Characterization of in vivo activated lymphocytes found in the peripheral blood of patients undergoing cardiac operation

J

THoRAc CARDIOVASC SURG

1987;93:109-14

Characterization of in vivo activated lymphocytes found in the peripheral blood of patients undergoing cardiac operation We studied in vivo lymphocyte activation in patients who underwent cardiac operation. A large number of deoxyribonucleic acid-synthesizing cells characterized by 3JI-thymidine uptake and morphologicaDy atypical lymphocyteswere found after operation in the peripheral blood samples of patients, the peak level occuring on the sixth or seventh postoperative day. Most of the deoxyribonucleic acid-synthesizing cells were in the B ceD fraction. The number of antibody secreting cells in the patients' peripheral blood samples was found increased from the normal level after operation. Immunoglobulin A-secreting ceUs were the most elevated fraction. The percentage of helper (OKT4) and suppressor (OKT8) ceUs in the postoperative samples was not significantly different from that in the preoperative samples. Only a few were la antigen positive cells, Co-culture of patients' B ceUs with their own T cells and with the T ceUs taken from two healthy controls revealed no signs of any helper or suppressor effect on these antibody-secreting cells. The results show that after cardiac operation the peripheral blood of patients contains an increased number of immunoglobulinsecreting ceUs. The regulatory T-lymphocytes seem not to have any effect in vitro on these in vivo activated cells.

Pauli Ryhanen, M.D., Jorma Ilonen, M.D.,* Heljii-Marja Surcel, M.Sci., Tapio Nurmi, Ph.D., Kari Poikonen, M.Sci., Kaisu Jiirvenpiiii, Lab.Tech., and AimoSalmi, M.D.,* Oulu, Finland

DeoXyribonUcleic acid (DNA)-synthesizing lymphocytes with atypical features have been found in peripheral blood of patients with certain infectious and autoimmune diseases,":' after blood transfusions,' and also after operation with and without signs of infection."? After cardiac operation the number of these cells in the peripheral blood of patients is significantly increased as early as the second postoperative day, with the peak level seen about a week after operation," The nature and function of these cells activated by operation, however, is

From the Departments of Anesthesiology, Medical Microbiology, and Radiotherapy, University of Oulu, Oulu, the National Public Health Institute, Oulu, and the Department of Virology, University of Turku, Turku, Finland. Dr. Poikonen was supported by a grant from Sigrid Juselius Foundation. Received for publication July 3, 1985. Accepted for publication Feb. 18, 1986. Address for reprints: Pauli Ryhanen, M.D., Department of Anesthesiology, University of Oulu, SF-90220 Oulu, Finland. 'Present address: Department of Medical Microbiology, University of Alberta, Edmonton, Alberta, Canada.

unknown. We designed a series of experiments to further characterize these activated cells. Methods Patients and control subjects. The series comprised a total of 45 patients who underwent elective cardiac operation with cardiopulmonary bypass, 28 patients for coronary bypass grafting and 17 for valve replacement. Thirty seven of these were men and eight were women; their ages ranged from 20 to 67 years (mean 51 years). The mode of anesthesia and the techniques of operation and perfusion were essentially similar to those described previously." In addition, two patients with infectious mononucleosis (positive reaction to Paul-Bunnell test) and two healthy adult volunteers were included in the series and served as control subjects. Laboratory methods. Samples of heparinized peripheral blood for different experiments were taken before and after operation as indicated. Peripheral blood mononuclear cells were separated by Lymphoprep gradient centrifugation (Nyegaard & Co., A/S, Oslo, Norway), washed three times with Hanks' balanced salt solution, and suspended in RPMI 1640 culture medium contain109

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Ryhanen et al.

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23456789 days

Fig. 1. Spontaneous lymphocyte proliferation (3H-thymidine uptake) before and after cardiac operation in seven patients. Significance of difference between preoperative and day 6 values, p < 0,01 (Wilcoxon test). Significance of difference between preoperative and day 7 values, p < 0.025 (Wilcoxon test). cpm, Amount of incorporated radioactivity into the synthesized DNA in in vitro cultured cells.

ing 10% fetal calf serum (FCS). Lymphocytes were then depleted from adherent cells by incubation in plastic Petri dishes at 37° C for 30 minutes in 5% carbon dioxide (C02) atmosphere. They were further enriched for T and B cells using 2-arninoethylisothiouronium bromide hydrobromide (AET)-treated sheep red blood cells. B (non-T) cell fraction was further characterized morphologically using May-Griinwald-Giemsa-stained smears prepared using routine laboratory methods. Spontaneous proliferative activity of lymphocytes. Spontaneous proliferation of lymphocytes was measured by incubating triplicate microtiter plate cultures (5 X 10" cells/well in RPMI 1640 supplemented with 10% AB serum) with 2 ~Ci/rnl of 3H-thymidine in a

