The Japanese Journal of Surgery (1992) 22:233-243
© Springer-Verlag 1992
Polymorphonuclear Leukocyte Function and Serum Opsonic Activity in Surgical Patients TOSHINAOINOUE, MITSURUOBATA, and YosHIo M~SmMA The SecondDepartment of Surgery, TokyoMedicaland Dental University,Tokyo,Japan
Abstract: Thirty-six patients who underwent major surgery were studied in order to clarify the perioperative changes in polymorpho-nuclear leukocyte (PMNL) function and serum opsonic activity. In patients without postoperative infection, the PMNL phagocytic-bactericidal capacity and plasma elastase levels significantly increased, while the serum opsonic index remarkably decreased just after surgery, however, all returned to the preoperative levels within 1 or 2 weeks. Conversely, in patients with postoperative infection, the PMNL bactericidal capacity and plasma elastase levels remained at high levels even after 1 or 2 weeks, while the PMNL phagocytic capacity and serum opsonic index substantially decreased after 2 weeks compared with the patients without postoperative infection. Plasma leukotriene B4, which is a potent chemo-attractant for PMNL, noticeably decreased in the patients with postoperative infection on the first postoperative day compared with that in the patients without postoperative infection. Our data suggests that the most important predisposing factors to postoperative infection may be a depressed PMNL phagocytic capacity and a lower serum opsonic activity after surgery, and that the increased PMNL bactericidal capacity and high plasma elastase levels during postoperative infection may contribute to the susceptibility to multiple organ failure. Words: polymorphonuclear leukocyte, phagocytic capacity, bactericidal capacity, opsonic index, elastase
in postoperative patients, often despite appropriate antibiotic therapy and adequate drainage. 1-4 Depression of the host defense mechanism, which is caused by humoral factor depletion, reticuloendothelial depression and the alteration of polymorphonuclear leukocyte (PMNL) function during infection, is a very important predisposing factor to bacterial infection, resulting in MOF. 5-7 PMNL may damage not only bacteria but also tissue by releasing lysosomal enzymes and oxygen free radicals. 7'8 Moreover, leukotriene B 4 (LTB4) is a potent chemo-attractant for PMNL and impairment of the leukotriene generation is thought to increase the risk of bacterial infection. 7-9 In the present study, we evaluated the perioperative alteration in PMNL function and serum opsonic activity in surgical patients, focusing on the PMNL phagocytic and bactericidal capacity, plasma PMNk elastase, plasma LTB4 and serum opsonic index. This was done to provide a control for the influence of the operation in the absence of postoperative infection. It was also undertaken to evaluate whether these measurements could be of value in either helping to establish an early diagnosis of postoperative infection and MOF or in predicting the prognosis.
Materials and Methods Introduction
Multiple organ failure (MOF) remains the most common cause of death after major operative procedures. Infection and its systemic manifestations are the most important causative factors in the evolution of MOF
A total 36 patients, comprised for 17 men and 19 women with a mean age of 54 years ranging from 34-78 years, who underwent major surgery at our surgical department during the period from April, 1989 through March, 1990, and ten healthy volunteers, comprised of 6 men and 4 women, with a mean age of 3'7 years ranging from 27-62 years, were included in this study. Of these patients, 22 had no preoperative complications apart from their primary disease, 5 had liver
Reprint requests to: Toshinao Inoue, MD, The Second Department of Surgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan (Received for publication on Dec. 15, 1990)
234 cirrhosis (Child's classification "B"), 4 far-advanced cancer and 5 a history of cancer chemotherapy, respectively. The 22 patients without preoperative complications were divided into two groups, namely, infected and noninfected, based upon the presence or absence of postoperative infection. Of these 22 patients, 6 developed such postoperative infections as peritonitis, intra-abdominal abscess, biliary tract infection and sepsis within 2 weeks of surgery. The remaining 16 noninfected patients served as controls to determine the influence of the operation. Most of these patients had undergone abdominal surgery for malignancies. All patients were uniformly anesthetized with endotracheal intubation and maintained with inhalation agents, narcosis and pancronium. The PMNL phagocytic capacity, PMNL bactericidal capacity, plasma PMNL elastase level and plasma LTB4 level were determined as PMNL function, and the serum opsonic index, plasma ,fibronectin level and serum complements (C3, C4, CH50) levels as the opsonic activity, in all patients preoperatively and on the 1st, 3rd, 7th and 14th postoperative days, respectively. Identical tests were performed on the 10 healthy volunteers to provide normal values.
T. Inoue et al.: PMNL Function and Serum Opsonic Activity Dulbecco's PBS containing 5 mM glucose and 0.1 per cent gelatin, and 10 ~tl of 25 ~tg/ml phorbol myristate acetate (PMA) (Sigma Chemical, USA), the sample was incubated at 37°C for 20 min. After adding 3 ml of 0.87 per cent NHaC1 lysing reagent, centrifugal isolation Was performed. The sediment to which 2ml of 3 mM EDTA-eontaining PBS was added, was measured using flow cytometry. The results were expressed as the number of fluorescent PMNL after PMA stimulation divided by the total PMNL count and multiplied by 100.
