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Effects of surgery and nutritional support on some lymphocyte and PMN leucocyte functions in man
CLINICAL NUTRITION
(1985) 4: 7 17 -72‘I
Effects of Surgery and Nutritional Support on Some Lymphocyte and PMN Leucocyte Functions in Man M.L.
Druart*, Y. A. Carpentier*, J. Duchateaut and F. Contrainez)
Department of Surgery* and Department of Immunology and Blood Transfusiont University of Brussels, 322 rue Haute, Brussels (Belgium) (reprint requests to YAC)
and Computer
Dept.+,
HBpltal Saint-Pierre.
Free
ABSTRACT
The purpose of the present study was to evaluate the effect on some leucocyte functions of 1) an elective surgical procedure; 2) nutritional repletion provided by parenteral alimentation (TPN). The rates of cellular proliferation and protein synthesis in lymphocyte cultures were measured by the incorporation of respectively 3H-thymidine and 3H-leucine; both measures were performed without and with additions of mitogenic agents. Random migration and chemotaxis of PMN leucocytes were measured under agarose. In 10 well-nourished patients, both lymphocyte proliferation and protein synthesis in stimulated cultures decreased after elective surgery, respectively by 50% (p < 0.01) and by 32% (p < 0.05) while random migration of PMN leucocytes was increased by 50% (p < 0.02). Stimulated lymphocyte proliferation and protein synthesis measured in IO nutritionally depleted non-cancer patients prior to TPN were lower in comparison to the values obtained in a control population (respectively p < 0.006 and p < 0.04). These parameters rose progressively during TPN and reached the normal range after 3 weeks. Before TPN, PMN leucocyte random migration was slower in depleted patients than in control subjects; this parameter reached normal values after one week of TPN, while chemotaxis tended to decrease. Both parameters were in the normal range after 3 weeks of TPN. Conclusions 1) an elective operation depresses lymphocyte functions but stimulates PMN leucocyte random migration in wellnourished patients; 2) in depleted patients, previously depressed leucocyte responses are restored within 3 weeks of adequate nutritional support.
INTRODUCTION
on various
leucocyte
functions.
Therefore,
lymphocyte
The interaction between malnutrition, infection and proliferation and protein synthesis as well ax PMN trauma is a common problem in surgical patients. leucocyte migration were measured before and after Increased susceptibility to bacterial [l], viral [2] and elective surgery in subjects with normal nutritional status. mycotic (31 infection as well as decreased skin test Similarly, these parameters were followed regularly before responses [4] have been reported after major surgery. and during IV nutritional support Similarly, prolonged survival of skin allografts (51 and non-cancer patients. xenografts [6] were observed in severely burned patients. Nutritional depletion, which is frequently encountered in surgical patients, is also known to impair the immune MATERIAL AND METHODS defences 171. In some of these patients, total parenteral
provided
to depleted
Selection of the patients
nutrition (TPN) is the only means for improving or restoring previously impaired nutritional status. Recent Elective surgery. The effect of surgery on some Immune studies have provided evidence that prolonged TPN could parameters was studied in 10 subjects (five males and five improve humoral and cellular immunity [8, 91. females). The mean age was 45 years. Nutritional status of The purpose of the present study was to separate the these subjects was normal, as indicated effect of a surgical injury from that of nutritional depletion concentration of various proteins (Table Table 1
Serum (% of Serum (% of Serum Serum
Nutritional
indices before and after surgery
prealbumin predictive value for age and sex) retinol binding protein predictive value for age and sex) alpha-I-glycoprotein”(mg/lOO ml) alpha-I-antitrypsin’(mgi100 ml)
in the 10 normal subjects’.
8ik
3
881t 635 -730 f
6 5 18
69k
6’
75k 5’ 6 98+ 298 + 15
a) Results are given as mean + SEM; b) Normal range: 44-l 16 mgiml: c) Normal range: 150-294 mgiml; d) p < 0.01 (significance of differences wth preoperarivr values, paired r-tcsrh) 317
by l),
serum delayed
218
I,YMPHOCY'I‘E AND
LwcocYx
FUNCTION
~ EFFE(~.S
hypersensitivity cutaneous (DHC) tests to four antigens (Table 2) and anthropometric measurements. Surgery was performed for gallstones (six cases) and uncomplicated duodenal ulcers (four cases) and consisted of respectively cholecystectomy and proximal vagotomy. Oral nutrition was re-initiated within 48 h of the operation. In patients with duodenal ulcer, cimetidine (1 g/day) was stopped at Table 2
Delayed skin tests before and after surgery in 10 normal subjects
Antigens
Before Surgery
After Surgery
P.P.D. Mumps Varidase P.H.A.
10 +
10 10 10 10
9 +; 1 9 +; 1 9 +; 1 -
+ + + +
least 4 days prior to surgery; IV cimetidine was administered in the postoperative period in only one patient. No patient was given steroid or other drug susceptible to modify the immune response. The postoperative course was uneventful in all these patients except for one who developed a wound infection on the seventh postoperative day. Table 3
Nutritional
0I: SI:K(;EKY
ANI) NU’I.KI’I.ION
Purenteral nutrition. The effect of TPN was studied in 10 nutritionally depleted non-cancer patients (six males and four females). The mean age was 52 years. Nutritional depletion was assessed on the basis of low serum protein levels (Table 3) and impaired DHC tests to four antigens (Table 4). The diagnoses were: inflammatory bowel disease (five cases), small bowel fistulae (three cases) and short bowel syndrome (two cases). None of these patients had evidence of altered hepatic or renal function, insulindependent diabetes and/or severe protein-losing enteropathy. Calorie intake was adjusted to provide 125% of the patient’s energy expenditure, the non-protein calorie intake being given in approximately equal parts by Intralipid@ 20% (Kabivitrum, Stockholm, Sweden) and glucose. Crystalline 1-aminoacid solution TravasoP 10% (Travenol, Lessines, Belgium) was given in order to provide 1 g nitrogen per 150 calories. Electrolytes, water- and fatsoluble vitamins, oligo- and trace elements were added according to the patient’s individual needs. Control groups. Two different groups of 10 normal individuals, each one perfectly matched for age and sex were studied in order to provide control values for both groups of patients.
