- Email: [email protected]
Immunologic effects of implantation of left ventricular assist devices
Immunologic Effects of Implantation of Left Ventricular Assist Devices M. Erren, B. Schlu¨ter, M. Fobker, G. Plenz, H. Baba, P. Willeke, R. Kwiotek, R. Junker, G. Assmann, H.H. Scheld, and M.C. Deng
M
ECHANICAL circulatory support with left ventricular assist devices (LVADs) is increasingly being used in patients with terminal chronic heart failure (CHF) as a bridge to transplantation.1–3 Recent studies have provided evidence that implantation of an LVAD causes immune alterations, with potential relevance for clinical management, both during the period of LVAD placement and the time after transplantation.4 A noteworthy observation is a prominent B cell activation, as evidenced by heightened production of anti-HLA antibodies5 and high levels of panel-reactive antibodies (PRA)6; the latter are triggering vascular rejections after transplantation.7 However, little is known about the precise etiopathogenesis of this phenomenon. It was the objective of our study to characterize the time course of B cells and of the B cell–stimulating factor, interleukin-6 (IL-6), in the peripheral blood of patients during the bridging period with LVADs. PATIENTS AND METHODS Demographic and Clinical Characteristics In this retrospective study between January 1995 and November 1997, 55 patients awaiting heart transplantation underwent LVAD implantation at our institution. The study protocol was in compliance with the rules and regulations of the ethics review board of Mu ¨nster University Hospital; study results were not available to physicians in charge of patient care. Patients consisted of 47 men and 8 women, ranging in age from 23 to 64 years (mean ⫾ SD: 43 ⫾ 10 years). Forty-four of them underwent treatment with the Novacor, and 11 the TCI HeartMate. The underlying diseases included congestive cardiomyopathy (n ⫽ 26), ischemic cardiomyopathy (n ⫽ 24), myocarditis (n ⫽ 2), postpartum cardiomyopathy (n ⫽ 1), a postrepair status of ascending aortic aneurysms (n ⫽ 1), and status after repair of tetralogy of fallot in childhood (n ⫽ 1). Inclusion criteria for LVAD insertion as a bridge to transplantation were cardiac index ⬍2 L/min 䡠 m2, systemic vascular resistance ⬎2100 dyne/s 䡠 cm⫺5, left atrial pressure ⬎20 mm Hg, systolic arterial pressure ⬍80 mm Hg, and urine output ⬍20 mL/h, with optimal drug therapy, corrected metabolism, and intra-aortic balloon pump support. Exclusion criteria for insertion were age ⬎65 years, chronic failure of more than two organs, and chronic infection. Four patients were being supported at the time of end of follow-up (November 1997). Fourteen of 55 (25%) patients died during LVAD support. Causes of death included multiorgan failure (n ⫽ 8, 25%), infection (n ⫽ 3, 2%), intracranial bleeding (n ⫽ 2, 4%), and ischemic cerebrovascular complications (n ⫽ 1, 2%). Actual infection-free survival during LVAD support was 87% at © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
100 days, 67% at 150 days, 60% at 200 days, and 50% at 250 and 300 days. The following groups were used in our study: (1) healthy volunteers recruited from medical students at our institution (n ⫽ 20); (2) cardiopulmonary bypass (CPB) patients who were undergoing cardiac surgery for coronary or valvular disease (n ⫽ 20); and (3) heart transplant patients who did not undergo previous LVAD insertion because pharmacologic therapy was sufficient (n ⫽ 40).
Surgical and Pharmacologic Treatment At the time of implantation, none of the patients was receiving immunosuppressive therapy. The same surgical, cardioplegic, and CPB regime was used in all patients. LVAD implantation was achieved during extracorporeal support with the beating heart, as previously reported.8 LVAD support lasted from 1 to 414 days (mean ⫾ SD: 149 ⫾ 72 days). Anticoagulation in the absence of bleeding early after surgery consisted of full intravenous heparinization. This was followed by platelet-inhibiting drugs (aspirin and/or dipyridamole) and, in Novacor patients, oral anticoagulation with phenprocoumon (international normalized ratio [INR] 3.0 to 4.0]. Basic oral medical therapy after LVAD implantation consisted of digoxin, angiotensin-converting enzyme inhibitors, and amiodaron.
