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Altered helper and suppressor lymphocyte populations in surgical patients
Altered Helper and Suppressor Lymphocyte Populations in Surgical Patients A Measure of Postoperative lmmunosuppression
John F. Hansbmugh, MD, FACS, Denver, Colorado Edward M. Bander, MD, Denver, Colorado Ramon Zapata-Sirvent, MD, Denver, Colorado Julia Anderson, BS, Denver, Colorado
The occurrence of immune suppression after general surgical operations remains controversial. It has been a widely held belief that surgery itself is immunosuppressive, and a number of reports [1-31 have demonstrated suppression of lymphocyte function after surgical procedures. It has been suggested that such suppression contributes to postoperative dissemination of carcinoma [4-71 and increases in p&operative infections [8,9]. However, some groups have been unable to document postsurgical immunosuppression by measurements of lymphocyte reactivity (IO-151 and delayed hypersensitivity (161, and immunoglobulin levels have been reported to be unchanged after surgery [I 71. Lymphopenia has been reported after surgical stress [18,19], but may reflect a redistribution of cells since the proportions of T and B cells remains constant. Because of the controversy surrounding the occurrence of immunosuppression associated with surgical procedures, we decided to study lymphocyte subpopulations in patients undergoing elective cholecystectomy. T and B lymphocytes and helper and suppressor subpopulations were identified and quantitated using specific monoclonal antibodies and immunofluorescence microscopy. Material and Methods Eleven patients were selected for study who were to undergo routine chokcystect.omy. They ranged in age from 18 to 72 years. Eight were female, and all patients were free of coexisting major medical problems. After informed FrcmUnrDepmmmtofSugery,UnkanyofcobraeoMedkaIschodand Heenhsciences centa. Denver.cobrado. Requests for reprintsshockIbe se&ass& to John F. liw&ough, MD. Depsrbnsnt of fhaay. hlvenfty of Callfomia. San Diego Medical Center. 225 Dlcklnson Street. San D~QII, California 92103.
vahm
140, sophlbu
1014
consent, blood was drawn by venipuncture on the day before surgery. Blood was again drawn on the first postoperative day, and in five patients who were available, blood was again drawn on the third or fourth postoperative day. One patient underwent common duct exploration. All patients had general anesthesia, and the duration of anesthesia and surgery averaged 2 hours 4 minutes (range 1 hour 25 minutes to 3 hours 15 minutes). Postoperative complications occurred in one patient (transient pancreatitis and jaundice). For cell preparation, venous blood was collected in heparinized tubes. Total and differential white blood cell counts were obtained by hand counting. Peripheral blood lymphocytes were separated by density gradient centrifugation in Ficoll-Hypaque gradients (Histopaque 1077@, Sigma, St. Louis, MO). Monocytes were identified by latex particle ingestion. Lymphocytes were washed and suspended in balanced salt solution and adjusted to a concentration of 1 X 10’ cells per milliliter. Using an indirect immunofluorescence technique a 100 ~1 aliquot (1 X 106 cells per milliliter) was incubated at 4°C for 30 minutes with one of three mouse antihuman monoclonal antibodies (Coulter Immunology, Hialeah, FL): OKT 11 (pan T cells), OKT 4 (T helper-inducer cells), or OKT 8 (T suppressor-cytotoxic cells); and a second incubation with fluorescein conjugated goat (F ab’Jpantimouse immunoglobulin at 4OC for 30 minutes. Another 100 ~1 was incubated at 4OC for 60 minutes with fluorescein conjugated goat (F ab’)p antihuman immunoglobulin (Kallestead, Austin, TX), recognizing immunoglobulin bearing B lymphocytes by direct immunofluorescence. Preparations were counted by fluorescent microscopy, counting 200 cells. The frequency of specifically labelled lymphocyte subpopulations was calculated by dividing the number of fluorescing cells by the number of total lymphocytes counted. Statistical analysis. The means of the percentages of OKT 4, OKT 8, and OKT 11 labelled cells, and the OKT 4 to OKT 8 ratios were compared on three different days
303
Hansbrough et al
TABLE I
Leukocyte Population in the Perioperative Period (mean f standard error of the mean)* 1 Day Preop
1 Day Postop
White blood cells
7.71 f
1.14
12.98 f 0.45
(103/ml) Lymphocytes
2.72 f 0.45
2.31 f 0.38
3-4 Days Postop 9.88 f 2.09 2.82 f
0.48
(103/ml) 88.38 f 10.91 f
T cells (%) B Cells (%)
83 f 14.38 f
1.05 1.11
1.33 1.38
82.83 f 1.88 12.67 f 2.2
l White blood counts obtained from patients undergoing cholecystectomy. Total T-ceil counts were determined by staining with OKT 11 monoclonal antibody followed by fluorescein conjugated goat antimouse immunoglobulin and total B-cell counts by staining with fluorescein conjugated antihuman immunoglobulin.