Plasma PMNL Elastase PMNL (Granulocyte) elastase was determined in plasma in complex with the ~l-proteinase inhibitor using.~an ELISA kit that is both specific for human ~lproteinase inhibitor and granulocyte elastase (Merck, FRG).
Plasma L TB4 Plasma L T B 4 concentrations were measured with a double radioimmunoassay using commercially available ~ntibodies as described by Kurimoto.14
PMNL Phagocytic Capacity Opsonic Index
The phagocytic capacity of PMNL was assayed with a flow cytometric method using fluorescent microspheres as described by Dunn and others. 1°-12 2.5 × 10 7 fluorescent monodispherse carboxylated microspheres with a DIA of 1.78-2.13~tm in a 2.5 per cent solution (Polyscience, USA) were added to 100~tl of heparinized blood drawn by venipuncture from the patients and incubated at 37°C for 30 min. After adding 3 ml of lysing reagent, being NH4CI 8.26 g + KHCO3 1.0 g + EDTA4Na 37 mg in 1 liter of d.w., centrifugal isolation was performed at 4°C at 1500 rpm for 5 rain. The sediment with the addition of 2 ml of 3 mM EDTAcontaining PBS was measured using flow cytometry. Results were expressed as the number of fluorescent PMNL accompanied with microspheres divided by the total PMNL count and multiplied by 100.
The serum opsonic index was determined by a chemiluminescence assay. For PMNL separation, red bloodcells in heparinized whole blood were hemolyzed with cold ammonium chloride (8 mg/ml), then washed twice in PBS and suspended in PBS to a cell concentration of 3 × 106/ml. Two hundred ~tl of 10-4M Luminol, 100 ~tl of 20 per cent serum samples drawn from the patients, and 200 ~tl of live 109CFU/ml E. coli were added to 500 ~tl of PMNL suspension. Luminoldependent chemiluminescence was determined in 500,000 PMNL using a luminescencereader (BLR-201, Aloka, Japan). Counts were recorded every 3 min for 90 min. The opsonic index was expressed as the ratio of the peak value of each patient's serum to the peak value of the healthy volunteers' serum.
PMNL Bactericidal Capacity
For PMNL bactericidal capacity, the superoxideproducing capacity of PMNL was measured by flow cytometry according to Bass et a1.,13 with some modifications. Two ml of 5 ~tM dichlorofluorescin diacetate (DCFH-DA) (Eastman-Kodak, USA) was added to 100~tl of heparinized blood drawn by venipuncture from the patients and incubated at 37°C for 15 min. After adding 0.5ml of 25mM EDTA-containing PBS-g, comprised of calcium- and magnesium-free
Plasma fibronectin concentrations were assayed with an immunoturbidimetric method using commercially available antisera (Behringwerke, Germany).
Serum Complement The total serum complement activity was determined with the modified method of Mayer 15 and expressed in 50 per cent hemolytic units (CHs0). Concentrations of
T. Inoue et al.: PMNL Function and Serum Opsonic Activity
Table 1. Preoperative PMNL functions in surgical patients Phagocytic Patients Capacity (%) No complications before surgery Postoperatively non-infected patients (n = 16) Postoperatively infected patients (n = 6) Liver cirrhosis (n = 5) Far-advanced cancer (n = 4) Cancer chemotherapy (n = 5) Healthy volunteers (n = 10)
*75.6 + 8.2 "71.6 **54.3 75.4 79.0 (81.8
+ 9.0 _+ 15.0 _+ 8.2 _+ 10.4 -+ 5.4)
Bactericidal Capacity (%) *'86.1 _+ 8.9 *88.6 -+ 8.5 **83.8 _+ 11.8 **76.8+ 18.3 "91.4 _+ 3.4 (94.7 + 1 . 3 )
156.8 _+ 81.4
**222.5_+ 65.5 210.0 + 148.8 **374.3+ 146.0 "199.2 -+ 110.2 (111.8+ 40.1)
"269.1-+ 29.3 "271.0 + 102.8 235.3 + 96.2 *285.3+ 49.4 (172.9 -+ 76.7)
* P < 0.05vs Healthy volunteers; ** P < 0.0l vs healthy volunteers Values are mean + SD
C3 and C4 in the serum samples were measured by a single radial immunodiffusion assay using commercially available antisera (Behringwerke, Germany).
values were 86.1 _+ 8.9 per cent in the postoperatively noninfected patients, and 88.6 _+ 8.5 per cent in the postoperatively infected patients. The mean bactericidal capacity was 94.7 _+ 1.3 per cent in the healthy volunteers. The preoperative mean bactericidal capacity in the surgical patients was substantially reduced compared with that of the healthy volunteers (P < 0.05 or P < 0.01). No significant difference was demonstrated between the postoperatively infected and noninfected patients. On the other hand, the mean preoperative plasma PMNL elastase and plasma LTB4 levels in most surgical patients were greater than those of the healthy volunteers. Regarding plasma elastase levels, there was a significant difference between the postoperatively infected and noninfected patients.