LVAD Devices Two different LVAD devices were used: 44 patients used the Novacor (Baxter Healthcare Corp, Oakland, Calif) and 11 used the HeartMate (Thermo Cardiosystems, Inc, Woburn, Mass). The HeartMate device has a textured inner surface allowing for the generation of a natural inner surface lining, whereas the Novacor device has a nontextured, smooth inner surface, which does not allow for the development of an inner surface.
From the Institute of Clinical Chemistry and Laboratory Medicine (M.E., B.S., M.F., P.W., R.K., R.J., G.A.), Department of Cardiothoracic Surgery (M.R., G.P., H.H.S.), Gerhard Domagk Institute of Pathology (H.B.), and Institute of Arteriosclerosis Research (M.E., B.S., F.B., R.J., G.A.), Westphalian WilhelmsUniversity of Mu¨nster, Mu¨nster, Germany; and Division of Circulatory Physiology (M.C.D.), Physicians and Surgeons of Columbia University, New York, New York, USA. Address reprint requests to Dr Michael Erren, Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Albert-Schweitzer-Strasse 33, D-48149 Mu¨nster, Germany. E-mail: [email protected]. 0041-1345/01/$–see front matter PII S0041-1345(00)02756-1 1965
Transplantation Proceedings, 33, 1965–1968 (2001)
1966
ERREN, SCHLU¨TER, ROTHENBURGER ET AL
Blood Sampling and Analysis Blood samples were taken by peripheral vein puncture before implantation of devices, and thereafter on a regular basis (Monday, Wednesday, Friday) at 6:00 to 7:00 AM. The results were assigned to the following timetable: preimplant, and 1 to 2, 3 to 4, 5 to 8, 9 to 16, 17 to 32, 33 to 64, 65 to 128 days after implantation. Laboratory measurements were performed for all parameters immediately after arrival of the samples in the laboratory (⬍3 hours after sampling). Flow cytometric determination of lymphocyte and monocyte subpopulations was performed by two-color fluorescence analysis on a Coulter-XL cytometer (Beckman-Coulter, Krefeld, Germany). Peripheral blood mononuclear cells (PBMCs) from 100-L EDTA blood samples were simultaneously stained directly with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies (Beckman-Coulter) to determine lymphocytic and monocytic surface antigens. Red blood cell lysis and leukocyte conservation was done with the Coulter Q-Prep system (Beckman-Coulter). Hematologic analysis for lymphocyte and monocyte counts was performed on a Technicon H3-autoanalyzer (Bayer, Leverkusen, Germany). Plasma IL-6 was measured on an Cobas Core II (Roche Diagnostics, Mannheim, Germany) with a commercially available enzyme-linked immunosorbent assay (Biosource, Ratingen, Germany), according to the manufacturer’s instructions. Serum immunoglobulins were measured turbidimetrically with the N antisera to human immunoglobulins on a BN II autoanalyzer (Dade-Behring, Marburg, Germany). All samples were analyzed in a blinded manner by a technician without knowledge of the patient characteristics.
Data Analysis Nonparametric tests were performed and data are reported as median ⫾ range because immune parameters were not normally distributed. If patients were analyzed more than once during a given time period the median of the results was determined and used for statistical analysis. Demographic and clinical results were analyzed by chi-square test. Significance of intragroup comparison was analyzed by Friedman test and Wilcoxon test. The Mann– Whitney U test was applied to test for significant intergroup differences. P ⬍ .05 was considered significant, and P ⬍ .1 indicated a trend. Statistical calculations were done using SPSS (Version 9, SPSS Inc, Chicago, Ill).