indices before TPN and during TPN in 10 nutritionally depleted non cancer patients’ Durrng TPN
Before TPN Week 1 Serum prealbumin (% or predictive value for age and sex) Serum retinol binding protein (% of predictive value for age and sex) Serum alpha-1-glycoprotein’ (mg1100 ml) Serum-alpha-1-antitrypsin” (mg1100 ml) C-reactive prorein’ (mg/lOO ml)
42
f
9
64.5 +
51
f
8
68
+lO
88.8 f 14
86
*
378.8 f 23 9.9 * 3
Week 3
Week 2
7
69
5
294.6 t 26’ 5.3 * 0.7
f
8’
81.2 +
7”
77.5 +
9’
88.5 f
6’
73
6.4
64.4 f
6.3’
+
255 ? 20’ 3.1 * 0.1’
246.6 + 22’ 2.8 + 0.4’:
a) Results are given as mean + SEM; b) Normal range: loo-150 mg/lOO ml; c) Normal range: 44-l 16 mg/lOO ml; d) Normal range: 150-294 mg/lOO ml; e) C-reactive protein 1 mg/lOO ml; r) p < 0.05; g) p < 0.01 (significance of differences pre-nutrition values, paired t-tests) Table 4 patients
Delayed skm tests before and during
Antigens
Before TPN
P.P.D.
IO-
Mumps Varidase P.H.A.
lo8-; 8-;
Total of positive tests Patients with 1 positive test Patients with > 1 positive test
TPN in 10 nutritionally
2 -L 2 +
depleted non cancer
Durmg TPN Week I
Week 2
Week 3
lolo6-; 4 + B-; 2 +
6-; lo4-; 4-;
6-; 4 + IO4-; 6 + 4-;6 +
4 + 6 + 6 +
4
6
16
16
4
2
3
4
0
2
2
2
with
CLINICAI.
Parameters
of nutritional
assessment
Two visceral proteins were measured: serum thyroxinbinding prealbumin (TBPA) and retinol binding protein (RBP); since the normal level of both these proteins are age and sex related [lo], the values are expressed as per cent of predicted concentration, as previously reported [ 111. Malnutrition was defined empirically as 1) < 90% of ideal weight for sex and height - 2) visceral protein level < 60% of the predicted values for age and sex. Two different acute phase proteins [alpha-1-glycoprotein ((y-1 GP), alpha-1-antitrypsin ((y-1 AT)] were measured in both groups of patients. In addition, C-reactive protein (CRP) was followed in the group of depleted patients. These three acute phase proteins are currently measured in our patients receiving nutritional support in order to separate effective repletion from the resolution of inflammatory processes [ 111. Plasma triglyceride levels were enzymatically determined according to Bucolo and David [ 121. All these measurements were performed prior to surgery or starting TPN and again on the fourth postoperative day in the first group and every week during nutritional repletion. Immune
parameters
The immunological parameters were measured prior to surgery and on the 4th postoperative day in the first group of patients and prior to TPN and after 1, 2 and 3 weeks of nutritional support in the second group of patients. They consisted of: Skin testing. All patients were skin tested for delayed hypersensitivity responsiveness with three recall antigens: PPD (Statens Serum Institute, Denmark, 5TU10.1 cc); mumps skin test antigen (Eli Lilly Company, 0.1 cc); streptokinase-streptodornase (Varidase, Lederle, 1000 U/cc) and phytohaemagglutinin (PHA-P-Wellcome, 10 pg/ml) as a non-specific T cell stimulus that mimicks in viva antigen specific triggering of T lymphocytes [ 13- 151. Syringes were prepared with 0.1 ml of each solution just before intradermal injection. The same investigator was in charge of the administration and the reading after 24 and 48 h. Skin test was considered positive if the induration at either reading was 5 mm or greater [16]. In vitro lymphocyte proliferation and protein synthesis. Separation of lymphocytes was performed by centrifugation according to Boyum [ 171. Lymphoblastic transformation in leucocyte cultures was studied by measuring the incorporation over 3 days of &i methyltritiated-thymidine (3.6 Ci/mmol; Institut des RadioElements, Fleurus, Belgium) firstly in the absence of mitogen and secondly in the presence of 1 pglml of PHA (PH4-P-Wellcome) or 25 pglml of concanavalin A (Con A; Sigma) [ 181. Protein synthesis was studied by measuring the incorporation of tritiated leucine (5 PCi) in lymphocyte
NU’I‘RI-L‘ION
219
cultures with and without stimulation by the mitogenic agents. The mean value for triplicated determinations has been considered for each measurement. In vitro PMN leucocyte random migration ant shemotaxis. Minor modifications were applied to the method described by Nelson et al. [19] for measuring the random migration and chemotaxis of PMN leucocytes. One percent agarose gel coated Petri dishes containing 0.1% human serum albumin in minimal essential medium (MEM, Difco, Belgium) were used. PMN leucocytes were separated as the Buffv-coat after whole blood spontaneous sedimentation (during 1 h) in the presence of 5% (volume/volume) dextran - EDTA (10 mmol, final concentration). They were adjusted at 10.lOh/ml after three washes in 5% fetal calf serum supplemented with MEM 10 ~1 aliquots of PMN leucocytes were deposited into six agarose punched wells and the migration assessed after 4 h. Three different procedures were performed to study random migration and chemotaxis: Patient’s leucocyte migration (Lp) in the presence of patient’s serum (Sp), patient’s leucocyte migration (Lp) in the presence of normal serum (Sn), normal leucocyte (Ln) migration in the presence of patient’s serum (Sp) were evaluated and expressed as the percentage of control values obtained when control leucocytes were studied in the presence of control serum. The mean value of triplicated measurements was used for each procedure. Staristical
analysis
Statistical significance was computed unpaired Student’s test.
using
paired
and
RESULTS Effects
of elecrive surgerv
Pre- and postoperative values of serum protein concentrations are shown in Table 1. Elective surgery had a significant effect on serum proteins, the level of acute phase reactants being higher and the concentration of visceral protein being lower in the postoperative period. The results of skin tests were not affected by surgery, as shown in Table 2. Preoperative values of the various lymphocyte and PMN leucocyte functions were very similar to those measured in the control group. Lymphocyte proliferation in stimulated and non-stimulated cultures was significantly decreased after surgery (Fig. 1). Protein synthesis in non-stimulated lymphocyte cultures was not affected by elective surgery (Fig. 2). However protein synthesis in stimulated cultures was significantly reduced after surgery (Fig. 2). Random migration of PMN leucocytes tended to increase
220
LYMPHOCYTE
AND LEUCOCYI‘E
FUNCTION
-
EFFECTS
OI; SURGERY
AND NU’I‘RI’I‘ION
cpm xIO-~
cpm x to-3 TTL
TTL
TTL
SP
PHA
PHA
ConA
5
_!