Preoperative PMNL Function in the Surgical Patients
Preoperative Opsonic Activity in the Surgical Patients
The PMNL phagocytic capacity measured as the percentage of PMNL with an uptake of microspheres was studied in all 36 patients. The preoperative value in the patients without preoperative complications was 75.6 _+ 8.2 per cent in the postoperatively noninfected patients and 71.6 + 9.0 per cent in the postoperatively infected patients, whereas it was 54.3 _+ 15.0 per cent in the patients with liver cirrhosis. The mean phagocytic capacity was 81.8 + 5.4 per cent in the healthy volunteers. The mean phagocytic capacity in the preoperative patients, especially in those with liver cirrhosis, was remarkably depressed compared with that of the healthy volunteers (P < 0.05 or P < 0.01). There was no demonstrative difference in preoperative phagocytic capacity between the postoperatively noninfected and infected patients. The PMNL bactericidal capacity was measured as the percentage of superoxide producing PMNLs in the PMA-stimulated PMNLs, the metabolic burst of which leads to a potent bactericidal system for ingested bacteria and other microorganisms. The preoperative
The mean preoperative opsonic indices were 0.93 + 0.15 in the postoperatively noninfected patients, 0.89 _+ 0.08 in the postoperatively infected patients, and 0.65 + 0.19 in the patients with liver cirrhosis. The mean preoperative opsonic index in the patients with liver cirrhosis was noticeably lower than that of the patients with no preoperative complications (P < 0.05). Moreover, the mean C3, C4 and CHs0 levels in the patients with liver cirrhosis were remarkably lower compared with those of the healthy volunteers (P < 0.05). There were no significant differences in fibronectin levels between the surgical patient groups.
Statistical Analysis Data are expressed as the mean _+ standard deviation in tables and figures. The comparison of mean values was done with the paired and unpaired Student's t-tests. Correlation coefficients were determined by linear regression and the significance of the regression analysis estimated using a t-test. Significance was accepted if the probability(P) was less than 0.05.
Influence of the Operation on PMNL Function in the Postoperatively Noninfected Patients The time course of PMNL function in the postoperatively noninfected patients is shown in Fig. 1. Thirteen complete sets of data were available. The mean PMNL phagocytic capacity was remarkably increased on the first and third postoperative days compared with before
T. Inoue et al.: PMNL Function and Serum Opsonic Activity
Table 2, Preoperative opsonic activity in surgical patients Patients Opsonic index C3 (mg/dl) No complications before surgery Postoperatively non-infected patients (n = 16) Postoperatively infected patients (n = 6) Liver cirrhosis (n = 5) Far-advanced cancer (n = 4) Cancer chemotherapy (n = 5) Healthy volunteers (n = 10)
0.93 + 0.15
65.8 + 19.0
25.0 + 6.0
31.8 + 7.9
29.6 + 5.3
0.89 + 0.08
75.5 + 7.5
25.0 + 1.1
39.4 +_ 7.4
31.2 + 7.5
0.65 + 0.19 0.85 + 0.12 0.88 + 0.06 (1.00)
**42.5 74.3 63.4 (76.3
+ 22.1 + 16.9 -+ 4.7 + 18.5)
"16.5 25.8 28.3 (28.0
+ + + +
6.1 10.1 3.3 8.0)
*'12.3 32.1 34.9 (35.6
_+ 3.9 + 8.6 -+ 2.8 -+ 7.6)
25.3 + 5.4 29.1 + 14.9 30.6 + 7.6 (31.4 _+ 7.4)
*P < 0.05 vs Healthy volunteers; **P < 0.01 vs healthy volunteers Values are mean _+ SD Phagocytic capacity
80. 70. 60' 50,
PbDl#OD3 PdDl4 Preo~.
Fig. l a - d . Influence of the operation on PMNL function in the postoperatively noninfected patients (n = 13) a Phagocytic capacity, b bactericidal capacity, c plasma elastase, and d plasma LTB4. *P < 0.05 Difference from the preoperative levels. **P < 0.01 Difference from the preoperative levels. Preop., Preoperative; POD, Postoperative day
Opsonic index 1,]'
1.0. 0,9. 0.8-
70 4030 (
Preo;. mbD1 P6D3
, C3 **
Fig. 2a,b. Influence of the operation on opsonic activity in the postoperatively non-infected patients (n = 13) a Opsonic index, b C3, C4 and CH50. *P < 0.05 Difference from the
preoperative levels. *~P < 0.01 Difference from the preoperative levels. Preop., Preoperafiv6; POD, Postoperative day
T. Inoue et al. : PMNL Function and Serum Opsonic Activity Fibronectin (m~/dl) 40- ~"
,,~ .0""~~"" "~m~~
operative days in the noninfected patients compared with before surgery. Regarding plasma fibronectin, the mean level after surgery showed a similar pattern to that of the opsonic index and complements, with an initial depression followed by an increase, reaching a plateau on the 7th postoperative day, and returning to the preoperative level on the 14th postoperative day (Fig. 3).