Fig 1. Time course of peripheral blood B cells before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
the observed changes were more pronounced. IL-6 levels before LVAD insertion were significantly higher (60 pg/ mL) than in healthy controls (⬍10 pg/mL, P ⬍ .05). Immediately after implantation, a sharp rise was observed, with peak levels at 4 to 8 hours after implantation up to 1000 pg/mL. At day 2, median levels of IL-6 were still markedly elevated (260 pg/mL). Thereafter, IL-6 concentrations decreased continuously to preimplantation values at day 32. At the end of the observation period, IL-6 levels were still elevated (20 pg/mL), compared with healthy controls or patients in the late phase after heart transplantation (both ⬍10 pg/mL, P ⬍ .05). Regarding the time course of the three immunoglobulin classes (IgG, IgM, IgA), different developments were
RESULTS
Before LVAD placement, peripheral blood B cell counts were significantly lower compared with healthy controls (170/L vs 264/L, respectively; P ⬍ .05). At the same time, lymphocyte counts were also markedly reduced (1520/L vs 2000/L, respectively, P ⬍ .05). After LVAD placement, B cell count gradually, but steadily, increased up to maximum levels of around 220/L at day 4 (Fig 1). Thereafter, B cell counts declined and reached preimplant levels (150 cells/ L) at approximately day 32 postimplantation. A further slow decrease to 140/L occurred up to the end of the observation period at day 128. Patients who underwent CPB without placement of an LVAD device showed a smaller B cell increase and returned to preoperative baseline levels as early as day 16. The time course of plasma IL-6 concentrations was remarkably similar to that of B cells (Fig 2), even though
Fig 2. Time course of interleukin-6 plasma levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
IMMUNOLOGIC EFFECTS
1967
to the type of implanted LVAD (ie, Novacor or TCI HeartMate). DISCUSSION
Fig 3. Time course of IgG serum levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
found. IgA serum levels increased immediately after implantation from 250 mg/dL (preimplant) to maximum levels of 420 mg/mL (days 16 to 32, P ⬍ .05), which is slightly above the upper limit of reference values (70 to 400 mg/dL); thereafter, up to the end of the observation period, IgA levels decreased slowly to 350 mg/dL. After a latency period of approximately 16 days, IgG showed an increase from 1150 mg/dL (preimplant) up to maximum values of 2250 mg/dL (days 33 to 64; P ⬍ .01), which is significantly higher than the upper limit of reference values (700 to 1600 mg/dL); at the end of the observation period IgG levels were only marginally reduced at 2010 mg/dL. In contrast, IgM concentrations at all timepoints were within reference limits and did not show any significant change (170 mg/dL). All immunologic effects observed did not differ according
Fig 4. Time course of IgM and IgA serum levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
Our data suggest that the elevation of B cells is attributable to a stimulation by IL-6, as this proinflammatory mediator is known to possess a strong stimulating effect on B cells (ie, activation, proliferation, differentiation)—therefore, it was formerly termed “B cell–stimulating factor.”9 Our assumption of a causal link between both parameters has been substantiated by the finding of a closely correlated time course of both parameters. B cell counts followed the development of IL-6 concentration with a consistent delay of 2 to 3 days, which suggesting that B cell proliferation was triggered and sustained by IL-6. Although our study lacked functional data regarding B cells, it is tempting to speculate that B cells were functionally activated under the influence of very high plasma levels of IL-6 during the early phase, and by a moderately, but persistently, elevated