Coil A 200
f
100 0 BEFORE (n=lO) values
AFTER (n 40)
CONTROL (n ~10)
+ SEM
Fig. 1 Effect of elective surgery (n= 10).
on lymphocyte
proliferation
after surgery (Fig. 3); however this change appeared to be statistically significant (p < 0.02) only when control cells were put in the presence of patient’s serum. Chemotaxis of PMN leucocytes was not significantly changed after surgery (Fig. 4). Effect
AFTER BEFORE (n--IO 1 (n=IO) values +_ SEM
of TPN on depleted patients
Before TPN, the plasma level of visceral proteins was below the normal range while the level of acute phase reactants was high. During TPN, the concentration of visceral proteins increased rapidly and markedly while the level of acute phase reactants decreased (Table 3). Plasma concentration of triglycerides was not modified by TPN. The values being 10.2 + 3.3; 12.1 f 2.4; 12.3 f 2.9; 12.7 + 3.4 mmol/l, respectively before and after 1, 2 and 3 weeks of TPN. As detailed in Table 4, these depleted patients showed relative or complete anergy to DHC tests. More positive tests were found during TPN. Lymphocyte proliferation in non-stimulated lymphocyte cultures was normal before TPN and remained unchanged during nutritional therapy. Lymphocyte proliferation in
Fig. 2
CONTROL (n-10)
Effect of surgery on protein synthesis by lymphoctyes.
stimulated lymphocyte cultures was lower in the group of depleted patients than in the control group (p < 0.006); this parameter rose towards the normal range after 2 weeks of TPN (Fig. 5). Protein synthesis in non-stimulated lymphocyte cultures was normal before starting TPN and was not affected by nutritional support. Protein synthesis in stimulated lymphocyte cultures of depleted patients was lower than in control cultures (p < 0.04). This parameter rose towards the normal range after 2 weeks of TPN (Fig. 6). The evolution of this parameter was parallel to the one observed for lymphocyte proliferation. Random migration of PMN leucocytes tended to be reduced in depleted patients (Fig. 7). However, the difference with normal values was significant only when patient’s cells were put in the presence of control serum. With the exception of the results obtained after 2 weeks of TPN, random migration tended to increase during TPN, so that no result remained significantly different from the control values. Chemotaxis of PMN leucocytes tended to be slower in depleted than in normal subjects (Fig. 8); however, the difference reached statistical significance only when patient’s cells were put in the presence of control serum. This difference was no longer observed after 3 weeks of TI’N.
(X.INI0.I.
DC
NI:‘I’RI~I‘ION
Control cells x control serum
lp
Patient cells x patient Serum
PxC Patient cells x control Serum
200
Cx P Control cells x patient Serum
I 5% - +I tg LT3 ;5 IO0 E? OE 00 20 25 s
0
P
c
BEFORE Fig. 3
Effect of surgery
on random migration
c
P PXC CXP AFTER (n=lO)
Control cells x control serum Patient cell5 x patient
UP
2 z
(n=to)
of PMN leucocytes.
UC Ol ’ 150
PXC CXP
q PxC
Patient cells x control serum
Cx P Control cells x patient 5erum i o6o8 _
c Fig. 4
Effect of surgery on chemotaxis
PXC CXP P BEFORE (n-10)
of PMN leucocytes
c
0.523 o,57,
P PXC CXP AFTER (n=lO)
221
LYMPHOCYTE
AND L.EUCOCY?‘E
FUNCI’ION
-
EFFEC’I‘S
OF SURGERY
cpm
AND NUI‘RI’I
PHA
xIO-~ normal
xl0
w cpm xIOe3
-3
-I
U
2
I-------------.------------------------normal
200
200
L -
Con A
TTL
( i4+2 6)
range
__ .
_ .
. _ .
.
. _
. .
normal
-1
_
_
00
0
0
Fig. 6
I TIME
2 (weeks)
( Ci + 2 5) _
_
IOO-
OLT
range
100
T _ _ _ _ _
Fig. 5
3
t
Z -
3
2
Con A
0 u
2
( G t26
0
5 E cpm
I
range
o,/
g a
0
ION
I 2 TIME (weeks)
3
Effect of TPN on stimulated protein synthesis (n= 10).
3
Effect of TPN on stimulated lymphocyte proliferation
(n= 10). DISCUSSION Surgery is known to alter some immune functions but this effect is rather limited where the postoperative course is uncomplicated and therefore bears little or no consequence [20, 211. The present study demonstrates decreased rates of cell proliferation in both non-stimulated and stimulated lymphocyte cultures during the postoperative course of subjects with normal nutritional status together with decreased protein synthesis in stimulated lymphocyte cultures. On the other hand, surgical trauma accelerates random migration of PMN leucocytes (by + 50%). This effect, reaching statistical significance only when control
cells are in the presence of patient’s serum, could be due at least partly to circulating factors such as C3l’A, Cls, Clq, C3, C4 and C5 as recently demonstrated [7]. Chemotaxis remains unchanged after surgery. The discrepancy between stimulated random migration and unchanged chemotaxis could result from a non-specific stimulation of PMN leucocytes by the inflammatory reaction that follows surgery. The severity of the inflammatory response is indicated by the increased plasma level of acute phase reactants. Nutritional support provided by TPN results in a quick increase in previously low visceral protein levels (TBPA, the RBP) as previously reported [ll]. However, concomitant decrease in the plasma level of acute phase reactants suggests that at least part of this response might be due to the resolution of inflammatory processes, and would not be specific for nutritional repletion. DHC tests revealed a high incidence of anergy in severely
CL.INICAI.