using the Student’s t test. Statistical significance was assumed at p <0.05. Results are shown as the mean f 1 standard error of the mean. Results Data for total white blood cells, lymphocyte counts and T- and B-lymphocyte counts in the peripheral blood are shown in Table I. These values demonstrate mild leukocytosis postoperatively without significant changes in the absolute lymphocyte counts or proportions of B and T cells (p >0.05). The results of the determinations of helper and suppressor lymphocyte counts are shown in Figures 1,2 and 3. In Figure 1, changes in lymphocyte subpopulations between the preoperative and 1 day postoperative samples are shown, as well as the changes in the five patients in whom blood was available for follow-up 3 to 4 days postoperatively. We found a decrease in the relative percentage of T-helper cells immediately after operation compared with preoperative levels (53 f 1.47 percent preopp eratively versus 46.2 f 1.5 postoperatively,
0.5 when compared with preoperative values). Thus, we found a larger decrease in helper cells than an increase in suppressor populations after surgery, although both changes were statistically significant. Figure 2 demonstrates values for T-helper to Tsuppressor ratios in individual patients, and Figure 3 demonstrates the mean values for the same ratios in all patients studied. All 11 patients experienced a decrease in the helper to suppressor ratio on the first postoperative day, with mean values falling significantly from preoperative levels (1.77 f 0.14 preoperatively versus 1.33 f 0.08 postoperatively, p 0.05). Comments
PreoP
1b!I-
1cIaY post op
days postop
Ftgure 1. Lymphocyte subpopulattons In pathts undergotng cholecystectomy. OKT 4 antlbody tdentffted helper T cells, and OK7 8 antlbody tdentttled suppressor T cells.
304
It has become clear that preoperative clinical anergy is an accurate predictor of sepsis and mortality in patients undergoing surgery or sustaining trauma [3,20]. However, although early studies suggested that surgical procedures themselves were associated with a pronounced depression of immunity, more recent reports have questioned whether surgery itself is immunosuppressive. A recent study examined the kinetics of the response of lymphocytes to phytohemagglutinin in operated patients. No clear effect of surgery on the reponse could be demonstrated, although there were wide variations in the results of individual determinations over time [15]. Part of the controversy surrounds the reproducibility of lymphocyte stimulation assays. Variations in stimulation values have been found to be as high as 50 to 100
The American Journal ol Surgery
Altered Lymphocyte
Populations
l3
0
2.5
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a
2.0
.lO 29
;
0 3 5
l2
l3
-11
lO*
-6
OD
1.3
79.11 04 9' '1
l6
I.0
r”
I
6.