Time Course of PMNL Functions in the Postoperatively Infected Patients Compared with the Noninfected Patients
Preol~e.P6 D1 P6D3
Fig. 3. Time course of the mean plasma fibronectin level in the postoperatively infected patients (e e, n = 6) compared with the noninfected patients (o . . . . o, n = 13). ~P < 0.01 Difference from the preoperative levels. *P < 0.01 Difference between groups. Preop., Preoperative; POD, postoperative day
surgery (p < 0.05). However, it returned to the preoperative levels on the 7th and 14th days. A transient increment in the rate of phagocytic capacity after surgery was demonstrated in the postoperatively noninfected patients. The mean PMNL bactericidal capacity was noticeably higher than the preoperative levels on the first postoperative day (p < 0.05), however, it returned to the preoperative levels over the ensuing week. The mean PMNL elastase level was remarkably increased on the first postoperative day compared with before surgery (p < 0.01), but returned to the preoperative level on the 7th postoperative day. The mean plasma LTB 4 concentration was substan~!ally increased on the 7th postoperative day compared with before surgery (p < 0.01), but returned to the preoperative level on the 14th postoperative day.
Influence of the Operation on Opsonic Activity in the Postoperatively Noninfected Patients The time course of opsonic activity in the postoperatively noninfected patients is shown in Fig. 2. Thirteen complete sets of data were available. The mean serum opsonic index was remarkably decreased on the first postoperative day, then became slightly increased on the 3rd and 7th postoperative days, and returned to the preoperative levels on the 14th postoperative day (Fig. 2). The mean complement levels increased after an initial slight depression on the 3rd and/or 7th post-
In the postoperatively infected patients, there was a marked decrease in PMNL phagocytic capacity on the 14th postoperative day compared with that of the noninfected patients (p < 0.05). On the other hand, the mean PMNL bactericidal capacity noticeably increased on the 3rd and 7th postoperative days compared with that of the noninfected patients (p < 0.05). However, no significant difference was demonstrated on the 14th postoperative day. The mean plasma PMNL elastase level was substantially increased after the 3rd postoperative day compared with that of the noninfected patients (p < 0.01), while the mean plasma LTB4 levels were remarkably decreased on the first postoperative day compared with that of the noninfected patients (p < 0.05) (Fig. 4).
Time Course of Opsonic Activity in the Postoperatively Infected Patients Compared with the Noninfected Patients In the postoperatively infected patients, the mean opsonic index was noticeably depressed on the 14th postoperative day compared with that of the noninfected patients (p < 0.01), and the mean C3 and CHs0 levels were remarkably decreased on the 1st (p < 0.05) and 3rd postoperative days in the former (p < 0.05), and on the 1st postoperative day in the latter (p < 0.01), respectively, compared with those of the noninfected patients (Fig. 5). Regarding plasma fibronectin levels, there was a marked depression which did not return to the preoperative level on the 14th postoperative day (Fig. 3).
PMNL Function in the Postoperatively Infected Patients (Table 3) The mean PMNL phagocytic capacity in the postoperatively infected patients was 71.6 + 9.0 per cent before surgery, 90.8 + 3.3 per cent at the onset of infection and 65.0 + 9.3 per cent at a later stage of infection, respectively. There was a significant increase in the PMNL phagocytic capacity at the onset of infec-
T. Inoue et al.: PMNL Function and Serum Opsonic Activity
Phagocytic ca >acity
80. 70. g0.