np
Patient cells x patlent serum
CxP Control BEFORE TPN (n-10) 4 f
,-’ _
cells x patient
serum AFTER TPN (n=lO)
4
0 P
Fig. 7
223
PxC Patient cells x control serum
RI
a
NLJ’L‘llITl~~N
P
PXC CXP
PXC CXP 1 week
P
PXC CXP 2 weeks
P PXC CXP 3 weeks
Effect of TPN on random migration of PMN leucocytes.
np I@
Patient cells x patient serum
Px C Patient cells x control serum CfP
Control cells x patient serum
BEFORE TPN (n=lO) ‘ l
S : +I
AFTER TPN (n=lO) t
f
in32 100 -,F p5 0:
50
5g $6 5
0 P
Fig. 8
PXC CXP
P
PXC CXP 1 week
P
PXC CXP 2 weeks
P
PXC CXP 3 weeks
Effect of TPN on chemotaxis of PMN leucocytes.
depleted non-cancer patients, even for varidase and PHA that both obviate the need for a previous sensitisation [ 13- 151. More positive tests were found during nutritional repletion. However, repeating these tests at an interval of 1 week can have a booster effect so that, their significance in relation to the nutritional status is strongly questioned [22-251. Nutritionally depleted patients exhibit decreased rates of lymphocyte proliferation in stimulated cultures but not in the non-stimulated ones, as previously reported [8]. Similarly, protein synthesis in stimulated lymphocyte cultures of depleted patients is lower than in those of normal subjects. Nutritional support provided by parenteral nutrition results in a progressive improvement of cell proliferation and protein synthesis in stimulated lymphocyte cultures. After 3 weeks of TPN, these parameters tend to or have reached the normal range. Both random migration and chemotaxis of PMN
leucocytes are to some extent, impaired in depleted patients; this effect seems mostly due to a cellular deficit. Both these parameters appear to be corrected after 3 weeks of TPN. The incorporation of exogenous fat in the TPN regimen of these depleted patients does not seem to have adversely affected the immunological parameters. A previous study has shown that in vim fat infusion and in virro addition of exogenous lipids has no effect on lymphocyte proliferation [26]. In another study, a direct relationship was found during fat infusion between impaired PMN leucocyte functions and increased plasma triglyceride concentration (271. In our patients, blood was drawn at least six hours after fat infusion was stopped and plasma triglyceride level was not significantly modified. In summary the present study shows that: 1) surgical injury depresses lymphocyte functions in normal subjects but stimulates random migration of PMN
224
LYMPHOCYTE
leucocytes specific 2)
concomitantly inflammatory
TPN
to
to
improves
leucocyte
and
regression
of non-specific
Whether proliferation
the
appearance
-
of
EFKX’I’S
a non-
response;
applied
progressively
FUNCI‘IOK
AND LEUCOCYl‘E
lymphocyte
depleted
previously functions
improvement
and
protein
synthesis
patients
depressed together
inflammatory
the
response tissues
nutritionally some
01:
in
PMN with
the
reactions. both of
stimulated
lymphocytes
in
SURGERY
AND
to nutritional
NU’I~RI~I’ION
therapy
can
be applied
to other
is still unknown.
ACKNOWLEDGEMENTS We thank Chantal Denys and Carol Destray for technical assistance, Mr. Francis Cantraine for assistance in statistical analyses and Carla Haelterman for preparation of the manuscript. This work was supported by the F.R.S.M. (grant 3.4555.81).
REFERENCES [II
PI (31
I41
[51
[61
[71
[81
F91
[lOI
[Ill
[I21
[I31
1141
Goodman J, Schaffner W, Collins H, et al 1968 Infection after cardiovascular surgery. New English Journal of Medicine 278: 117 Seaman A J, Stara A 1962 Febrile postcardiotomy lymphocyte splenomegaly. Annals of Surgery 156: 956 Smith D R 1964 A syndrome resembling infectious mononucleosis after open heart surgery. British Medical Journal 1: 945 l’ietsch J B, Meakins J L, MacLean L D 1977 The delayed hypersensitivity responses: application in clinical surgery. Surgery 82: 349-355 Kay G D 1957 Prolonged survival of a skin homograft in a patient with very extensive burns. Annals of the New York Academy of Sciences 64: 767 Polk H C Jr 1968 Prolongation of xenograft survival in patients with pseudomonas sepsis: a clarification. Surgical Forum 19: 514 Dudrick S J, Long J M, Steiger E, et al 1970 Intravenous hyperalimentation. Medical Clinics of North America 54: 577-591 Carpentier Y A, Druart M L, Delespesse G 1981 Influence of total parenteral nutrition (TPN) on some immune parameters in malnourished surgical patients. Acta Chirurgica Belgica 2-3: 149-155 Alexander J W, Dionigi R, Stinnet J D, et al 1978 Detection of defects in host defence mechanisms and their repair in the infection-prone patient. In: Burke J F (ed) The Infection-Prone Hospital Patients, Little Browns Co, Boston p 51 Ingenbleek Y, Van Den Schriek H G, Denayer I’, et al 1975 Albumin, transferrin and the thyroxin-binding prealbuminlretinol binding protein (TBPA-RBI’) complex in assessment of malnutrition. Clinica Chimica Acta 63: 61-67 Carpentier Y A, Barthel J, Bruyns J 1982 Plasma protein concentration in nutritional assessment. Proceedings of the Nutrition Society 41: 405-417 Bucolo G, David H 1973 Quantitative determination of serum triglycerides by the use of enzymes. Clinical Nutrition 19: 476-482 Blaese R M, Weiden I’, Oppenheim J J, et al 1973 l’hytohaemagglutinin as a skin test for the evaluation of cellular immune competence in man. Journal of Laboratory and Clinical Medicine 81: 538-548 Bonporte R J, Topilsky M, Stitbach L E, et al 1972 l’hytohaemagglutinin skin test: a possible in arno measure
Submission date 19 Dec. 84. Accepted after revision 28 May 85.