-I-
'7
6 100' '2 t1*5:7 '4
11
ll
5
0.s I I
I I
I I
1 day
1 day
PreoP
postop
3-4
days
postop
1 day
1 day
3-4days
PreoP
postop
postop
F&r@ 3. Mean values of T-he&u lo T-supprww ratfos (OK7 I/OKT 6) A the 11 patfents undergofngchofecysfectomy.
r8i&S(oKT F&ne2hdfvMdv~ofT~toT~ 4/M 8) In 11 paifents undergoingchofecystectomy.
percent in normal subjects [21-241. Payne et al [15] estimated that lymphocyte transformation tests in more than 400 surgical patients would be required before significant immunosuppressive effects could be detected. Limitations exist for the use of stimulation tests of lymphocytes as measures of immune competence, however [25]. The various tests are performed in vitro and depend on interactions and undoubtedly signals between various cell populations, including monocyte and lymphocyte populations [26,27]. Since some cell populations probably die rapidly in culture conditions, true in vivo lymphocyte reactivity may well be altered after even short periods of culture. Direct measurement of lymphocyte subpopulations, utilizing staining and quantitation by antibodies specific for different cell types, may prove to be more accurate and dependable measurements of cellular immune competence than in vitro stimulation assays. In animal studies with burned mice, we have demonstrated that the helper to suppressor lymphocyte ratio is an accurate reflection not only of in vivo cell-mediated immunity, as measured by delayed hypersensitivity, but also of the modulatory effects of certain drugs on the immune response (28-301. In those experiments, changes in the degree of contact hypersensitivity were closely correlated with changes in lymphocyte subpopulations using indirect immunofluorescent microscopy to measure helper and suppressor lymphocyte populations.
T-lymphocyte functions play pivotal roles in not only cell-mediated immunity but also antibody production [31] and the inflammatory response [32,33]. Both helper and suppressor T-lymphocyte populations are integrally related with such functions [34], and suppressor cell activity has been associated with impaired host defenses in many disease states. Elevated suppressor lymphocyte activity and numbers have been described in burn and trauma patients [35-371, although Miller et al [38,39] have described a suppressor monocyte population. It appears that the balance between various monocyte, T-helper, and T-suppressor populations plays an important role in host defense, If the ratio of Thelper to T-suppressor lymphocytes or facilitory monocytes to suppressor monocytes, or both becomes unbalanced toward excessive suppression, the result is immunoincompetence and increased host susceptibility to infection 1391. The evidence presented herein suggests that uncomplicated surgical procedures result in a shift of the immunoregulatory system toward suppression. The contribution to immunosuppression of factors associated with the surgical procedure remain to be determined. The length and complexity of surgery, associated blood loss and hypotension, and duration and type of anesthesia are factors that must be examined. However, our data suggest that direct measurement of helper and suppressor cell populations in peripheral blood may prove to be more accurate and reproducible indicators of immune status in surgical and traumatized patients than in vitro measurements of lymphocyte reactivity.
305
Hansbrough et al
Summary Although a wealth of evidence has suggested that cell-mediated immunity is suppressed after simple surgical trauma, there have been contradictory results using stimulation assays of lymphocyte function. We quantitated T-lymphocyte subsets in 11 patients undergoing routine cholecystectomy by immunofluorescence microscopy using specific monoclonal antibodies. T-helper to T-suppressor cell ratios were calculated on the preoperative day and the first postoperative day in all patients, and on the third or fourth postoperative day in five patients. Helper to suppressor ratios decreased in all patients on the first postoperative day (p >O.Ol), but returned to within normal limits on subsequent days. Changes were due more to decreases in helper cells than to increases in suppressor cells, although