Plasma levels of immunosuppressive mediators during cardiopulmonary bypass

The aim of this study was to evaluate plasma levels of two mediators with immunosuppressive properties, complement fraction C3a (C3a) and transforming growth factor-β1 (TGF-β1), during extracorporeal circulation. The proliferation index after phytohaemagglutinin (PHA) stimulation of isolated peripheral blood mononuclear cells was also investigated. Sixteen patients undergoing hypothermic (n = 8, group 1) and normothermic (n = 8, group 2) cardiopulmormry bypass (CPB) were enrolled in this study. As a control, we evaluated four patients undergoing thoracovascular operations without CPB. Blood samples were collected before CPB but after anaesthesia, every 30 min during CPB, at the end of CPB and 10 min after protamine administration. Both C3a and TGF-β1 increased significantly during CPB and after protamine administration in the hypothermic as well as the normothermic group. In the latter case the increase of C3a and TGF-β1, although more prominent, was not significantl higher than in the former group. Conversely, the proliferation, index of peripheral mononuclear cells had already decreased 30 min after CPB was started and remained depressed throughout the CPB time. These results suggest a possible role of C3a and TGF-β1 in the immunological changes occurring during extracorporeal circulation.

extracorporeal circulation. The proliferation index after phytohaemagglutinin (PHA) stimulation of isolated peripheral blood mononuclear cells was also investigated. Sixteen patients undergoing hypothermic (n 8, group 1) and normothermic (n=8, group 2) cardiopulmormry bypass (CPB) were enrolled in this study. As a control, we evaluated four patients undergoing thoracovascular operations without CPB. Blood samples were collected before CPB but after anaesthesia, every 30 rain during CPB, at the end of CPB and 10 rain after protamine administration. Both C3a and TGF-I increased significantly during CPB and after protamine administration in the hypothermic as well as the normothermic group. In the latter case the increase of C3a and TGF-I, although more prominent, was not significantl higher than in the former group. Conversely, the proliferation, index of peripheral mononuclear cells had already decreased 30 rain after CPB was started and remained depressed throughout the CPB time. These results suggest a Introduction Current knowledge on cardiopulmonary bypass (CPB) suggests that complement activation and release of inflammatory mediators from activated platelets and polymorphonucleates into the bloodstream, is one of the most important pathophysiological changes occurring during extracorporeal circulation, and probably is responsible for a number of postoperative derangements such as the increase of capillary permeability, systemic vasodilatation and multiple organ dysfunction. > Recently, it has been reported that the mediators released during CPB may also play a determinant role in the biochemical and clinical events, leading to the postoperative immunosuppression in cardiac surgery patients throughout a functional and quantitative decrease of Tand Blymphocytes. 4 Experimental investigations showed that T-helper number, mitogen responsiveness and interleukin-2 production significantly decreased on the first postoperative day and remained depressed even on day 7. 5 Although (C) 1996 Rapid Science Publishers high levels of cortisol may be a cause of the impairment of the immune system with a redistribution of circulating lymphocytes to lymphoid organs and bone marrow, 6 other inhibitory factors may be released during the CPB technique.
As suggested by our previous study, 7 using isolated T-lymphocytes, an inhibition of proliferation and cytokine production appears 30 min after the onset of CPB. To evaluate the causes of this early immunological defect during CPB, we have focused our attention on the plasma levels of two mediators with immunosuppressive effects, complement fraction C3a (C3a), and transforming growth factor-[31 (TGF-31).
It is known that C3a, a soluble fragment derived from complement, suppresses antigeninduced T-cell proliferation, antibody production, T-cell and natural killer (NK) cell mediated cytotoxicity, as reported by studies in vitro and in vivo. 8 TGF-I, a cytokine mainly released from the alpha granules of the activated platelets, is a multifunctional mediator that affects cell growth and differentiation, immunoresponses, angio-genesis and tissue repair. 9 The aim of this study was to determine whether CPB was associated with a change of C3a and TGF-[31 plasma concentration and possibly with the decrease of in vitro T-cell proliferation. In recent years, a number of investigators have suggested the superiority of normothermic heart surgery compared to hypothermic heart surgery to reduce the adverse consequences of intraoperative myocardial ischaemia and cardiopulmonary bypass. Both approaches have shown advantages and disadvantages, and data are not conclusive. To evaluate the possible differences in the activation of the inflammatory system, we have performed our study in normothermic (35-37C) and hypothermic (30C) CPB.