[I51
[I61
[I71
1181
[I91
[201
[211
[221
J231
[241
[251
[261
[271
of cell-mediated immunity. Journal of Pediatrics 8 1: 775-780 Lawlor G J, Stiehm E R, Kaplan M S, et al 1973 Phytohaemagglutinin skin test in the diagnosis of cellular immunodeficiency. Journal of Allergy and Clinical Immunology 52: 31-37 MacLean L D, Meakins J L, Taguchi K, et al 1975 Host resistance in sepsis and trauma. Annals of Surgery 182: 207-217 Boyum A 1968 Separation of leucocytes from blood and bone marrow. Scandinavian Journal and Clinical Laboratory Investigation 21 (suppl 97): l-7 Delespesse G, Duchateau J, Keenes B, et al 1974 Cellmediated immunity in diabetes mellitus. Clinical and Experimental Immunology 18: 461-467 Nelson R D, Quie P G, Simmons R L 1975 Chemotaxis under agarose: A new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leucocytes and monocytes. Journal of Immunology 115: 1650-1656 Cullen B F, Van Belle G 1975 Lymphocyte transformation and changes in leucocyte count: effect of anesthesia and operation. Anesthesiology 43: 563-569 Salo M S 1978 Efect of anesthesia and surgery on the number of and mitogen induced transformation of T and B lymphocytes. Annals of Clinical Research 10: 1-13 Miller C L 1978 Immunological assays as measurements of nutritional status: a review. Journal of I’arenteral and Enteral Nutrition 2: 554-566 Woods H F, Newton D J, Kay R, et al 1981 The nutritional assessment of hospital patients - a critical review. Acta Chirurgica Scandinavica suppl 507: 171-180 Elia M, Martin S, Price C, et al 1984 Effect of starvation and elective surgery on hand dynamometry and circulating concentration of various proteins. Clinical Nutrition 2: 173-179 Harries A D, Jones L A, Heatly R V, et al 1984 Precision of anthropometric measurements: the value of mid-arm circumference. Clinical Nutrition 2: 193-196 Carpentier Y A, Druart M L, Delespesse G 1981 Influence of TPN on some immune parameters in malnourished surgical patients. Acta Chirurgica Belgica 2-3: 149- 155 Nordenstrom J, Jarstrand C, Wiernik A 1979 Decreased chemotactic and random migration of leucocytes during Intralipid infusion. American Journal of Clinical Nutrition 32: 2416-2422
(1985) 4: 7 17 -72‘I
Effects of Surgery and Nutritional Support on Some Lymphocyte and PMN Leucocyte Functions in Man M.L.
Druart*, Y. A. Carpentier*, J. Duchateaut and F. Contrainez)
Department of Surgery* and Department of Immunology and Blood Transfusiont University of Brussels, 322 rue Haute, Brussels (Belgium) (reprint requests to YAC)
and Computer
Dept.+,
HBpltal Saint-Pierre.
Free
ABSTRACT
The purpose of the present study was to evaluate the effect on some leucocyte functions of 1) an elective surgical procedure; 2) nutritional repletion provided by parenteral alimentation (TPN). The rates of cellular proliferation and protein synthesis in lymphocyte cultures were measured by the incorporation of respectively 3H-thymidine and 3H-leucine; both measures were performed without and with additions of mitogenic agents. Random migration and chemotaxis of PMN leucocytes were measured under agarose. In 10 well-nourished patients, both lymphocyte proliferation and protein synthesis in stimulated cultures decreased after elective surgery, respectively by 50% (p < 0.01) and by 32% (p < 0.05) while random migration of PMN leucocytes was increased by 50% (p < 0.02). Stimulated lymphocyte proliferation and protein synthesis measured in IO nutritionally depleted non-cancer patients prior to TPN were lower in comparison to the values obtained in a control population (respectively p < 0.006 and p < 0.04). These parameters rose progressively during TPN and reached the normal range after 3 weeks. Before TPN, PMN leucocyte random migration was slower in depleted patients than in control subjects; this parameter reached normal values after one week of TPN, while chemotaxis tended to decrease. Both parameters were in the normal range after 3 weeks of TPN. Conclusions 1) an elective operation depresses lymphocyte functions but stimulates PMN leucocyte random migration in wellnourished patients; 2) in depleted patients, previously depressed leucocyte responses are restored within 3 weeks of adequate nutritional support.
INTRODUCTION
on various
leucocyte
functions.
Therefore,
lymphocyte
The interaction between malnutrition, infection and proliferation and protein synthesis as well ax PMN trauma is a common problem in surgical patients. leucocyte migration were measured before and after Increased susceptibility to bacterial [l], viral [2] and elective surgery in subjects with normal nutritional status. mycotic (31 infection as well as decreased skin test Similarly, these parameters were followed regularly before responses [4] have been reported after major surgery. and during IV nutritional support Similarly, prolonged survival of skin allografts (51 and non-cancer patients. xenografts [6] were observed in severely burned patients. Nutritional depletion, which is frequently encountered in surgical patients, is also known to impair the immune MATERIAL AND METHODS defences 171. In some of these patients, total parenteral
provided
to depleted
Selection of the patients
nutrition (TPN) is the only means for improving or restoring previously impaired nutritional status. Recent Elective surgery. The effect of surgery on some Immune studies have provided evidence that prolonged TPN could parameters was studied in 10 subjects (five males and five improve humoral and cellular immunity [8, 91. females). The mean age was 45 years. Nutritional status of The purpose of the present study was to separate the these subjects was normal, as indicated effect of a surgical injury from that of nutritional depletion concentration of various proteins (Table Table 1
Serum (% of Serum (% of Serum Serum
Nutritional
indices before and after surgery
prealbumin predictive value for age and sex) retinol binding protein predictive value for age and sex) alpha-I-glycoprotein”(mg/lOO ml) alpha-I-antitrypsin’(mgi100 ml)
in the 10 normal subjects’.