The Journal of Thoracic and Cardiovascular Surgery

humidified 5% CO 2 atmosphere. The cells were harvested after 24 hours, and incorporated radioactivity was measured with a liquid scintillation counter. The results are expressed as the median of the counts per minute (cpm) values of the triplicate cultures. Determination of antibody secreting cells. The number of immunoglobulin (IgM, IgG, and IgA) secreting cells was determined with an enzyme-linked irnmunosorbent assay-plaque method'? with some modifications. In brief, 96 well plates (Immunoplate I, Nunclon Delta, Roskilde, Denmark) were coated with antihuman IgM, IgG, and IgA (Dakopatts, Glostrup, Denmark). Antisera were diluted 1: 100 with carbonatebicarbonate buffer, pH 9.6, and 50 ~I was added to each well. After I hour of incubation at 3]0 C, the plates were washed three times with washing buffer (phosphate-buffered saline solution [PBS] plus 0.05% Tween 20), and once with distilled water. Thereafter, 100 ~I of appropriately diluted cell suspensions was added to duplicate wells, and the plates were incubated for 3 hours at 37° C. After incubation the plates were washed three times with washing buffer. Fifty microliters of alkaline phosphatase-eonjugated anti-human IgM, IgG, and IgA (Orion Dianostica, Espoo, Finland) diluted 1: 100 in PBS plus 0.05% Tween 20 were then added to the wells, and the plates were incubated overnight at 4° C. The following morning the plates were rewashed again, and 50 ~I of 5-bromo-4-chloro-3-indolyl-phosphate, a substrate for alkaline phosphatase, was added to each well. The substrate was prepared (1.2 mg/ml) in a buffer containing 95.8 rnl 2-amino-2-methyl-l-propa· nol, 150 mg MgCl 2 X 6 H 20, 0.1 rnl Triton X40S, and 1.0 gm sodium azide per liter. To four parts of the substrate solution, heated to 42° C, one part of melted (100° C) 3% agarose (Type 1; Sigma Chemical Co., St. Louis, Mo.) in distilled water was added. The mixture was vortexed and applied quickly to the plates. The plates were incubated again overnight in moist chambers at room temperature. During incubation blue spots appeared on the bottom of the wells, indicating areas in which immunoglobulin secreting cells had been attached. The spots were counted using a stereomicroscope. Results are expressed as antibody secreting cells per l.Q6 live cells. Co-cultures. Separated T and B cells as well as unfractionated mononuclear cells were suspended in RPMI 1640 with 5% FCS. Tissue culture tubes, each containing 8 X 105 T cells and 2 X 105 B cells in 1 ml of cell suspension, were placed in an incubator together with tubes containing unfractionated cells (I X 106/ml) and kept for either 18 or 66 hours at 37° C in a humidified atmosphere containing 5% CO 2 in air. All

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In vivo activated lymphocytes in peripheral blood 1 1 1

January 1987

Table I. Peripheral blood lymphocyte proliferation in 12 patients before and 7 days after cardiac operation and in two patients with infectious mononucleosis 'Hsthymidine uptake (cpm) Before operation PBMC

Patient 1

45

2

77

3 4

5

78 90 59

6

109

7

63 185 158 147 66 308

8 9

10 11 12 Mean SD Median

115

75 84 12

n

After operation PBMC

Non- T cell features (%)

1055 825 1812 3438 803 970 1190 862 1619 1154 1498 849 1340* 740

T cells

B cells

Atypical

685 19 . 165 194 98 116 118 384 149 238 565 236 204 157 12

3072 2560 7856 6523 1760 3708 2402 4973 5255 4615 4218 1370 4033 1941 3963 12

51.0 60.0 45.0 52.0 29.0 61.0 54.0 36.0 35.0 46.0 40.0 48.0 46.4

4047 460

725 146

99

1104

12

10.0

47 12

I

Myeloid

1.0 2.0 4.0

I

Lymphocytes

o

o o

4.0 5.0 8.0 3.0 3.0 1.0 2.6 2.4 2.5

12

23.0 17.0 14.0 40.5 65.0 36.0 27.0 53.0 40.0 41.0 32.0 43.0 36.0 14.6 38 12

Mononucleosis 1

2 Legend: PBMC

= peripheral

blood mononuclear cells. cpm

= amount of incorporated

11.0 17.0

4.0

o

46.0 82.0

radioactivity into the synthesized DNA in in vitro cultured cells.