Materials and Methods
Sixteen patients undergoing extracorporeal circulation for coronary artery bypass disease were enrolled in this study. The patients were randomized in two groups: group 1 (eight patients) received hypothermic CPB and group 2 (eight patients) received normothermic CPB. There was no significant difference between the two groups with respect to the age of the patients and CPB times. The patients' characteristics are reported in Table 1. As a control, blood samples were drawn at the indicated times after anaesthesia in four patients undergoing thoracovascular operation without CPB. The influence of anaesthesia was eliminated by taking the baseline value after its induction, just before CPB or thoracovascular operation. It must be mentioned that, for ethical reasons, we could not check the influence of protamine in these controls.
Anaesthesia was uniform in all cases and considered of a standard combination of fentanyl citrate, diazepam, pancuronium bromide and isoflurane CPB equipment consisted of a nonpulsatile roller pump, a membrane oxygenator and an arterial filter. Myocardial protection was performed in group 1 with anterograde cold blood cardioplegic solution as described by Buckberg, 2 and in group 2 with continuous intermittent normothermic blood cardioplegia as reported by Lichtenstein. 3 In patients undergoing cold CPB, the temperature of the circulating blood was cooled to 30C, whereas those in the warm group were kept between 35 and 37C. Pump flows were maintained at 2.51/min/ m 2 in normothermic CPB, and about 2.21/min/ m 2 in hypothermic CPB. Administration of heparin before cannulation and subsequent neutralization after bypass with protamine sulfate were performed in a standard fashion.
Whole-blood samples (5 ml) for C3a and TGFfl determination were drawn before CPB (T1) but after anaesthesia at 30 (T2), 60 (T3) and 90 (T4) min during CPB, immediately before the end of CPB (T5), and 10 min after protamine administration (T6). After platelets count, blood-EDTA was immediately centrifuged and the plasma was stored at -80C until assayed. C3a was measured in the plasma samples by radioimmunoassay 4  A three-cycle automatic washing was roufinely performed. Negative plasma samples, in the absence or presence of haemoglobin, were spiked with the cytokine standards to assess the reliability and precision of the various assays.
Yields ranged between 86 and 107%. For in vitro T-cell isolation, samples of venous blood (10ml) were obtained from each patient of both groups at the following times: before the institution of CPB, at 30 and 60 min during CPB and at the end of CPB.
Briefly, 2 x 105 cells/well were incubated in 2001 of culture medium in the presence or absence of PHA for 40 h. After removing 100 1 of medium, 10 gl of a solution of MTT (5 mg/ml) was added to each well and incubated at 37C. normothermia; p < 0.01), and it remained high during the CPB period (Fig. 1). In the controls without CPB the plasma level of C3a increased towards the end of the operation but it tended to normalize quickly ( There was a slight increase during CPB, followed by a decreased level after protamine administration (Fig. 3). Without CPB (control group) platelet levels did not vary ( Table 2).
To evaluate the effect of CPB on cell proliferation, PBMC were isolated from blood samples during CPB and a similar number of cells were cultured in the presence or absence of PHA (5 Ig/ml). At variance with the controls, the proliferation index (PI) was greatly affected during CPB, as shown in Fig. 4. PI significantly decreased as soon as 30 min after CPB commenced (PI at 30 min, 1.26 4-0.08 vs. 1.59 _+ 0.09 of baseline value, group 1; PI at 30 rain, 1.36 4-0.04 vs. 1.74 4-0.06 of baseline value, group 2; p< 0.01); at the end of CPB the PI decreased further (1.23 4-0.05 group 1, p < 0.05; 1.58 4-0.06 group 2, p < 0.05). There was also a slight, but not significant decrease of  the PI during the thoracovascular operation (Table 2).