8ik
3
881t 635 -730 f
6 5 18
69k
6’
75k 5’ 6 98+ 298 + 15
a) Results are given as mean + SEM; b) Normal range: 44-l 16 mgiml: c) Normal range: 150-294 mgiml; d) p < 0.01 (significance of differences wth preoperarivr values, paired r-tcsrh) 317
by l),
serum delayed
218
I,YMPHOCY'I‘E AND
LwcocYx
FUNCTION
~ EFFE(~.S
hypersensitivity cutaneous (DHC) tests to four antigens (Table 2) and anthropometric measurements. Surgery was performed for gallstones (six cases) and uncomplicated duodenal ulcers (four cases) and consisted of respectively cholecystectomy and proximal vagotomy. Oral nutrition was re-initiated within 48 h of the operation. In patients with duodenal ulcer, cimetidine (1 g/day) was stopped at Table 2
Delayed skin tests before and after surgery in 10 normal subjects
Antigens
Before Surgery
After Surgery
P.P.D. Mumps Varidase P.H.A.
10 +
10 10 10 10
9 +; 1 9 +; 1 9 +; 1 -
+ + + +
least 4 days prior to surgery; IV cimetidine was administered in the postoperative period in only one patient. No patient was given steroid or other drug susceptible to modify the immune response. The postoperative course was uneventful in all these patients except for one who developed a wound infection on the seventh postoperative day. Table 3
Nutritional
0I: SI:K(;EKY
ANI) NU’I.KI’I.ION
Purenteral nutrition. The effect of TPN was studied in 10 nutritionally depleted non-cancer patients (six males and four females). The mean age was 52 years. Nutritional depletion was assessed on the basis of low serum protein levels (Table 3) and impaired DHC tests to four antigens (Table 4). The diagnoses were: inflammatory bowel disease (five cases), small bowel fistulae (three cases) and short bowel syndrome (two cases). None of these patients had evidence of altered hepatic or renal function, insulindependent diabetes and/or severe protein-losing enteropathy. Calorie intake was adjusted to provide 125% of the patient’s energy expenditure, the non-protein calorie intake being given in approximately equal parts by Intralipid@ 20% (Kabivitrum, Stockholm, Sweden) and glucose. Crystalline 1-aminoacid solution TravasoP 10% (Travenol, Lessines, Belgium) was given in order to provide 1 g nitrogen per 150 calories. Electrolytes, water- and fatsoluble vitamins, oligo- and trace elements were added according to the patient’s individual needs. Control groups. Two different groups of 10 normal individuals, each one perfectly matched for age and sex were studied in order to provide control values for both groups of patients.
indices before TPN and during TPN in 10 nutritionally depleted non cancer patients’ Durrng TPN
Before TPN Week 1 Serum prealbumin (% or predictive value for age and sex) Serum retinol binding protein (% of predictive value for age and sex) Serum alpha-1-glycoprotein’ (mg1100 ml) Serum-alpha-1-antitrypsin” (mg1100 ml) C-reactive prorein’ (mg/lOO ml)
42
f
9
64.5 +
51
f
8
68
+lO
88.8 f 14
86
*
378.8 f 23 9.9 * 3
Week 3
Week 2
7
69
5
294.6 t 26’ 5.3 * 0.7
f
8’
81.2 +
7”
77.5 +
9’
88.5 f
6’
73
6.4
64.4 f
6.3’
+
255 ? 20’ 3.1 * 0.1’
246.6 + 22’ 2.8 + 0.4’:
a) Results are given as mean + SEM; b) Normal range: loo-150 mg/lOO ml; c) Normal range: 44-l 16 mg/lOO ml; d) Normal range: 150-294 mg/lOO ml; e) C-reactive protein 1 mg/lOO ml; r) p < 0.05; g) p < 0.01 (significance of differences pre-nutrition values, paired t-tests) Table 4 patients
Delayed skm tests before and during
Antigens
Before TPN
P.P.D.
IO-
Mumps Varidase P.H.A.
lo8-; 8-;
Total of positive tests Patients with 1 positive test Patients with > 1 positive test
TPN in 10 nutritionally
2 -L 2 +
depleted non cancer
Durmg TPN Week I
Week 2
Week 3
lolo6-; 4 + B-; 2 +
6-; lo4-; 4-;
6-; 4 + IO4-; 6 + 4-;6 +
4 + 6 + 6 +
4
6
16
16
4
2
3
4
0
2
2
2
with
CLINICAI.
Parameters
of nutritional
assessment
Two visceral proteins were measured: serum thyroxinbinding prealbumin (TBPA) and retinol binding protein (RBP); since the normal level of both these proteins are age and sex related [lo], the values are expressed as per cent of predicted concentration, as previously reported [ 111. Malnutrition was defined empirically as 1) < 90% of ideal weight for sex and height - 2) visceral protein level < 60% of the predicted values for age and sex. Two different acute phase proteins [alpha-1-glycoprotein ((y-1 GP), alpha-1-antitrypsin ((y-1 AT)] were measured in both groups of patients. In addition, C-reactive protein (CRP) was followed in the group of depleted patients. These three acute phase proteins are currently measured in our patients receiving nutritional support in order to separate effective repletion from the resolution of inflammatory processes [ 111. Plasma triglyceride levels were enzymatically determined according to Bucolo and David [ 121. All these measurements were performed prior to surgery or starting TPN and again on the fourth postoperative day in the first group and every week during nutritional repletion. Immune
parameters
The immunological parameters were measured prior to surgery and on the 4th postoperative day in the first group of patients and prior to TPN and after 1, 2 and 3 weeks of nutritional support in the second group of patients. They consisted of: Skin testing. All patients were skin tested for delayed hypersensitivity responsiveness with three recall antigens: PPD (Statens Serum Institute, Denmark, 5TU10.1 cc); mumps skin test antigen (Eli Lilly Company, 0.1 cc); streptokinase-streptodornase (Varidase, Lederle, 1000 U/cc) and phytohaemagglutinin (PHA-P-Wellcome, 10 pg/ml) as a non-specific T cell stimulus that mimicks in viva antigen specific triggering of T lymphocytes [ 13- 151. Syringes were prepared with 0.1 ml of each solution just before intradermal injection. The same investigator was in charge of the administration and the reading after 24 and 48 h. Skin test was considered positive if the induration at either reading was 5 mm or greater [16]. In vitro lymphocyte proliferation and protein synthesis. Separation of lymphocytes was performed by centrifugation according to Boyum [ 171. Lymphoblastic transformation in leucocyte cultures was studied by measuring the incorporation over 3 days of &i methyltritiated-thymidine (3.6 Ci/mmol; Institut des RadioElements, Fleurus, Belgium) firstly in the absence of mitogen and secondly in the presence of 1 pglml of PHA (PH4-P-Wellcome) or 25 pglml of concanavalin A (Con A; Sigma) [ 181. Protein synthesis was studied by measuring the incorporation of tritiated leucine (5 PCi) in lymphocyte