N0 100 0
LTB, (pg/ml) Bactericidal capacity
~eo~.F;ODI~OD) POb7 d
Fig. 4a-d. Time course of the PMNL function in the postoperatively infected patients (o *, n = 6) compared with the noninfected patients (o . . . . o, n = 13). a Phagocytic capacity, b bactericidal capacity, c plasma elastase, and d plasma LTB4. *P < 0.05 Difference between groups. **P < 0.01 Difference between groups. Preop., Preoperative; POD, Postoperative day
0.8 0.7, 0.6 0T Preol~, PbDIF~OD3 Pob7
60" ~0' ~ -----~ ~
Preop;. F;ODI #OD3
Preo~.P'ODIl~OO3 PdD7 C OHio
(mgldl) 100" 80-
Fig. 5a-d. Time course of opsonic activity in the postoperatively infected patients (o o, n = 6) compared with the noninfected patients (o . . . . o, n = 13). A Opsonic index, B C3, C C4 and, D CHs0 (D). *P < 0.05 Difference between groups. **P < 0.01 Difference between groups. Preop., Preoperative; POD, Postoperative day
T. Inoue et al.: PMNL Function and Serum Opsonic Activity
Table 3. PMNL functions in the patients with postoperative infection Before surgery At the onset At a later stage (n=6) ( n = 6) (n=5) Phagocytic capacity (%) Bactericidal capacity (%) PMNL Elastase (gg/1) LTB4 (pg/ml)
71.6 _+ 9.0 88.6 -+ 8.5 222.5 _+ 65.5 269.1 _+ 29.3
*90.8 95.8 *593.6 200.0
_+ 3.3 -+ 1.5 + 402.1 _+ 73.9
65.0 90.0 *349.0 304.2
+ 9.3 + 3.8 _+ 112.8 + 106.4
* P < 0.05 vs before surgery; ** P < 0.01 vs before surgery Values are mean _+ SD
Table 4. Opsonic activity in the patients with postoperative infection
Before surgery (n=6) Opsonic index C3 (mg/dl) C4 (mg/dl) CHs0 (U/ml) Fibronectin (mg/dl)
0.89 75.5 25.0 39.4 31.2
_+ 0.08 + 7.5 _+ 1.1 _+ 7.4 + 7.5
At the onset (n=6) 0.93 **58.0 24.0 37.7 23.1
_+ 0.08 -+ 2.9 _+ 3.7 _+ 3.9 + 10.4
At a later stage (n:5) **0.68 *43.3 *'17.3 *23.8 *'17.4
+ 0.09 _+ 17.1 _+ 5.7 + 11.7 + 5.6
* P < 0.05vs Before surgery; **P < 0.01vs before surgery Values are mean + SD
tion compared with before surgery (p < 0.01). On the other hand, the mean P M N L phagocytic capacity at a later stage of infection was low, though there was not a marked difference compared with before surgery. The mean P M N L bactericidal capacity in the postoperatively infected patients was 88.6 + 8.5 per cent before surgery, 95.8 _+ 1.9 per cent at the onset of infection and 90.0 + 3.8 per cent at a later stage of infection, respectively. At the onset and at a later stage of infection there were no demonstrative differences compared with before surgery. The mean P M N L elastase level at the onset and at a later stage of infection was substantially increased compared with that before surgery (p < 0.05).
plasma P M N L elastase levels, plasma LTB4 levels and PMNL function. The total peripheral white blood cell counts substantially correlated with the P M N L phagocytic capacities (r = 0.39, p < 0.01), P M N L bactericidal capacities (r = 0.30, p < 0.01) and PMNL elastase levels (r = 0.60, p < 0.01). Moreover, the PMNL phagocytic capacities noticeably correlated with the PMNL bactericidal capacities (r = 0.55, p < 0.01). However, no correlation could be found between the peripheral total white blood cell counts and plasma LTB4 levels, between the PMNL elastase levels and plasma LTB4 levels, or between the PMNL function capacities and plasma LTB4 levels (Fig. 6).
Linear Regression Analysis for Opsonic Activity Opsonic Activity in the Postoperatively Infected Patients (Table 4) The mean opsonic indices were 0.89 + 0.08 before surgery, 0.93 + 0.08 at the onset of postoperative infection, and 0.68 + 0.09 at a later stage of infection, respectively. There was a marked decrease in the opsonic index at a later stage of infection compared with that before surgery (p < 0.01). The mean C3, C4, CHs0 and fibronectin levels at the later stage were also significantly lower compared with those before surgery (p < 0.05 or p < 0.01).
Linear regression analyses were performed to compare the serum complement levels and plasma fibronectin levels with the serum opsonic indices. There were demonstrative correlations between the serum opsonic index and C3 (r = 0.46, p < 0.01) (Fig. 7), and between the serum opsonic index and CHso (r = 0.6l, p < 0.01) (Fig. 8). Moreover, marked correlations of plasma fibronectin with the opsonic index (r = 0.66, p < 0.05), C3 (r = 0.78, p < 0.01), C4 (r = 0.79, p < 0.01), and CHs0 (r = 0.68, p < 0.05) were also demonstrated.
Linear Regression Analysis for PMNL Function
Linear regression analyses were performed to compare the total peripheral white blood cell counts with the
PMNL, which ingests and kills bacteria by producing oxygen radicals as well as other chemical mediators,
T. Inoue et al.: PMNL Function and Serum Opsonic Activity WBC (cells/#l~ •
oo• •••o •o •
o " ;
r'=0.60 P 0,01 • • • n=94
15,000. 10,000 5,000
Phagocytic capacity WBC (cells/#l)
• • • •
y=165.7x-6372,4 r=0.30 P
"" "" ".: -! ""'":
Bactericidal • capacity
do 6"5 7'o 7g ~0 ~5 go ~5 100(%)
_.: . . ;
• • Bactericidal capacity
~0 ~5 io i5 ~0 ~'5 go ~5 160(°~o)
Fig. 6a-d. Linear regression analysis for PMNL functions, a Correlation of phagocytic capacity, b bactericidal capacity, and e plasma elastase levels with total white blood cell counts, d Correlation of phagocytic capacity with bactericidal capacity
_ - ~ ' ~ , 4 .
~ ~ ~...-.~"~." .
