of T-lymphocyte function was observed 1 week after the operation. Impairment of B-lymphocyte function, how-

ever, was reported later by Eskola et a179who reported that the secretion of immunoglobulins (IgG, IgM, and IgA) by PWM-stimulated lymphocytes was depressed in the period immediately after cardiac surgery. Normal secretory capacity was restored 6 to 7 days after surgery. These findings contrast with those in the report by Ryhaenen et also who observed an increase in DNA-synthesizing and immunoglobulin-secreting cells after cardiac surgery. The distribution of the various subpopulations of lymphocytes after cardiac surgery and CPB has recently received attention. Using monoclonal antibodies, T$nnesen al*’ compared the distribution of T lymphocytes (OKT3), T-helper cells (OKT4), T-suppressor cells (OKT8), and B lymphocytes (Bl) before surgery and 1 and 6 days after. Coronary artery bypass grafting and CPB were found to reduce the number of T lymphocytes, T-helper cells, and T-suppressor cells, whereas the number of B cells was unaffected. Normalization was observed at day 6. The mechanisms accounting for the reduction in T-lymphocyte function after cardiac surgery and CPB remain to be elucidated, but they must occur in the blood cells because no factor-suppressing proliferation can be shown in plasma samples obtained at the end of operation.** Although many studies have suggested that the impairment of lymphocyte function shown by in vitro assays may contribute to the occurrence of postoperative infections, no study has documented this assumption. Kress et al” used a skin test, that included seven standardized antigens as an in vivo test for both the afferent and efferent limbs of cell-mediated immunity. Cardiac surgery and CPB caused an immediate deterioration of the skin test, lasting for days. The rate of incidence of septic complications was significantly higher among patients with preoperative anergy, and the rate of postoperative anergy was much higher than for noncardiac major surgery, These results suggest that CPB has a deleterious effect on cellular immunity that contributes to the outcome. Natural Killer Cells

Natural killer cells (NK cells) are a heterogenous subpopulation of lymphoid cells that are not strictly B or T lymphocytes.83 NK cells show spontaneous cytotoxicity against a variety of

IMMUNOLOGY

AND CPB

253

target cells such as virus-infected cells, tumor cells, and primitive normal cells.” Major surgery during general anesthesia is followed by a depression of NK cell activity lasting for a few days.84*85 Ryhaenen et als6 reported that NK cell activity was depressed 2 days after cardiac surgery and CPB compared with preoperative values. Normal activity was found at day 7 after the operation. Whether the depression was the result of a decrease in the number of NK cells or inhibition of their function could not be elucidated from their data. Tdnnesen et al” examined NK cell activity and number before, during, and after cardiac surgery and CPB. Induction of anesthesia was followed by a reduction in NK cell activity. CPB elicited an acute increase in NK cell activity, whereas a reduction, lasting for 1 to 3 days, occurred after the operation. The number of circulating NK cells in peripheral blood was reduced on the first day after the procedure. No differences in NK cell activity or number were observed comparing two different anesthetic techniques with supposedly different degrees of attenuation of the endocrine stress response. CPB would be expected to acutely reduce NK cell activity because of peristaltic pumping and the deleterious effect of cell-air interfaces. NK cell activity is acutely increased by epinephrine,” and the enhancement of NK cell activity reported by T#nnesen et al” could reflect an overwhelming effect on epinephrine released during CPB. Whether or not the transitory decrease in NK cell activity observed after cardiac surgery and CPB contributes to postoperative infections remains to be clarified. IMMUNOGLOBULINS