level during the late phase.10 Under these conditions, there was an increased transformation of B cells to plasma cells, leading to increased production of immunoglobulins. The total plasma levels of immunoglobulins may be regarded as a surrogate marker for the production of specific antibodies. The elevated levels of immunoglobulins in our LVAD patients are therefore in line with other studies, which showed increased levels of immunoglobulin-producing cells (ie, plasma cells) after CPB11,12 and a higher incidence of PRA in patients after LVAD implantation.13 The latter was shown to be accompanied by an increased risk for vascular rejection in case of heart transplantation.7 The real risk for early graft failure after a positive crossmatch appears to reside in the IgG fraction of donorspecific antibodies.7 Therefore, it is of interest that our LVAD patients demonstrated the most pronounced increase in immunoglobulin serum levels of this subclass, thereby reaching concentrations significantly above the upper limit of reference values. Based on our findings, the elevated PRA observed after LVAD implantation may be caused by two different mechanisms: on one hand, immunocompetent B cells are unspecifically stimulated by the inappropriate activation of the proinflammatory cascade (eg, surgical trauma,14,15 cardiopulmonary bypass,16,17 metabolic alterations,18 hormonal stress19), whereas, on the other hand, the humoral immune system is specifically activated by foreign antigens (unfiltered cellular and/or soluble blood products, immunogenic components of the LVAD).20 Therefore, in addition to current strategies (restricted use of transfusions, hereby using filtered products exclusively) it may be beneficial: (1) to prophylactically shorten the CPB time,21 perform less traumatic surgery,22 and use smaller assist devices23 (eg, DeBakey LVAD); and (2) to interfere therapeutically with the proinflammatory pathway (eg, antibodies against TNF-␣, IL-1, IL-6 or its receptors.24,25 In conclusion, we suggest that the implantation of
1968
LVADs activates the proinflammatory pathway and thereby leads to an upregulation of the B cell system. To further clarify this issue, investigations regarding the functional status of the B cell system are required (eg, expression of activation and adhesion molecules, proliferation, apoptosis, cytokine, and immunoglobulin production). ACKNOWLEDGMENTS The authors gratefully acknowledge the statistical advice of Dr A. Heinecke, Institute for Medical Informatics and Biomathematics, University of Mu ¨nster, and the excellent technical assistance of U. Ehmann, K. Meyer zu Himmern, K. Grussel, and R. Kwiotek, Institute of Clinical Chemistry and Laboratory Medicine, University of Mu ¨nster.
REFERENCES 1. Parameshwar J, Wallwork J: Int J Cardiol 62(suppl 1), 1997 2. Keon WJ, Olsen DB: Can J Cardiol 12:1017, 1996 3. Kocandrle V, Fabian J, Firt P: Cor Vasa 29:325, 1987 4. Smith JD, Danskine AJ, Laylor RM, et al: Transplant Immunol 1:60, 1993 5. Massad MG, Cook DJ, Schmitt SK, et al: Ann Thorac Surg 64:1120, 1997 6. Tsau PH, Arabia FA, Toporoff B, et al: ASAIO J 44:M634, 1998 7. Itescu S, Tung TC, Burke EM, et al: Circulation 98:786, 1998 8. Scheld HH, Hammel D, Schmid C, et al: Thorac Cardiovasc Surg 44:62, 1996 9. Van Damme J, Cayphas S, Van Snick J: Eur J Biochem 168:543, 1987