;-o.,~6 " P 0.01 '~=gg-'
• % •
•o• 0. I
Fig. 7a,b. Linear regression analysis for opsonic activity. a Correlation of C3, and b CH,~0with opsonic index
plays a fundamentally important role in the host defense against bacterial infection. Despite the increase in the PMNL number after major surgery, patients who undergo major surgery have a high risk of developing bacterial infections, which may possibly be caused by an impairment in PMNL function and a depressed opsonic activity during the postoperative period. Studies of host defense mechanisms performed on patients suffering from burns, trauma and intraabdominal infections have revealed remarkably depressed PMNL chemotaxis, PMNL microbicidal activity and delayed cutaneous hypersensitivity reactions. 16-21 These depressed functions have been associated with an increased rate of sepsis. However, general surgical patients have not been as thoroughly studied and therefore, the influence of surgery on PMNL function is still not established. Specifically, some reports have demonstrated the PMNL dysfunction, 8'22-~5 while others have not. 26'27 Thus, we investigated the per•operative alteration in PMNL function and opsonic activity. In the present study, although the PMNL phagocytic and bactericidal capacities were already diminished in most of the surgical patients before surgery compared with in the healthy volunteers, the PMNL phagocyticbactericidal capacities and elastase levels in the postoperatively noninfected patients were noticeably increased just after surgery, and returned to the preoperative levels after 1 or 2 weeks. These results
T. Inoue et al. : PMNL Function and Serum Opsonic Activity
r =0.66 p< 0.05
n=15 ~ibronectin (m6/a~)
(mg/[I)40 30 20
• ~ ~
¢'~,* • • • 10
, , 30
n=]7 Fib..... tin (mg/d~)
r=0.68 p< o.o~ n=18
suggest that the PMNL bacteria-killing function is activated by surgical stress in the postoperative period. In the postoperatively infected patients, the PMNL bactericidal capacity and PMNL elastase secretion were activated on the 3rd and 7th postoperative days compared with in the noninfected patients. The PMNL phagocytic capacity on the 14th postoperative day was significantly depressed in the infected patients, whereas the PMNL elastase secretion was noticeably activated in the infected patients compared with in the noninfected patients. At a later stage of postoperative infection, both the PMNL bactericidal capacity and PMNL elastase secretion showed a tendency to increase compared with the preoperative levels, whereas the PMNL phagocytic capacity presented a tendency to decrease. PMNL activation appears to be very important for carrying out the host immune response, however, it may induce multiple organ injury because, with ongoing stimulation, PMNL release not only elastase but also superoxide radical, both of which are efficient in killing bacteria and in inflicting own organ injury. 7 Therefore, the results seen at the later stage of postoperative infection, such as the activation of PMNL elastase release and of superoxide radical release and the decrease in PMNL phagocytic capacity, may indicate the patient's susceptibility to his or her own organ injury instead of killing bacteria. Many previous studies on PMNL function after surgery or in infected patients have shown a depression in PMNL function such as chemotaxis, lysosomal enzyme content, and respiratory burst. However, there have been few reports showing the increased respiratory burst in patients after surgery and with infection. Hill et al. 28 and Barbour et al. 29 have shown that extraabdominal infections associated with low mortality are accompanied by enhanced rather than reduced chemotactic activity, bactericidal activity, and respira-
Fig. 8. Linear regression analysis for opsonic activity, a Correlation of opsonic index, b C3, e C4, and cl CHs0 with fibronectin
tory burst. On the other hand, Alexander et al. 3° have shown that patients with more severe infections, associated with a marked high mortality rate, show a depression in PMNL function. Except for the postoperative transient increment in PMNL function, our results are almost consistent with those of many previous reports with regard to the decreased PMNL function and increased PMNL elastase in infected patients. The differences in the time of obtaining samples for study, severity of infection, patient selection, and methodology may be responsible for the differences between our study and previous studies. A depression in PMNL chemotactic activity has been demonstrated after surgery, which was explained by Christou et al. 31'32 and Bowers et al. 33 to be due to the presence of serum chemotactic inhibitors or to the decrease in serum chemotactic agents. Although PMNL chemotactic activity was not assessed in our study, LTB4 was measured as a chemotactic agent, because LTB4 released from PMNL generates the action of PMNL aggregation, stimulation and chemotaxis. Thus, LTB4 is a potent chemo-attractant for PMNL and the impairment of LTB4 generation is thought to increase the risk of bacterial infection, s,9 In our study, the plasma LTB4 levels in the noninfected patients increased on the 7th .postoperative day compared with the preoperative levels. On the other hand, a marked decrease in plasma LTB4 levels in the postoperatively infected patients was demonstrated on the first postoperative day with a corresponding lowered opsonic index and many other opsonic factors. Our results indicate that LTB4 levels usually increase as a consequence of surgical stress and that patients with postoperatively depressed LTB4 levels are at high risk of developing infection after surgery. These tendencies to decreased LTB4 levels and decreased opsonic activ-