NU’I‘RI-L‘ION
219
cultures with and without stimulation by the mitogenic agents. The mean value for triplicated determinations has been considered for each measurement. In vitro PMN leucocyte random migration ant shemotaxis. Minor modifications were applied to the method described by Nelson et al. [19] for measuring the random migration and chemotaxis of PMN leucocytes. One percent agarose gel coated Petri dishes containing 0.1% human serum albumin in minimal essential medium (MEM, Difco, Belgium) were used. PMN leucocytes were separated as the Buffv-coat after whole blood spontaneous sedimentation (during 1 h) in the presence of 5% (volume/volume) dextran - EDTA (10 mmol, final concentration). They were adjusted at 10.lOh/ml after three washes in 5% fetal calf serum supplemented with MEM 10 ~1 aliquots of PMN leucocytes were deposited into six agarose punched wells and the migration assessed after 4 h. Three different procedures were performed to study random migration and chemotaxis: Patient’s leucocyte migration (Lp) in the presence of patient’s serum (Sp), patient’s leucocyte migration (Lp) in the presence of normal serum (Sn), normal leucocyte (Ln) migration in the presence of patient’s serum (Sp) were evaluated and expressed as the percentage of control values obtained when control leucocytes were studied in the presence of control serum. The mean value of triplicated measurements was used for each procedure. Staristical
analysis
Statistical significance was computed unpaired Student’s test.
using
paired
and
RESULTS Effects
of elecrive surgerv
Pre- and postoperative values of serum protein concentrations are shown in Table 1. Elective surgery had a significant effect on serum proteins, the level of acute phase reactants being higher and the concentration of visceral protein being lower in the postoperative period. The results of skin tests were not affected by surgery, as shown in Table 2. Preoperative values of the various lymphocyte and PMN leucocyte functions were very similar to those measured in the control group. Lymphocyte proliferation in stimulated and non-stimulated cultures was significantly decreased after surgery (Fig. 1). Protein synthesis in non-stimulated lymphocyte cultures was not affected by elective surgery (Fig. 2). However protein synthesis in stimulated cultures was significantly reduced after surgery (Fig. 2). Random migration of PMN leucocytes tended to increase
220
LYMPHOCYTE
AND LEUCOCYI‘E
FUNCTION
-
EFFECTS
OI; SURGERY
AND NU’I‘RI’I‘ION
cpm xIO-~
cpm x to-3 TTL
TTL
TTL
SP
PHA
PHA
ConA
5
_!
Coil A 200
f
100 0 BEFORE (n=lO) values
AFTER (n 40)
CONTROL (n ~10)
+ SEM
Fig. 1 Effect of elective surgery (n= 10).
on lymphocyte
proliferation
after surgery (Fig. 3); however this change appeared to be statistically significant (p < 0.02) only when control cells were put in the presence of patient’s serum. Chemotaxis of PMN leucocytes was not significantly changed after surgery (Fig. 4). Effect
AFTER BEFORE (n--IO 1 (n=IO) values +_ SEM
of TPN on depleted patients
Before TPN, the plasma level of visceral proteins was below the normal range while the level of acute phase reactants was high. During TPN, the concentration of visceral proteins increased rapidly and markedly while the level of acute phase reactants decreased (Table 3). Plasma concentration of triglycerides was not modified by TPN. The values being 10.2 + 3.3; 12.1 f 2.4; 12.3 f 2.9; 12.7 + 3.4 mmol/l, respectively before and after 1, 2 and 3 weeks of TPN. As detailed in Table 4, these depleted patients showed relative or complete anergy to DHC tests. More positive tests were found during TPN. Lymphocyte proliferation in non-stimulated lymphocyte cultures was normal before TPN and remained unchanged during nutritional therapy. Lymphocyte proliferation in
Fig. 2
CONTROL (n-10)
Effect of surgery on protein synthesis by lymphoctyes.
stimulated lymphocyte cultures was lower in the group of depleted patients than in the control group (p < 0.006); this parameter rose towards the normal range after 2 weeks of TPN (Fig. 5). Protein synthesis in non-stimulated lymphocyte cultures was normal before starting TPN and was not affected by nutritional support. Protein synthesis in stimulated lymphocyte cultures of depleted patients was lower than in control cultures (p < 0.04). This parameter rose towards the normal range after 2 weeks of TPN (Fig. 6). The evolution of this parameter was parallel to the one observed for lymphocyte proliferation. Random migration of PMN leucocytes tended to be reduced in depleted patients (Fig. 7). However, the difference with normal values was significant only when patient’s cells were put in the presence of control serum. With the exception of the results obtained after 2 weeks of TPN, random migration tended to increase during TPN, so that no result remained significantly different from the control values. Chemotaxis of PMN leucocytes tended to be slower in depleted than in normal subjects (Fig. 8); however, the difference reached statistical significance only when patient’s cells were put in the presence of control serum. This difference was no longer observed after 3 weeks of TI’N.
(X.INI0.I.
DC
NI:‘I’RI~I‘ION
Control cells x control serum
lp
Patient cells x patient Serum
PxC Patient cells x control Serum
200
Cx P Control cells x patient Serum
I 5% - +I tg LT3 ;5 IO0 E? OE 00 20 25 s
0
P
c
BEFORE Fig. 3
Effect of surgery
on random migration
c
P PXC CXP AFTER (n=lO)
Control cells x control serum Patient cell5 x patient
UP
2 z
(n=to)
of PMN leucocytes.