ERREN, SCHLU¨TER, ROTHENBURGER ET AL 10. Hilbert DM, Cancro MP, Scherle PA, et al: J Immunol 143:4019, 1989 11. Tajima K, Yamamoto F, Kawazoe K, et al: Ann Thorac Surg 55:625, 1993 12. Siminelakis S, Bossinakou I, Antoniou F, et al: J Cardiothorac Vasc Anesthesiol 10:893, 1996 13. John R, Lietz K, Burke E, et al: Circulation 100(suppl 19):II229, 1999 14. Hirota M, Ogawa M: Nippon Geka Gakkai Zasshi 97:721, 1996 15. Sakamoto K, Arakawa H, Mita S, et al: Cytokine 6:181, 1994 16. Deng MC, Wiedner M, Erren M, et al: Eur J Cardiothorac Surg 9:22, 1995 17. Furunaga A: Nippon Kyobu Geka Gakkai Zasshi 42:2200, 1994 18. Kotani G, Usami M, Kasahara H, et al: Kobe J Med Sci 42:187, 1996 19. Yamauchi H, Kobayashi E, Yoshida T, et al: Cytokine 10:549, 1998 20. Dowling RD, Jones JW, Carroll MS, et al: Transplant Proc 30:1110, 1998 21. Roth Isigkeit A, Borstel TV, Seyfarth M, et al: Clin Exp Immunol 118:242, 1999 22. Bitterman H, Kinarty A, Lazarovich H, et al: J Clin Immunol 11:184, 1991 23. Grzelak I, Olszewski WL, Zaleska M, et al: J Clin Immunol 16:159, 1996 24. Volk HD, Reinke P, Krausch D, et al: Intens Care Med 22(suppl 4), 1996 25. Read RC: J Antimicrob Chemother 41(suppl A), 1998
M
ECHANICAL circulatory support with left ventricular assist devices (LVADs) is increasingly being used in patients with terminal chronic heart failure (CHF) as a bridge to transplantation.1–3 Recent studies have provided evidence that implantation of an LVAD causes immune alterations, with potential relevance for clinical management, both during the period of LVAD placement and the time after transplantation.4 A noteworthy observation is a prominent B cell activation, as evidenced by heightened production of anti-HLA antibodies5 and high levels of panel-reactive antibodies (PRA)6; the latter are triggering vascular rejections after transplantation.7 However, little is known about the precise etiopathogenesis of this phenomenon. It was the objective of our study to characterize the time course of B cells and of the B cell–stimulating factor, interleukin-6 (IL-6), in the peripheral blood of patients during the bridging period with LVADs. PATIENTS AND METHODS Demographic and Clinical Characteristics In this retrospective study between January 1995 and November 1997, 55 patients awaiting heart transplantation underwent LVAD implantation at our institution. The study protocol was in compliance with the rules and regulations of the ethics review board of Mu ¨nster University Hospital; study results were not available to physicians in charge of patient care. Patients consisted of 47 men and 8 women, ranging in age from 23 to 64 years (mean ⫾ SD: 43 ⫾ 10 years). Forty-four of them underwent treatment with the Novacor, and 11 the TCI HeartMate. The underlying diseases included congestive cardiomyopathy (n ⫽ 26), ischemic cardiomyopathy (n ⫽ 24), myocarditis (n ⫽ 2), postpartum cardiomyopathy (n ⫽ 1), a postrepair status of ascending aortic aneurysms (n ⫽ 1), and status after repair of tetralogy of fallot in childhood (n ⫽ 1). Inclusion criteria for LVAD insertion as a bridge to transplantation were cardiac index ⬍2 L/min 䡠 m2, systemic vascular resistance ⬎2100 dyne/s 䡠 cm⫺5, left atrial pressure ⬎20 mm Hg, systolic arterial pressure ⬍80 mm Hg, and urine output ⬍20 mL/h, with optimal drug therapy, corrected metabolism, and intra-aortic balloon pump support. Exclusion criteria for insertion were age ⬎65 years, chronic failure of more than two organs, and chronic infection. Four patients were being supported at the time of end of follow-up (November 1997). Fourteen of 55 (25%) patients died during LVAD support. Causes of death included multiorgan failure (n ⫽ 8, 25%), infection (n ⫽ 3, 2%), intracranial bleeding (n ⫽ 2, 4%), and ischemic cerebrovascular complications (n ⫽ 1, 2%). Actual infection-free survival during LVAD support was 87% at © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
100 days, 67% at 150 days, 60% at 200 days, and 50% at 250 and 300 days. The following groups were used in our study: (1) healthy volunteers recruited from medical students at our institution (n ⫽ 20); (2) cardiopulmonary bypass (CPB) patients who were undergoing cardiac surgery for coronary or valvular disease (n ⫽ 20); and (3) heart transplant patients who did not undergo previous LVAD insertion because pharmacologic therapy was sufficient (n ⫽ 40).