*p < 0.005 compared with preoperative value by Wilcoxon test.

combinations of patient and control T and B cells were used. Lymphocyte subpopulations. A modified indirect immunofluorescence method II was used to determine the percentage of T cells, with each of the markers defined by the following monoclonal antibodies: OKT4, OKT8, OKTll and OKIal (Ortho Diagnostics Inc., Raritan, N. J.). The two-color immunofluorescence method was used to determine the percentage of HLADR (Ia antigen) positive helper or suppressor T cells. In brief, 0.2 ml of cell suspension (2.5 X 106 celljml) was incubated for 30 minutes at 4 0 C with 5 JLI of fluorescein (FITC)--conjugated monoclonal antibodies (Leu 3a and Leu 2a, Becton-Dickinson, Rutherford, N.J.). The cells were washed twice with PBS supplemented with 5% FCS and 0.02% sodium azide, reincubated with 20 JLI of biotin-eonjugated anti-DR antibody (Becton-Dickinson Inc), and washed as above. The cells were then incubated again for 30 minutes at 4 C with 1 JLI of rhodamine-eonjugated avidin (Becton-Dickinson). The cells were suspended in 20 JLI of glycerol-PBS (I : 1) after twowashings, and the number of cells positive for FITC and rhodamine-staining cells was microscopically counted with alternate light microscopy and appropriate fluorescence illumination. 0

Statistics. The Wilcoxon test and the Mann-Whitney U test were used for statistical analysis of the results. Results Spontaneous proliferation of lymphocytes. High spontaneous proliferative activity, measured as 3H_ thymidine uptake, was found in patients' peripheral blood samples taken after cardiac operation, the peak level occurring on the sixth or seventh postoperative day (Fig. 1). The activity values on either of these differ significantly (p < and p < 0.025) from preoperative values. When lymphocytes obtained on the seventh postoperative day in a group of patients were segregated into T- and B-Iymphocyte enriched fractions, most of the 3H-thymidine incorporating cells were found to be in the B cell enriched fraction, whereas in the patients with infectious mononucleosis, included for comparison, the T cell fraction showed the greatest proliferative activity (Table I). A great number of cells morphologically identified as atypical lymphocytes were found in the non-T cell (B cell) fraction, whereas myeloid cell contamination was small. Antibody secreting ceUs. Additional experiments were performed in eight patients to further characterize

om

The Journal of Thoracic and Cardiovascular

1 1 2 Ryhanen et al.

Surgery

Table II. Number of antibody secreting cells (ASe) in the peripheral blood samples taken I and 7 days after cardiac operation in eight patients ASC/IO' viable cells Day 7

Day I Patient

IgM

1 2 3 4 5 6 6 8

0 0 0 50 40 90 110 10 25 8

Median n

I

IgG 6 0 0 50 20 0 10 10 8 8

I

IgA

IgM

41 100 1,575 160 490 100 200 660 180 8

714 2,108 583 1,130 130 50 710 210 647" 8

I

IgG

I

1,200 1,506 1,506 6,190 450 470 820 1,400 1,300" 8

IgA 5,500 3,675 9,770 36,990 4,230 540 5,000 7,950 5,250" 8

"p < 0.001 compared with day I value by Wilcoxon test.

Table ill. Effect of I8-hour co-culture of patients' B cells with their own T cells and controls' T cells on the number of antibody secreting cells (ASe) after cardiac operation in eight patients ASC/IO' viable cells (median) Day 1* (n = 8) Cell populations

IgM

T 1 + s, T 2+Bp Tp++Bp

35 30 20

I

IgG 12.5 10.0 10.0

Day 7* (n = 8)

I

IgA

IgM

415 170 150

380 220 260

I

IgG 1,008 1,226 1,485

I

IgA 5,374.0 5,379.5 7,266.5

Legend: Statistical comparison made in different immunoglobulin classesbetween the combinationsof T, + B, and T 1 + B, and between the combinationsof T, + B, and T, + B, showed no statistical significance (p > 0.1, Mann-Whitney U test). I = Control subject I. 2 = Control subject 2. p = Patient. 'Blood samples were taken I and 7 days after operation.

the activated cells. The number of antibody secreting cells in peripheral blood samples was increased (p < O.OOI) in all immunoglobulin classes on the seventh postoperative day compared with the values from the first postoperative day, which were in the normal range (Table II). To study the possible effect of helper and suppressor cells in vitro on antibody secreting cells, patients' B cells were co-cultured for 18 hours with their own separated T cells and the T cells from two healthy adult control subjects. The numbers of antibody secreting cells were similar whether the control or the patient T cells were used (Table III). Thus no significant (p> 0.1) helper or suppressor effect was observed. The increase of coculture time to 66 hours (data not shown) had no influence on the function of the helper and suppressor cells, but the number of immunoglobulin synthesizing cells decreased in all combinations with the longer incubation time.