changes in both populations were statistically significant. The measurement of T-cell subsets by antibodyspecific labeling and immunofluorescence microscopy may prove to be a more sensitive, quantifiable, and reproducible assay of immune function in surgical or traumatized patients than use of stimulation assays. Measurements of specific helper and suppressor lymphocyte populations may prove useful in predicting morbidity and mortality, and may also help in studying the effect of immunomodulating agents on the immune response. References 1. Riddle PR, Berenbaum MC. Post-operative depression of the lymphocyte response to phytohaemagglutinin. Lancet 1967;1:746-6. 2. Park SK, Brody JI, Wallace HA, Blakemore WS. Immunosuppressive effect of surgery. Lancet 1971;1:53-5. 3. Lundy J, Ford CM. Surgery, trauma and immune suppression. Evolving the mechanism. Ann Surg 1963;197:434-8. 4. Editorial: Post-operative immunosuppression. LanCet 1974; 2:817-8. 5. Howard RJ, Simmons RL. Acquired immunologic deficiencies after trauma and surgical procedures. Surg Gynecot Obstet 1974;139:771-82. 6. Lundy J, Lovett EJ, Hamilton S, Conran P. Halothane, surgery, immunosuppression and pulmonary metasteses. Cancer 1978;41:827-30. 7. Lundy J, Lovetl EJ, Hamilton S, Conran P. Immune impairment and metastatic tumor growth. Cancer 1979;43:945-51. 8. Wang BS, Heacock EH, Mannick JA. Characterization of suppressor cells generated in mice after surgical trauma. Clin lmmunol lmmunopathol 1982;24:161-70. 9. Wang BS, Heacock EH, Wu AVO, Mannick JA. Generation of suppressor cells in mice after surgical trauma. J Clin Invest 1980;66:200-9. 10. Vose JM, Moudgil GC. Post-operative depression of antibody dependent lymphocyte cytotoxiclty following minor surgery and anaesthesia. Immunology 1976;30: 123-8. 11. Jubert AV, Lee ET, Hersh EM, McBride CM. Effects of surgery, anaesthesia and intraoperative blood loss on immunocompetence. J Surg Res 1973;15:399-403. 12. Espanol T, Todd GB, Soothill JF. The effect of anaesthesia on the lymphocyte response to phytohaemagglutinin. Clin Exp lmmunol 197418:73-Q.
306
13. Rynahen P. Effects of anaesthesia and operative surgery on the immune response of patients of different ages. Stand J lmmunol 1977;6:1194. 14. Cohen BE, Gill G, Cullen PR, Morris PJ. Reversal of postoperative immunosuppression in many by Vitamin A. Surg Gynecol Obstet 1979; 149:658-62. 15. Payne J, Hayden P, Meyer HJ, Walls RS. Observed differences in postoperative lymphocyte transformation are explained by patient and population variations. Am J Surg 1984;147: 237-42. 16. Pietsch JB, Meakins JL, Cotto D, MacLean LD. Delayed hypersensitivity responses: the effect of surgery. J Surg Res 1977;22:228-30. 17. Slade MS, Simmons RL, Yunis E, Greenberg LJ. Immunodepression after major surgery in normal patients. Surgery 1975;78:363-72. 18. Miller GC, Pritchard DJ, Ritts RE. lvins JC, Pierre RV. Effect of surgery on the quality of lymphocyte subpopulations. J Surg Res 1976;21:144-8. 19. Bolton PM, Kirov SM, Donald KJ. The effects of major and minor trauma on the lymphocyte kinetics in mice. Aust J Exp Biol Med Sci 1979;57:479-92. 20. Johnson WC, Ulrich F, Meguid MM, et al. Role of delayed hypersensitivity in predicting postoperative morbidity and mortality. Am J Surg 1979;137:536-42. 21. Boudet RA. Morse PA, Watts LM. An analysis of phytohaemagglutinin lymphoblastogenesis in whole blood. Surg Gynecol Obstet 1978;146:609-16. 22. Maclntyre OR, Cole AF. Variation in the response of normal lymphocytes to PHA. Int Arch Allergy lmmunol 1969;35: 105-18. 23. Allen HJ. Frequency distribution analysis of the normal lyrnphocyte response to phytohaemagglutinin. lmmunol Commun 1974;3:557-91. 24. Graybill JR, Alford RH. Variability of sequential studies of lymphocyte blastogenesis in normal adults. Clin Exp Immunol 1976;25:28-35. 25. Ninnemann JL. lmmunosuppression following thermal injury through B cell activation of suppressor T cells. J Trauma 1980;20:206-13. 26. Rosenstreich DL. The macrophage requirement for mttogenic activation of T-lymphocytes. In: Dppenheim JJ, Rosenstreich DL, eds. Mitogen in Immunobiology. New York: Academic Press, 1976:365-94. 