T. Inoue et al. : PMNL Function and Serum Opsonic Activity
ity just after surgery may make clear the defects in PMNL chemotactic activity after surgery. Moreover, these remarkable deficiencies of chemotactic agents and opsonin may be a consequence of the initial infection after surgery in spite of the increased PMNL bactericidal capacity and high plasma elastase levels. Opsonization by serum factors and the ingestion and killing by phagocytic cells are known to be absolute requirements for resistance to bacterial pathogens. Plasma fibronectin may be an important non-specific opsonin for the reticuloendothelial (RE) clearance of particulate matter and bacteria. A deficiency of plasma fibronectin levels was associated with a depression in R E function. 34-37 In the present study we determined the opsonic index, plasma fibronectin and serum complement levels for opsonic activity. The levels of these factors are the result of a balance between component production, consumption and destruction. We excluded the patients with liver dysfunction from the postoperative time course analysis and therefore, the low levels of serum complement could either be due to postoperative consumption or to the postoperative failure of production by the liver. In our study the mean complement (C3, C4 and CH50) levels increased on the 3rd and/or 7th postoperative days in the noninfected patients after an initial slight depression compared with before surgery. These complements appeared to act as acutephase proteins, usually increasing in concentration as a consequence of surgical stress except where a consumptive process occurred. 38 The opsonic index remarkably decreased on the first postoperative day and at a later stage of infection, and this decrease in the opsonic index correlated well with the plasma fibronecfin and serum complement levels. These results indicate that the opsonic index reflects fibronectin, complements and other serum opsonic factor levels. Moreover, our results of serum opsonic activity after surgery are in accordance with those of reports by other authors. 38,39 Although cellular host resistance to infection, such as PMNL phagocytic-bactericidal capacity and PMNL elastase secretion, increased transiently in our study, humoral host resistance to infection (opsonic activity), such as the opsonic index, fibronectin and complement levels, decreased just after surgery. These defects in the opsonic activity may contribute to the patient's susceptibility to infection immediately after surgery. Moreover, despite the exclusion of patients with preoperative complications from the influence of the operation in the absence of infection, the PMNL phagocytic and bactericidal capacities were already diminished in most of the surgical patients before surgery. This PMNL dysfunction which can be due to the underlying disease may also be an important factor predisposing postoperative infection in surgical
patients. It could be possible that patients who undergo major surgery may be at high risk of developing bacterial infection after surgery because of the lower level of chemotactic agents, serum opsonic activity and PMNL dysfunction. At a later stage of postoperative infection, both PMNL and humoral host resistance to infection decreased. In most patients with severe infection, a depression in PMNL function and opsonic activity may precede the clinical diagnosis of M O F by several days, and control of the infection may lead to improved PMNL function and opsonic activity. From the present study we conclude that numerous defects in the PMNL and humoral host resistance to infection can be observed at a later stage of postoperative severe infection. Thus, the monitoring of PMNL function and opsonic activity during the postoperative course is beneficial for making an early diagnosis of postoperative infection and for predicting tlae risk of MOF. However, our results do not necessarily clarify the cause and effect relationship between the observed PMNL function and infection and therefore, further studies are needed to investigate its relationship and determine the method of preventing defects in the host defense. References