AND CARDIOPULMONARY BYPASS

One of the major tasks of circulating immunoglobulins is the recognition of infectious agents and subsequent action to facilitate the ingestion and killing of the offender by K cells, neutrophils, monocytes, and fixed macrophages. Changes in serum immunoglobulins were one of the earliest recognized side effects of CPB. Hairston et alg8 noticed decreases in levels of IgG, IgA, and IgM during perfusion; normal levels were restored after 5 to 7 days. Major surgery not involving CPB did not elicit such changes. Ryhaenen et aIs9 confirmed the postoperative depression in immu-

noglobulin levels and found that parenteral nutrition did not prevent these changes. This indicated that denaturation of proteins rather than hypercatabolism caused the postoperative depletion of immunoglobulins. Detailed analysis of serum IgM during CPB has shown that not only does the blood concentration decrease during bypass, but a significant amount of IgM circulates in an aggregated form.26 This may be of clinical significance because aggregated IgM is one of the known activators of the complement system.26 The reasons for the changes in serum levels of immunoglobulins during CPB have not been firmly established. The reductions in IgG, IgA, and IgM seem to closely follow the changes in hematocrit,76 indicating that hemodilution is the major cause. Analysis of post-CPB plasma showed an impaired uptake of bacteria by normal polymorphonuclear leukocytes. Normal opsonic capacity was found if IgG concentrations were adjusted to pre-CPB levels. These results show that CPB causes a quantitative, but not functional, decrease in IgG.76 Comparison of bubble and membrane oxygenators has shown equal decreases in IgG, IgA, and IgM during bypass. The serum opsonizing capacity for endotoxin and serum bactericidal activity towards Seratia marcescens were decreased in both groups, mainly because of hemodilution, although they were additionally affected by bubble oxygenation.34 This could be an expression of direct blood-air contact. Although the changes in immunoglobulins during CPB have been suggested to alter the host defense against infections, this theory needs further clarification. CIRCULATING ENDOTOXINS AND CARDIOPULMONARY

BYPASS

Endotoxins (lipopolysaccharides, LPS) are derived from the cell walls of gram-negative bacteria and are widely distributed in the environment. They are stable and difficult to eliminate; this constitutes a major problem for the manufacture of pyrogen-free utensils and fluids. LPS are known activators of various biological cascades such as complement, coagulation, reactive oxygen species, endorphins, and the monocytemacrophage system leading to generation of cachetin, interleukin- 1, and other lymphokines.gO Using the limulus amebocyte lysate test

254

combined with a new highly sensitive rocket immunoelectrophoresis, Andersen et algl reported, in 1987, that CPB was associated with the occurrence of circulating LPS. All patients tested negative for presence of LPS and had no signs of infection before surgery. During the cardiac operation, a substantial amount of LPS was found in samples from the extracorporeal circuit, the pulmonary artery, and the cardiac suction lines. The concentration of LPS decreased progressively after termination of bypass until it reached zero on the seventh postoperative day. A positive result for the limulus amebocyte test was documented in several of the fluids administered during the procedure, including the cardioplegic fluids, the priming fluids for the extracorporeal circuit, banked blood, and ice used for cooling of the heart. Based on these findings, it seemed likely that the occurrence of circulating LPS during cardiac surgery was caused by the CPB procedure. Simultaneously, Rocke et al’* confirmed that CPB is followed by an increase in LPS concentration in systemic venous blood. They found that plasma LPS levels increased progressively during bypass. Upon release of the aortic crossclamp, an additional increase occurred after 5 to 15 minutes. The peak LPS concentration correlated with both the duration of bypass and the time of aortic cross-clamping. These data were interpreted to suggest that the increase in LPS during and after bypass reflects “flush-out” of stagnant blood loaded with LPS from the splanchnic system after reestablishment of normal circulation.‘* Absorption of LPS from the intestines during CPB could contribute to its increased concentration, as supported by a recent study by Andersen et aLs3 who reported an increase in peripheral venous and intestinal arcade vein plasma LPS after aortic cross-clamping in patients undergoing aortofemoral bypass surgery. Therefore, splanchnic vasoconstriction during CPB and subsequent reperfusion may be expected to result in systemic absorption of LPS from the gut. Administration of methylprednisolone, 30 mg/kg, at the time of anesthetic induction resulted in higher plasma LPS levels during bypass than in untreated controls.g4 This indicates that