27. Gershon RK, Leibhaber SA. Ryn S. T-cell regulation of T-cell responses to antigen. Immunology 1974;26:909-23. 28. Peterson V, Hansbrough JF, Zapata-Sirvent R, Bender E, Wang X. Topical cerium nitrate prevents postburn immunosuppression. Proceedings of the 16th Congress of the American Burn Association, San Francisco, 1984. 29. Hansbrough JF, Peterson V, Zapata-Sirvent R, Bender E, Claman H. Restoration of immunity in burned mice by cimetidine. Proceedings of the 16th Congress of the American Burn Association, San Francisco, 1984. 30. Hansbrough JF, Peterson V, Zapata-Sirvent R, Claman H. Studies of post-burn immunosuppression using an animal model. II. Restoration of cell-mediated immunity by immunomodulating drugs. Surgery 1984;95:290-7. 31. Katz DH, Benacerraf B. The regulatory influence of activated T cells on B cell responses to antigen. Adv lmmunol 1972; 15:1-94. 32. David JR, Renold HG. Macrophage activation of lymphocyte mediators and studies on the interaction of macrophage inhibitory factor (MIF) with its target cell. In: Nelson DS, ed. lmmunobiology of the Macrophage. New York: Academic Press, 1976:401-26. 33. Gallin JI. Abnormal chemotaxis: cellular and humoral components. In: Bellanti JA, Dayton DH, eds. The phagocytic cell in host resistance. New York: Raven Press, 1975:227. 34. Waldmann TA, Broder S. Suppressor cells in the regulation of
The American Journal of Surgery
Altered
the immune response. in: Schwartz RS, ed. Progress in Clinical Immunology V. 3. New York: Grune and Stratton, 1977:155-99. 35. Ninnemann JL. fmmunologic defenses against infection: alterations following thermal injuries. J Burn Care Rehab 1983;3:355-98. 36. Antonacci AC, Good RA. T-cell subpopulations following thermal injury. Surg Gynecol Obstet 1992;155:1-8. 37. Ninnernann JL. Immune depression in burn and trauma patients.
volume 149, SeptMkr
1984
Lymphocyte
Populations
The role of circulating suppressors. In: Ninneman JL, 8d. Traumatic Injury: infection and other immunologic sequelae. Baltimore: University Park Press, 198333-56. 38. Miller SE, Miller CL, Trunkey DD. The immune consequences of trauma. Surg Cl North America 1982;62:167-82. 39. Miller SE, Trunkey DD, Miller CL. The immunologic effects of trauma. In: Ninneman JL, ed. Traumatic Injury: infection and other immunologic sequelae. Baltimore: University Park Press, 1983: 17-32.
307
John F. Hansbmugh, MD, FACS, Denver, Colorado Edward M. Bander, MD, Denver, Colorado Ramon Zapata-Sirvent, MD, Denver, Colorado Julia Anderson, BS, Denver, Colorado
The occurrence of immune suppression after general surgical operations remains controversial. It has been a widely held belief that surgery itself is immunosuppressive, and a number of reports [1-31 have demonstrated suppression of lymphocyte function after surgical procedures. It has been suggested that such suppression contributes to postoperative dissemination of carcinoma [4-71 and increases in p&operative infections [8,9]. However, some groups have been unable to document postsurgical immunosuppression by measurements of lymphocyte reactivity (IO-151 and delayed hypersensitivity (161, and immunoglobulin levels have been reported to be unchanged after surgery [I 71. Lymphopenia has been reported after surgical stress [18,19], but may reflect a redistribution of cells since the proportions of T and B cells remains constant. Because of the controversy surrounding the occurrence of immunosuppression associated with surgical procedures, we decided to study lymphocyte subpopulations in patients undergoing elective cholecystectomy. T and B lymphocytes and helper and suppressor subpopulations were identified and quantitated using specific monoclonal antibodies and immunofluorescence microscopy. Material and Methods Eleven patients were selected for study who were to undergo routine chokcystect.omy. They ranged in age from 18 to 72 years. Eight were female, and all patients were free of coexisting major medical problems. After informed FrcmUnrDepmmmtofSugery,UnkanyofcobraeoMedkaIschodand Heenhsciences centa. Denver.cobrado. Requests for reprintsshockIbe se&ass& to John F. liw&ough, MD. Depsrbnsnt of fhaay. hlvenfty of Callfomia. San Diego Medical Center. 225 Dlcklnson Street. San D~QII, California 92103.