Surgical and Pharmacologic Treatment At the time of implantation, none of the patients was receiving immunosuppressive therapy. The same surgical, cardioplegic, and CPB regime was used in all patients. LVAD implantation was achieved during extracorporeal support with the beating heart, as previously reported.8 LVAD support lasted from 1 to 414 days (mean ⫾ SD: 149 ⫾ 72 days). Anticoagulation in the absence of bleeding early after surgery consisted of full intravenous heparinization. This was followed by platelet-inhibiting drugs (aspirin and/or dipyridamole) and, in Novacor patients, oral anticoagulation with phenprocoumon (international normalized ratio [INR] 3.0 to 4.0]. Basic oral medical therapy after LVAD implantation consisted of digoxin, angiotensin-converting enzyme inhibitors, and amiodaron.
LVAD Devices Two different LVAD devices were used: 44 patients used the Novacor (Baxter Healthcare Corp, Oakland, Calif) and 11 used the HeartMate (Thermo Cardiosystems, Inc, Woburn, Mass). The HeartMate device has a textured inner surface allowing for the generation of a natural inner surface lining, whereas the Novacor device has a nontextured, smooth inner surface, which does not allow for the development of an inner surface.
From the Institute of Clinical Chemistry and Laboratory Medicine (M.E., B.S., M.F., P.W., R.K., R.J., G.A.), Department of Cardiothoracic Surgery (M.R., G.P., H.H.S.), Gerhard Domagk Institute of Pathology (H.B.), and Institute of Arteriosclerosis Research (M.E., B.S., F.B., R.J., G.A.), Westphalian WilhelmsUniversity of Mu¨nster, Mu¨nster, Germany; and Division of Circulatory Physiology (M.C.D.), Physicians and Surgeons of Columbia University, New York, New York, USA. Address reprint requests to Dr Michael Erren, Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Albert-Schweitzer-Strasse 33, D-48149 Mu¨nster, Germany. E-mail: [email protected]. 0041-1345/01/$–see front matter PII S0041-1345(00)02756-1 1965
Transplantation Proceedings, 33, 1965–1968 (2001)
1966
ERREN, SCHLU¨TER, ROTHENBURGER ET AL
Blood Sampling and Analysis Blood samples were taken by peripheral vein puncture before implantation of devices, and thereafter on a regular basis (Monday, Wednesday, Friday) at 6:00 to 7:00 AM. The results were assigned to the following timetable: preimplant, and 1 to 2, 3 to 4, 5 to 8, 9 to 16, 17 to 32, 33 to 64, 65 to 128 days after implantation. Laboratory measurements were performed for all parameters immediately after arrival of the samples in the laboratory (⬍3 hours after sampling). Flow cytometric determination of lymphocyte and monocyte subpopulations was performed by two-color fluorescence analysis on a Coulter-XL cytometer (Beckman-Coulter, Krefeld, Germany). Peripheral blood mononuclear cells (PBMCs) from 100-L EDTA blood samples were simultaneously stained directly with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies (Beckman-Coulter) to determine lymphocytic and monocytic surface antigens. Red blood cell lysis and leukocyte conservation was done with the Coulter Q-Prep system (Beckman-Coulter). Hematologic analysis for lymphocyte and monocyte counts was performed on a Technicon H3-autoanalyzer (Bayer, Leverkusen, Germany). Plasma IL-6 was measured on an Cobas Core II (Roche Diagnostics, Mannheim, Germany) with a commercially available enzyme-linked immunosorbent assay (Biosource, Ratingen, Germany), according to the manufacturer’s instructions. Serum immunoglobulins were measured turbidimetrically with the N antisera to human immunoglobulins on a BN II autoanalyzer (Dade-Behring, Marburg, Germany). All samples were analyzed in a blinded manner by a technician without knowledge of the patient characteristics.