UC Ol ’ 150
PXC CXP
q PxC
Patient cells x control serum
Cx P Control cells x patient 5erum i o6o8 _
c Fig. 4
Effect of surgery on chemotaxis
PXC CXP P BEFORE (n-10)
of PMN leucocytes
c
0.523 o,57,
P PXC CXP AFTER (n=lO)
221
LYMPHOCYTE
AND L.EUCOCY?‘E
FUNCI’ION
-
EFFEC’I‘S
OF SURGERY
cpm
AND NUI‘RI’I
PHA
xIO-~ normal
xl0
w cpm xIOe3
-3
-I
U
2
I-------------.------------------------normal
200
200
L -
Con A
TTL
( i4+2 6)
range
__ .
_ .
. _ .
.
. _
. .
normal
-1
_
_
00
0
0
Fig. 6
I TIME
2 (weeks)
( Ci + 2 5) _
_
IOO-
OLT
range
100
T _ _ _ _ _
Fig. 5
3
t
Z -
3
2
Con A
0 u
2
( G t26
0
5 E cpm
I
range
o,/
g a
0
ION
I 2 TIME (weeks)
3
Effect of TPN on stimulated protein synthesis (n= 10).
3
Effect of TPN on stimulated lymphocyte proliferation
(n= 10). DISCUSSION Surgery is known to alter some immune functions but this effect is rather limited where the postoperative course is uncomplicated and therefore bears little or no consequence [20, 211. The present study demonstrates decreased rates of cell proliferation in both non-stimulated and stimulated lymphocyte cultures during the postoperative course of subjects with normal nutritional status together with decreased protein synthesis in stimulated lymphocyte cultures. On the other hand, surgical trauma accelerates random migration of PMN leucocytes (by + 50%). This effect, reaching statistical significance only when control
cells are in the presence of patient’s serum, could be due at least partly to circulating factors such as C3l’A, Cls, Clq, C3, C4 and C5 as recently demonstrated [7]. Chemotaxis remains unchanged after surgery. The discrepancy between stimulated random migration and unchanged chemotaxis could result from a non-specific stimulation of PMN leucocytes by the inflammatory reaction that follows surgery. The severity of the inflammatory response is indicated by the increased plasma level of acute phase reactants. Nutritional support provided by TPN results in a quick increase in previously low visceral protein levels (TBPA, the RBP) as previously reported [ll]. However, concomitant decrease in the plasma level of acute phase reactants suggests that at least part of this response might be due to the resolution of inflammatory processes, and would not be specific for nutritional repletion. DHC tests revealed a high incidence of anergy in severely
CL.INICAI.
np
Patient cells x patlent serum
CxP Control BEFORE TPN (n-10) 4 f
,-’ _
cells x patient
serum AFTER TPN (n=lO)
4
0 P
Fig. 7
223
PxC Patient cells x control serum
RI
a
NLJ’L‘llITl~~N
P
PXC CXP
PXC CXP 1 week
P
PXC CXP 2 weeks
P PXC CXP 3 weeks
Effect of TPN on random migration of PMN leucocytes.
np I@
Patient cells x patient serum
Px C Patient cells x control serum CfP
Control cells x patient serum
BEFORE TPN (n=lO) ‘ l
S : +I
AFTER TPN (n=lO) t
f
in32 100 -,F p5 0:
50
5g $6 5
0 P
Fig. 8
PXC CXP
P
PXC CXP 1 week
P
PXC CXP 2 weeks
P
PXC CXP 3 weeks
Effect of TPN on chemotaxis of PMN leucocytes.
depleted non-cancer patients, even for varidase and PHA that both obviate the need for a previous sensitisation [ 13- 151. More positive tests were found during nutritional repletion. However, repeating these tests at an interval of 1 week can have a booster effect so that, their significance in relation to the nutritional status is strongly questioned [22-251. Nutritionally depleted patients exhibit decreased rates of lymphocyte proliferation in stimulated cultures but not in the non-stimulated ones, as previously reported [8]. Similarly, protein synthesis in stimulated lymphocyte cultures of depleted patients is lower than in those of normal subjects. Nutritional support provided by parenteral nutrition results in a progressive improvement of cell proliferation and protein synthesis in stimulated lymphocyte cultures. After 3 weeks of TPN, these parameters tend to or have reached the normal range. Both random migration and chemotaxis of PMN
leucocytes are to some extent, impaired in depleted patients; this effect seems mostly due to a cellular deficit. Both these parameters appear to be corrected after 3 weeks of TPN. The incorporation of exogenous fat in the TPN regimen of these depleted patients does not seem to have adversely affected the immunological parameters. A previous study has shown that in vim fat infusion and in virro addition of exogenous lipids has no effect on lymphocyte proliferation [26]. In another study, a direct relationship was found during fat infusion between impaired PMN leucocyte functions and increased plasma triglyceride concentration (271. In our patients, blood was drawn at least six hours after fat infusion was stopped and plasma triglyceride level was not significantly modified. In summary the present study shows that: 1) surgical injury depresses lymphocyte functions in normal subjects but stimulates random migration of PMN
224
LYMPHOCYTE
leucocytes specific 2)
concomitantly inflammatory
TPN
to
to
improves
leucocyte
and
regression
of non-specific
Whether proliferation
the
appearance
-
of
EFKX’I’S
a non-
response;
applied
progressively
FUNCI‘IOK
AND LEUCOCYl‘E
lymphocyte
depleted
previously functions
improvement
and
protein
synthesis
patients
depressed together
inflammatory
the
response tissues
nutritionally some
01:
in
PMN with
the
reactions. both of
stimulated
lymphocytes
in
SURGERY
AND
to nutritional
NU’I~RI~I’ION
therapy
can
be applied
to other
is still unknown.
ACKNOWLEDGEMENTS We thank Chantal Denys and Carol Destray for technical assistance, Mr. Francis Cantraine for assistance in statistical analyses and Carla Haelterman for preparation of the manuscript. This work was supported by the F.R.S.M. (grant 3.4555.81).
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I41
[51
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F91
[lOI
[Ill
[I21
[I31
1141
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Submission date 19 Dec. 84. Accepted after revision 28 May 85.
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