vahm
140, sophlbu
1014
consent, blood was drawn by venipuncture on the day before surgery. Blood was again drawn on the first postoperative day, and in five patients who were available, blood was again drawn on the third or fourth postoperative day. One patient underwent common duct exploration. All patients had general anesthesia, and the duration of anesthesia and surgery averaged 2 hours 4 minutes (range 1 hour 25 minutes to 3 hours 15 minutes). Postoperative complications occurred in one patient (transient pancreatitis and jaundice). For cell preparation, venous blood was collected in heparinized tubes. Total and differential white blood cell counts were obtained by hand counting. Peripheral blood lymphocytes were separated by density gradient centrifugation in Ficoll-Hypaque gradients (Histopaque 1077@, Sigma, St. Louis, MO). Monocytes were identified by latex particle ingestion. Lymphocytes were washed and suspended in balanced salt solution and adjusted to a concentration of 1 X 10’ cells per milliliter. Using an indirect immunofluorescence technique a 100 ~1 aliquot (1 X 106 cells per milliliter) was incubated at 4°C for 30 minutes with one of three mouse antihuman monoclonal antibodies (Coulter Immunology, Hialeah, FL): OKT 11 (pan T cells), OKT 4 (T helper-inducer cells), or OKT 8 (T suppressor-cytotoxic cells); and a second incubation with fluorescein conjugated goat (F ab’Jpantimouse immunoglobulin at 4OC for 30 minutes. Another 100 ~1 was incubated at 4OC for 60 minutes with fluorescein conjugated goat (F ab’)p antihuman immunoglobulin (Kallestead, Austin, TX), recognizing immunoglobulin bearing B lymphocytes by direct immunofluorescence. Preparations were counted by fluorescent microscopy, counting 200 cells. The frequency of specifically labelled lymphocyte subpopulations was calculated by dividing the number of fluorescing cells by the number of total lymphocytes counted. Statistical analysis. The means of the percentages of OKT 4, OKT 8, and OKT 11 labelled cells, and the OKT 4 to OKT 8 ratios were compared on three different days
303
Hansbrough et al
TABLE I
Leukocyte Population in the Perioperative Period (mean f standard error of the mean)* 1 Day Preop
1 Day Postop
White blood cells
7.71 f
1.14
12.98 f 0.45
(103/ml) Lymphocytes
2.72 f 0.45
2.31 f 0.38
3-4 Days Postop 9.88 f 2.09 2.82 f
0.48
(103/ml) 88.38 f 10.91 f
T cells (%) B Cells (%)
83 f 14.38 f
1.05 1.11
1.33 1.38
82.83 f 1.88 12.67 f 2.2
l White blood counts obtained from patients undergoing cholecystectomy. Total T-ceil counts were determined by staining with OKT 11 monoclonal antibody followed by fluorescein conjugated goat antimouse immunoglobulin and total B-cell counts by staining with fluorescein conjugated antihuman immunoglobulin.