Data Analysis Nonparametric tests were performed and data are reported as median ⫾ range because immune parameters were not normally distributed. If patients were analyzed more than once during a given time period the median of the results was determined and used for statistical analysis. Demographic and clinical results were analyzed by chi-square test. Significance of intragroup comparison was analyzed by Friedman test and Wilcoxon test. The Mann– Whitney U test was applied to test for significant intergroup differences. P ⬍ .05 was considered significant, and P ⬍ .1 indicated a trend. Statistical calculations were done using SPSS (Version 9, SPSS Inc, Chicago, Ill).
Fig 1. Time course of peripheral blood B cells before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
the observed changes were more pronounced. IL-6 levels before LVAD insertion were significantly higher (60 pg/ mL) than in healthy controls (⬍10 pg/mL, P ⬍ .05). Immediately after implantation, a sharp rise was observed, with peak levels at 4 to 8 hours after implantation up to 1000 pg/mL. At day 2, median levels of IL-6 were still markedly elevated (260 pg/mL). Thereafter, IL-6 concentrations decreased continuously to preimplantation values at day 32. At the end of the observation period, IL-6 levels were still elevated (20 pg/mL), compared with healthy controls or patients in the late phase after heart transplantation (both ⬍10 pg/mL, P ⬍ .05). Regarding the time course of the three immunoglobulin classes (IgG, IgM, IgA), different developments were
RESULTS
Before LVAD placement, peripheral blood B cell counts were significantly lower compared with healthy controls (170/L vs 264/L, respectively; P ⬍ .05). At the same time, lymphocyte counts were also markedly reduced (1520/L vs 2000/L, respectively, P ⬍ .05). After LVAD placement, B cell count gradually, but steadily, increased up to maximum levels of around 220/L at day 4 (Fig 1). Thereafter, B cell counts declined and reached preimplant levels (150 cells/ L) at approximately day 32 postimplantation. A further slow decrease to 140/L occurred up to the end of the observation period at day 128. Patients who underwent CPB without placement of an LVAD device showed a smaller B cell increase and returned to preoperative baseline levels as early as day 16. The time course of plasma IL-6 concentrations was remarkably similar to that of B cells (Fig 2), even though
Fig 2. Time course of interleukin-6 plasma levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
IMMUNOLOGIC EFFECTS
1967
to the type of implanted LVAD (ie, Novacor or TCI HeartMate). DISCUSSION
Fig 3. Time course of IgG serum levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
found. IgA serum levels increased immediately after implantation from 250 mg/dL (preimplant) to maximum levels of 420 mg/mL (days 16 to 32, P ⬍ .05), which is slightly above the upper limit of reference values (70 to 400 mg/dL); thereafter, up to the end of the observation period, IgA levels decreased slowly to 350 mg/dL. After a latency period of approximately 16 days, IgG showed an increase from 1150 mg/dL (preimplant) up to maximum values of 2250 mg/dL (days 33 to 64; P ⬍ .01), which is significantly higher than the upper limit of reference values (700 to 1600 mg/dL); at the end of the observation period IgG levels were only marginally reduced at 2010 mg/dL. In contrast, IgM concentrations at all timepoints were within reference limits and did not show any significant change (170 mg/dL). All immunologic effects observed did not differ according
Fig 4. Time course of IgM and IgA serum levels before and after implantation of the left ventricular assist devices. The data from the box plots are given as median plus the 25th/75th percentile. The bars represent the total range of values.