Discussion CPB causes a number of inflammatory and immunologic responses, documented in a series of studies, aby the increased plasma levels of several compounds which are not readily correlated with the physiopathological modifications. Clinical observation on the increased susceptibility to infections in postoperative cardiac patients has suggested a CPB related Tand Bcell immunodeficiency. 7 Profound lymphopenia with drastic reduction in T-helper-lymphocytes was reported from 24 h to 3-4 days after operation, 17 and other investigators found changes in the CD4/CD8 ratio as early as 2 h after CPB. Our recent study, 7 performed during CPB, showed that lymphocyte function was already suppressed 30 min after CPB had commenced, and remained throughout the CPB time. This interesting experimental finding suggests that the cause of the immunosuppression could be due to very early events mainly occurring during extracorporeal circulation.
It is well known that the complement system is rapidly activated through the contact of blood with foreign materials of the o_xygenator, and 8 18,19 recently it has been reported' that complement by-products play an important modulatory .role in the inductive phase of the immune response, throughout a cooperation between immobilized C3 split products (that stimulate Tand B-cell activation) and the soluble C3a product, which inhibit lymphocyte proliferation. Moreover, other studies have shown that soluble complement fragments generated during decomplementation may be immunosuppressive, s Thus, the high levels of C3a during CPB determined in the present study ( Fig. 1) agree with the possibility of an immunosuppressive effect.
Perhaps the best known effect of TGF-131 is on the cells of the immune system: TGF-[I inhibits Tand B-cell proliferation without affecting IL-2 or IL-2 receptor expression, and also inhibits NK 20 21 cell activity.
Data reported in our study indicate that during extracorporeal circulation, the decrease of platelets and their degranulation is concomitant with the release of TGF-]]I from the alpha granules, with its consequent increase in the bloodstream. Because TGF-I mediates the immunodepressive effects at levels in the fentomolar ranges, it appears reasonable to postulate that binding of TGF-fll to circulating immune cells may lead later to their hyporesponsiveness to PHA, as shown by the. decrease of the PI in Fig. 4. Furthermore, it is well known that prot-amine administration causes an activation of the classic pathway of the complement system. The concomitant increase of circulating levels of C3a and TGF-[31 and the fall of platelet count, 10 min after protamine administration, are presumably linked to complement changes and platelet degranulation.
As far as the comparison between normothermic and hypothermic CPB is concerned, it has been reported previously that hypothermia causes an inhibition of complement activation and platelet degranulation. Experimental studies > have further shown that hypothermia decreases the capacity of serum to generate C3a and C4a in response to a known complement activator (zymosan), with a reversible defect in platelet function. Our investigation is in agreement with this results, because we have found a consistent reduction, although not significant, in plasma levels of C3a and TGF-J31 during hypothermic CPB in comparison to the normothermic technique. This thermic effect is particularly evident in platelet activation and degranulation which slightly affected the C3a production. Further studies are needed to clarify the optimal temperature for the preservation of the circulation homeostasis.
In conclusion, our study suggests that the increased levels of plasma C3a and TGF-J31 seem temporarily related to the inhibition of T-cell proliferation observed during CPB. Because the immunosuppressive effects of these molecules are reported for in vitro and in vivo studies, we can speculate that these mediators as well as others not evaluated here, are responsible for the immunodepression observed in cardiac patients undergoing CPB.
A final comment concerns the pleiotropic activity of TGF-122 so that it can exert either protective or damaging effects during CPB. As an example, TGF-[31 may be associated with the thermotolerance. Thermotolerance can generally be defined as the ability of an organism to withstand an otherwise lethal stress due to pretreatment with a nonlethal stress. The stress could result from a number of insults such as hyperthermia and oxidative injury which can induce expression of heat stress proteins (HSP) as an acute response. Furthermore, the well recognized role of TGF-[31 as a mediator in tissue repair suggests that its increased generation after the perturbation of cellular homeostasis could be beneficial in promoting recovery of cells and healing. 22 Thus we are now planning to evaluate an immunoadjuvating treatment carried out before and after CPB to prevent or reduce the immunosuppression that may favour septic complications after surgery.