using the Student’s t test. Statistical significance was assumed at p <0.05. Results are shown as the mean f 1 standard error of the mean. Results Data for total white blood cells, lymphocyte counts and T- and B-lymphocyte counts in the peripheral blood are shown in Table I. These values demonstrate mild leukocytosis postoperatively without significant changes in the absolute lymphocyte counts or proportions of B and T cells (p >0.05). The results of the determinations of helper and suppressor lymphocyte counts are shown in Figures 1,2 and 3. In Figure 1, changes in lymphocyte subpopulations between the preoperative and 1 day postoperative samples are shown, as well as the changes in the five patients in whom blood was available for follow-up 3 to 4 days postoperatively. We found a decrease in the relative percentage of T-helper cells immediately after operation compared with preoperative levels (53 f 1.47 percent preopp eratively versus 46.2 f 1.5 postoperatively,
PreoP
1b!I-
1cIaY post op
days postop
Ftgure 1. Lymphocyte subpopulattons In pathts undergotng cholecystectomy. OKT 4 antlbody tdentffted helper T cells, and OK7 8 antlbody tdentttled suppressor T cells.
304
It has become clear that preoperative clinical anergy is an accurate predictor of sepsis and mortality in patients undergoing surgery or sustaining trauma [3,20]. However, although early studies suggested that surgical procedures themselves were associated with a pronounced depression of immunity, more recent reports have questioned whether surgery itself is immunosuppressive. A recent study examined the kinetics of the response of lymphocytes to phytohemagglutinin in operated patients. No clear effect of surgery on the reponse could be demonstrated, although there were wide variations in the results of individual determinations over time [15]. Part of the controversy surrounds the reproducibility of lymphocyte stimulation assays. Variations in stimulation values have been found to be as high as 50 to 100
The American Journal ol Surgery
Altered Lymphocyte
Populations
l3
0
2.5
z a
a
2.0
.lO 29
;
0 3 5
l2
l3
-11
lO*
-6
OD
1.3
79.11 04 9' '1
l6
I.0
r”
I
6.
-I-
'7
6 100' '2 t1*5:7 '4
11
ll
5
0.s I I
I I
I I
1 day
1 day
PreoP
postop
3-4
days
postop
1 day
1 day
3-4days
PreoP
postop
postop
F&r@ 3. Mean values of T-he&u lo T-supprww ratfos (OK7 I/OKT 6) A the 11 patfents undergofngchofecysfectomy.
r8i&S(oKT F&ne2hdfvMdv~ofT~toT~ 4/M 8) In 11 paifents undergoingchofecystectomy.
percent in normal subjects [21-241. Payne et al [15] estimated that lymphocyte transformation tests in more than 400 surgical patients would be required before significant immunosuppressive effects could be detected. Limitations exist for the use of stimulation tests of lymphocytes as measures of immune competence, however [25]. The various tests are performed in vitro and depend on interactions and undoubtedly signals between various cell populations, including monocyte and lymphocyte populations [26,27]. Since some cell populations probably die rapidly in culture conditions, true in vivo lymphocyte reactivity may well be altered after even short periods of culture. Direct measurement of lymphocyte subpopulations, utilizing staining and quantitation by antibodies specific for different cell types, may prove to be more accurate and dependable measurements of cellular immune competence than in vitro stimulation assays. In animal studies with burned mice, we have demonstrated that the helper to suppressor lymphocyte ratio is an accurate reflection not only of in vivo cell-mediated immunity, as measured by delayed hypersensitivity, but also of the modulatory effects of certain drugs on the immune response (28-301. In those experiments, changes in the degree of contact hypersensitivity were closely correlated with changes in lymphocyte subpopulations using indirect immunofluorescent microscopy to measure helper and suppressor lymphocyte populations.