1. Pollock AV (1979) Surgical wound sepsis. Lancet 1:1283-1286 2. Polk HC, Shields CL (1977) Remote organ failure: A valid sign of intra-abdominal infection. Surgery 81:310-313 3. Border JR, Chenier R, McMenamyRH (1976) Multiple systems organ failure: Muscle fuel deficit with visceral protein malnutrition. Surg Clin North Am 56:1147-1168 4. Solomkin JS, Bauman MP, Nelson RD, Simmons RL (1981) Neutrophils dysfunction during the course of intra-abdominal infection. Ann Surg 194:9-17 5. MacLean LD, Meakins JL, Taguchi K, Duignan JP, Dhillon KS, Gordon J (1975) Host resistance in sepsis and trauma. Ann Surg 182:207-216 6. Everson NW, Stacey RL, Wood RFM, Bell PRF (1979) The reversal of surgically induced reticuloendothelial depression. Clin Exp Immunol 37:169-173 7. Carrico CJ, Meakins JL, Marshall JC, Fry D, Maier RV (1986) Multiple-organ-failuresyndrome. Arch Surg 121:196-208 8. Utoh J, Yamamoto T, Utsunomiya T, Kambara T, Goto H, Miyauchi Y (1988) Effect of surgery on neutrophil functions, superoxide and leukotriene production. Br J Surg 75:682-685 9. Ford-HutchinsonAW, Bray MA, Doig MV, ShipleyME, Smith MJH (1980) Leukotriene B, a potent chemokineticand aggregating substance released from polymorphonuclear leukocytes. Nature (Lond) 286:264-265 10. Dunn PA, Tyrer HW (1981) Quantitation of neutrophil phagocytosis, using fluorescent latex beads: Correlation of microscopy and flow cytometry. J Lab Clin Med 98:374-381 11. Steinkamp JA, Wilson JS, Saunders GC, Stewart CC (1982) Phagocytosis: Flow cytometric quantitation with fluorescent microspheres. Science215:64-66 12. Bjerknes R and BassCe CF (1983) Human leukocyte phagocytosis of zymosan particles measured by flow cytometry. Acta Pathol Microbiol Immunol Scand [C] 91:341-348
T. Inoue et al.: PMNL Function and Serum Opsonic Activity 13. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M (1983) Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation, J Immunol 130:1910-1917 14. Kurimoto F, Sakurai H (1987) Radioimmunoassay for arachidonic acid metabolite (in Japanese). Igakunoayumi 143:323-325 15. Mayer MM (1971) Complement and complement fixation. In: Kabat EA, Mayer MM (eds) Experimental immunochemistry, 2rid edn. Charles C Thomas, Springfield, pp 162-240 16. Alexander JW, Meakins JL (1972) A physiological basis for the development of opportunistic infections in man. Ann Surg 176:273-287 17. Alexander JW, Wixon D (1970) Neutrophil dysfunction and sepsis in burn injury. Surg Gynecol Obstet 130:431-438 18. Alexander JW, Ogle CK, Stinnett JD, Macmillan BG (1978) A sequential, prospective analysis of immunologic abnormalities and infection following severe thermal injury. Ann Surg 188: 809-816 19. Meakins JL, Christen NV, Shizgal HM, Maclean LD (1979) Therapeutic approaches to anergy in surgical patients. Ann Surg 190:286-296 20. Meakins JL, Pietsch JB, Bubenick O (1977) Delayed hypersensitivity: Indicator of failure of host defense in sepsis and of acquired trauma. Ann Surg 186:241-250 21. Christou NV, Meakins JL (1979) Delayed hypersensitivity in surgical patients: A mechanism for anergy. Surgery 86:78-85 22. Stanley TH, Hill GE, Portas MR, Hogan NA, Hill HR (1976) Neutrophil chemotaxis during and after general anesthesia and operation. Anesth Analg 55:668-673 23. Cullen BF, Hume RB, Chretien PB (1975) Phagocytosis during general anesthesia in man. Anesth Analg 54:501-5045 24. Davies JM, Sheppard K, Fletcher J (1983) The effect of surgery on the activity of neutrophil granule proteins. Br J Haematol 53:5-13 25. E1-MaallemH, Fletcher J (1981) Effects of surgery on neutrophil granulocyte function. Infect Immun 32:38-41 26. van Dijk WC, Verbrugh HA, van Rijswijk REN, Vos A, Verhoef J (1982) Neutrophil function, serum opsonic activity, and delayed hypersensitivity in surgical patients. Surgery 92: 21-29
243 27. Duignan JP, Collins PB, Johnson AH, Bouchier-Hayes D (1986) The association of impaired neutrophil chemotaxis with postoperative surgical sepsis. Br J Surg 73:238-240 28. Hill HR, Gerrard JM, Hogan NA, Quie PG (1974) Hyperactivity of neutrophil leukotactic responses during active bacterial infection. J Clin Invest 53:996-1002 29. Barbour AG, Crain DA, Solberg CO, Hill HR (1980) Chemiluminescence by polymorphonuclear leukocytes from patients with active bacterial infection. J Infect Dis 141:14-26 30. Alexander JW, Stinnett JD, Ogle CK (1979) A comparison of immunologic profiles and their influence on bacteremia in surgical patients with a high risk of infection. Surgery 86:94-104 31. Christou NV, Meakins JL (1979) Neutrophil function in surgical patients: Two inhibitors of granulocyte chemotaxis associated with sepsis. J Surg Res 26:355-364 32. Christou NV, Meakins JL (1979) Neutrophil function in anergic surgical patients: Neutrophil adherence and chemotaxis. Ann Surg 190:557-564 33. Bowers TK, O'Flaherty J, Simmons RL, Jacob HS (1977) Postsurgical granulocyte dysfunction: Studies in healthy kidney donors. J Lab Clin Med 90:720-727 34. Scott RL, Sohmer PR, MacDonald MG (1982) The effect of starvation and repletion on plasma fibronectin in man. JAMA 248:2025-2027 35. Howard L, Dillon B, Saba TM, Hoffman S, Cho E (1984) Decreased plasma fibronectin during starvation in man. J Parenter Enter Nutr 8:237-244 36. Chadwick SJD, Sire AJW, Dudley HAF (1986) Changes in plasma fibronectin during acute nutritional deprivation in healthy human subjects. Br J Nutr 55:7-12 37. Lanser ME, Saba TM (1983) Correction of serum opsonic defects after burn and sepsis by opsonic fibronectin administration. Arch Surg 118:338-342 38. Alexander JW, McClellan MA, Ogle CK, Ogle JD (1976) Consumptive opsoninopathy: Possible pathogenesis in lethal and opportunistic infections. Ann Surg 184:672-678 39. Bjornson AB, Altemeier WA, Bjorson HS (1980) Complement opsonins and the immune response to bacterial infection in burned patients. Ann Surg 191:323-329.