T-lymphocyte subpopulations. The T cell fraction was further characterized by lymphocyte surface markers in five patients who underwent cardiac operation (Table IV). The percentage of helper (OKT4) and suppressor (OKT8) cells in postoperative samples was not different (p> 0.1) from that seen before operation. Because the percentage of activated (OKIa positive) T cells showed a tendency to rise after operation, we performed a further experiment in six other patients to identify activated helper and suppressor cells. The results of this experiment show that no significant (p > 0.1) T cell activation occurs in these patients after operation (Table V). Discussion

Cardiac operation with cardiopulmonary bypass is known to damage different components of blood, such as red blood cellsI 2 and thrombocytes." We have previously shown that cardiac operation results in a

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In vivo activated lymphocytes in peripheral blood 1 1 3

January 1987

Table IV. Median percentage and range of T-lymphocytes with different surface makers in five patients before and 1 and 7 days after cardiac operation After operation Day I

Before operation Surface marker

Median

OKTl1 OKT4 OKT8 OKT4/0KT8 OKla1

87.8 44.5 25.8 1.8 4.8

I

Range

Median

81.9-96.0 36.5-62.6 18.1-38.0 1.0-3.1 0.9-10.0

87.4 40.5 31.9 1.4 8.0

I

Day 7 Range

Median

76.5-94.0 34.7-48.5 19.5-46.6 0.8-2.5 1.0-18.0

91.6 55.5 26.7 2.3 10.1*

Range

I

85.0-95.5 46.6-67.3 16.5-35.7 1.4-4.1 4.4-13.0

*p < 0.1 compared with the value before operation. by Wilcoxon test.

decline in lymphocyte count and impairment of their function' and in a quantitative decrease in serum immunoglobulin (IgM, IgG, and IgA) and complement (e) and C 4 ) concentrations." At the same time, however, an increased amount of morphologically atypical lymphocytesspontaneously synthesizing DNA appear in the peripheral blood of patients. Our findings indicate that most of the activated lymphocytes are B cells. The results obtained from the small groups studied for variousT cell markers do not allow far-reaching conclusions, but clearly show that no significant T cell activation was found and that T helper/suppressor ratios also remained within the normal range without any major deviations. Differentation of B cells into antibody secreting cells was found. All immunoglobulins were synthesized, but the number of IgA secreting cells was most prominent. This immunoglobulin class also dominates the low spontaneous immunoglobulin secretion found in the normal state." The observed increase in immunoglobulin synthesis of all major heavy chain subclasses suggests a polyclonal nature of B cell activation. These cells were activated in vivo to produce immunoglobulins, and seem not to be affected in vitro by helper or suppressor cells. The largest part of the postoperativeincrease in activated lymphocytes has been considered to be a response to blood transfusion, 16 products of tissue decomposition," and certain infective factors."? These cells, however, have also been found to increase in patients undergoing operations but not receiving transfusions." The results of our study do not support those of Roses and co-workers," who suggested that the appearance of atypical lymphocytes after cardiac operations reflects a T cell rather than a B cell immunoresponse to blood transfusion. On the contrary, our results suggest that the occurrence of activated lymphocytes can be regarded as an expression of a polyclonal antibody response. This polyclonal activation of B cells has also been found in alcoholic patients," and

Table V. Median percentage and range of activated (Ia-positivet helper and suppressor cells in six patients before and 7 days after cardiac operation Surface marker

Leu Leu Leu Leu

3a 3a + Ia 2a 2a + [a

Before operation Median

53.0 0.25 31.0 0.25

I

After operation. Day 7

Range

Median

28.0-56.5 0-2.0 15.0-36.0 0-6.0

52.5 2.25 21.5 2.0

I

Range

50.0-67.0 0.5-3.5 16.5-35.0 0.5-4.0

Legend: Statistical comparison made between values before and after operation showed no statistical significance (p > om, Wilcoxon test).

extracts of oral bacteria associated with periodontal disease can induce nonspecific antibody production in cell culture." The nature of B cell activating factors and the role of specific antigens in patients undergoing cardiac operation remain to be clarified. The determination of antibody specificities secreted might elucidate the nature of activation, although it must be noted that, for example, in viral infections antibodies are also synthesized against other agents than those causing the infection. 21 Our results show that after cardiac operation the peripheral blood of patients contains activated cells that are mostly B-lymphocytes secreting immunoglobulins of all major heavy chain subclasses. These cells are not affected in vitro by the regulatory T-lymphocytes.

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Thoracic and Cardiovascular Surgery

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