T-lymphocyte functions play pivotal roles in not only cell-mediated immunity but also antibody production [31] and the inflammatory response [32,33]. Both helper and suppressor T-lymphocyte populations are integrally related with such functions [34], and suppressor cell activity has been associated with impaired host defenses in many disease states. Elevated suppressor lymphocyte activity and numbers have been described in burn and trauma patients [35-371, although Miller et al [38,39] have described a suppressor monocyte population. It appears that the balance between various monocyte, T-helper, and T-suppressor populations plays an important role in host defense, If the ratio of Thelper to T-suppressor lymphocytes or facilitory monocytes to suppressor monocytes, or both becomes unbalanced toward excessive suppression, the result is immunoincompetence and increased host susceptibility to infection 1391. The evidence presented herein suggests that uncomplicated surgical procedures result in a shift of the immunoregulatory system toward suppression. The contribution to immunosuppression of factors associated with the surgical procedure remain to be determined. The length and complexity of surgery, associated blood loss and hypotension, and duration and type of anesthesia are factors that must be examined. However, our data suggest that direct measurement of helper and suppressor cell populations in peripheral blood may prove to be more accurate and reproducible indicators of immune status in surgical and traumatized patients than in vitro measurements of lymphocyte reactivity.
305
Hansbrough et al
Summary Although a wealth of evidence has suggested that cell-mediated immunity is suppressed after simple surgical trauma, there have been contradictory results using stimulation assays of lymphocyte function. We quantitated T-lymphocyte subsets in 11 patients undergoing routine cholecystectomy by immunofluorescence microscopy using specific monoclonal antibodies. T-helper to T-suppressor cell ratios were calculated on the preoperative day and the first postoperative day in all patients, and on the third or fourth postoperative day in five patients. Helper to suppressor ratios decreased in all patients on the first postoperative day (p >O.Ol), but returned to within normal limits on subsequent days. Changes were due more to decreases in helper cells than to increases in suppressor cells, although changes in both populations were statistically significant. The measurement of T-cell subsets by antibodyspecific labeling and immunofluorescence microscopy may prove to be a more sensitive, quantifiable, and reproducible assay of immune function in surgical or traumatized patients than use of stimulation assays. Measurements of specific helper and suppressor lymphocyte populations may prove useful in predicting morbidity and mortality, and may also help in studying the effect of immunomodulating agents on the immune response. References 1. Riddle PR, Berenbaum MC. Post-operative depression of the lymphocyte response to phytohaemagglutinin. Lancet 1967;1:746-6. 2. Park SK, Brody JI, Wallace HA, Blakemore WS. Immunosuppressive effect of surgery. Lancet 1971;1:53-5. 3. Lundy J, Ford CM. Surgery, trauma and immune suppression. Evolving the mechanism. Ann Surg 1963;197:434-8. 4. Editorial: Post-operative immunosuppression. LanCet 1974; 2:817-8. 5. Howard RJ, Simmons RL. Acquired immunologic deficiencies after trauma and surgical procedures. Surg Gynecot Obstet 1974;139:771-82. 6. Lundy J, Lovett EJ, Hamilton S, Conran P. Halothane, surgery, immunosuppression and pulmonary metasteses. Cancer 1978;41:827-30. 7. Lundy J, Lovetl EJ, Hamilton S, Conran P. Immune impairment and metastatic tumor growth. Cancer 1979;43:945-51. 8. Wang BS, Heacock EH, Mannick JA. Characterization of suppressor cells generated in mice after surgical trauma. Clin lmmunol lmmunopathol 1982;24:161-70. 9. Wang BS, Heacock EH, Wu AVO, Mannick JA. Generation of suppressor cells in mice after surgical trauma. J Clin Invest 1980;66:200-9. 10. Vose JM, Moudgil GC. Post-operative depression of antibody dependent lymphocyte cytotoxiclty following minor surgery and anaesthesia. Immunology 1976;30: 123-8. 11. Jubert AV, Lee ET, Hersh EM, McBride CM. Effects of surgery, anaesthesia and intraoperative blood loss on immunocompetence. J Surg Res 1973;15:399-403. 12. Espanol T, Todd GB, Soothill JF. The effect of anaesthesia on the lymphocyte response to phytohaemagglutinin. Clin Exp lmmunol 197418:73-Q.
306
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