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methylprednisolone inhibits the clearance of LPS, possibly by affecting the Fc-receptor function of monocytes and macrophages.” The clinical significance of the transitory endotoxemia elicited by CPB remains to be clarified, and neither in the series by Andersen et al” nor in the series reported by Rocke et al’* were overt side effects observed. CLINICAL IMPLICATIONS AND THERAPEUTIC POSSIBILITIES

From the present comprehensive review, it is evident that CPB is associated with an array of changes in the immune system affecting not only cellular but also humoral factors. The first subset of changes includes those that are primarily a part of the normal host defense against infections. CPB elicits either an immediate or shortly delayed immunodepression through decreases in serum immunoglobulin concentrations and impairment of serum opsonizing capacity. NK cell activity, granulocyte chemotaxis, and phagocytosis, as well as killing capacity of bacteria, are all depressed either during or immediately after CPB. These deleterious effects on host defense may explain the increased incidence of infections among patients undergoing surgery for cardiac diseases using CPB, compared with those undergoing cardiac surgery not involving ECC.g6,g7 The second subset of changes elicited by CPB has been denoted the “whole-body” inflammatory response.40 This includes complement activation with generation of anaphylatoxins C3a and C5a, stimulation of polymorphonuclear leukocytes, resulting in increased adherence and leukoaggregation, and release of reactive oxygen species and lysosomal enzymes, of which granulocyte elastase is thought to play a major role. Clinical studies have shown that this inflammatory response, as measured by the degree of complement activation, is related to the occurrence of postoperative cardiac, renal, and pulmonary dysfunction, as well as to the overall rate of morbidity.3*44 Both light- and electron-microscopic studies of lung biopsy samples from patients who have undergone CPB have shown pathological changes consistent with an acute inflammatory response. The pulmonary capillaries are plugged with leukocytes, and the endothelial cells are the site of swelling due to intracellu-

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lar edema. The mitochondria and the endoplasmic reticulum show ultrastructural changes, and there is pronounced interstitial edema.g8*‘00 These changes may result in the need for prolonged ventilatory support,44 widening of the arterial-toend-tidal carbon dioxide tension difference,“’ or frank noncardiogenic pulmonary edema after cardiac surgery performed with CPB.“’ A more detailed knowledge of the biocompatibility of materials used for extracorporeal technology may be one way of mitigating the deleterious effects elicited by CPB. Research with various types of dialyzer membranes has produced materials such as polycarbonate and cellulose-acetate, which have improved biocompatibility, resulting in attenuated complement activation. 18-20Experience with dialyzer re-use has shown improved biocompatibility because of binding of C3b to the dialyzer membrane.lo3 Incorporation of C3b into materials used for production of cardiotomy reservoirs and oxygenators could possibly enhance biocompatibility. Unselective passivation of foreign surfaces by precoating with albumin seems attractive, but studies evaluating this approach have not been published. Pharmacologic manipulation of the immune response elicited by CPB has received some attention. Complement activation as assessed by changes in plasma C3a35 or C3c and C3dg4 may be inhibited by administration of methylprednisolone, 30 mg/kg, before the institu-

tion of bypass. Glucocorticosteroids may also inhibit the release of granulocyte elastase from isolated leukocytes stimulated by endotoxin.104 This may be of importance if the recently reported increase in circulating endotoxin during CPB has any clinical significance. It is doubtful that a single dose of corticosteroids given before institution of bypass will have any adverse effect on the rate of postoperative infections, and it has been reported that neither granulocyte chemotaxis nor the skin test is adversely affected by perioperative dexamethasone prophylaxis.” Although it seems that prophylactic treatment with corticosteroids may have several beneficial effects attenuating the systemic inflammatory response caused by CPB, well-planned, randomized studies on perioperative morbidity and outcome are lacking. Any improvement by treatment with protease inhibitors (aprotinin) and drugs that act either extracellularly or intracellularly to counteract the deleterious effects of reactive oxygen species (superoxide dismutase, catalase, vitamins E and C, mannitol, chlorpromazine) await documentation. Improvement of patient care will probably be achieved through better understanding of the initial mechanisms eliciting the systemic inflammatory response during CPB, and development of materials with enhanced biocompatibility used for extracorporeal technology.

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