Our data suggest that the elevation of B cells is attributable to a stimulation by IL-6, as this proinflammatory mediator is known to possess a strong stimulating effect on B cells (ie, activation, proliferation, differentiation)—therefore, it was formerly termed “B cell–stimulating factor.”9 Our assumption of a causal link between both parameters has been substantiated by the finding of a closely correlated time course of both parameters. B cell counts followed the development of IL-6 concentration with a consistent delay of 2 to 3 days, which suggesting that B cell proliferation was triggered and sustained by IL-6. Although our study lacked functional data regarding B cells, it is tempting to speculate that B cells were functionally activated under the influence of very high plasma levels of IL-6 during the early phase, and by a moderately, but persistently, elevated level during the late phase.10 Under these conditions, there was an increased transformation of B cells to plasma cells, leading to increased production of immunoglobulins. The total plasma levels of immunoglobulins may be regarded as a surrogate marker for the production of specific antibodies. The elevated levels of immunoglobulins in our LVAD patients are therefore in line with other studies, which showed increased levels of immunoglobulin-producing cells (ie, plasma cells) after CPB11,12 and a higher incidence of PRA in patients after LVAD implantation.13 The latter was shown to be accompanied by an increased risk for vascular rejection in case of heart transplantation.7 The real risk for early graft failure after a positive crossmatch appears to reside in the IgG fraction of donorspecific antibodies.7 Therefore, it is of interest that our LVAD patients demonstrated the most pronounced increase in immunoglobulin serum levels of this subclass, thereby reaching concentrations significantly above the upper limit of reference values. Based on our findings, the elevated PRA observed after LVAD implantation may be caused by two different mechanisms: on one hand, immunocompetent B cells are unspecifically stimulated by the inappropriate activation of the proinflammatory cascade (eg, surgical trauma,14,15 cardiopulmonary bypass,16,17 metabolic alterations,18 hormonal stress19), whereas, on the other hand, the humoral immune system is specifically activated by foreign antigens (unfiltered cellular and/or soluble blood products, immunogenic components of the LVAD).20 Therefore, in addition to current strategies (restricted use of transfusions, hereby using filtered products exclusively) it may be beneficial: (1) to prophylactically shorten the CPB time,21 perform less traumatic surgery,22 and use smaller assist devices23 (eg, DeBakey LVAD); and (2) to interfere therapeutically with the proinflammatory pathway (eg, antibodies against TNF-␣, IL-1, IL-6 or its receptors.24,25 In conclusion, we suggest that the implantation of
1968
LVADs activates the proinflammatory pathway and thereby leads to an upregulation of the B cell system. To further clarify this issue, investigations regarding the functional status of the B cell system are required (eg, expression of activation and adhesion molecules, proliferation, apoptosis, cytokine, and immunoglobulin production). ACKNOWLEDGMENTS The authors gratefully acknowledge the statistical advice of Dr A. Heinecke, Institute for Medical Informatics and Biomathematics, University of Mu ¨nster, and the excellent technical assistance of U. Ehmann, K. Meyer zu Himmern, K. Grussel, and R. Kwiotek, Institute of Clinical Chemistry and Laboratory Medicine, University of Mu ¨nster.
REFERENCES 1. Parameshwar J, Wallwork J: Int J Cardiol 62(suppl 1), 1997 2. Keon WJ, Olsen DB: Can J Cardiol 12:1017, 1996 3. Kocandrle V, Fabian J, Firt P: Cor Vasa 29:325, 1987 4. Smith JD, Danskine AJ, Laylor RM, et al: Transplant Immunol 1:60, 1993 5. Massad MG, Cook DJ, Schmitt SK, et al: Ann Thorac Surg 64:1120, 1997 6. Tsau PH, Arabia FA, Toporoff B, et al: ASAIO J 44:M634, 1998 7. Itescu S, Tung TC, Burke EM, et al: Circulation 98:786, 1998 8. Scheld HH, Hammel D, Schmid C, et al: Thorac Cardiovasc Surg 44:62, 1996 9. Van Damme J, Cayphas S, Van Snick J: Eur J Biochem 168:543, 1987
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