ADONIS
Clin. exp. Immunol. (1991) 85, 297-301
000991049100233Y
Serum CD14 levels in polytraumatized and severely burned patients C. KR(JGER*1, C. SCHUTT*, U. OBERTACKEt, T. JOKAt, F. E. MULLERt, J. KNOLLER, M. KOLLER, W. KONIG & W. SCHONFELD Medical Microbiology and Immunology, Ruhr University, Bochum, *Ernst Moritz Arndt University, Geifswald, tDepartment of Medical Immunology, University Clinic of Accident Surgery, Essen, and $University Clinic Bergmannsheil, Department of Plastic Surgery and Burn Unit, Bochum, Germany
(Acceptedfor publication 5 February 1991)
SUMMARY Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS-binding protein (LBP) and thus mediates LPS-induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean+ s.e.m.) were 3-7 + 0 05 yg/ml (n = 30, 25-50 years of age) as determined by ELISA (detection limit 20 ng/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n = 16) significantly decreased levels (1 -7 + 0 3) were detected immediately after the trauma, which increased to 4-9+0 3 yg/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score > 45 points), whereas a return to normal levels was observed in patients with an injury score of < 45 points. In addition, the levels of the high-density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area> 35%) within the second week post trauma when clinical signs of septicaemia were evident. Keywords polytrauma sepsis lipopolysaccharide CD14
INTRODUCTION Infectious processes determine the fate of patients with polytrauma and severe burns (Antonacci, 1986; DeCamp & Demling, 1988). Two general principles are recognized which essentially influence the outcome of the disease: the mechanisms of the specific and non-specific host defence system, and the pathogenicity of the bacterial pathogen (Arturson, 1985; Green & Faist, 1988; Finlay & Falkow, 1989; Konig et al., 1990). In this regard, the lipopolysaccharide (LPS), a component of Gram-negative bacteria, is recognized as one major pathogenicity factor (Westphal, 1975; Morrison & Ulevitch, 1978). In vitro and in vivo experiments indicate clearly that LPS triggers inflammatory cells, such as monocytes, macrophages, granulocytes and lymphocytes, for the release and production of inflammatory mediators and cytokines (Morrison & Ryan, 1987; Movat et al., 1987). In vivo application of LPS leads to typical symptoms of Gram-negative septicaemia such as fever, shock and neutrophilia, which are supposed to be induced by Correspondence: Dr med. Wolfgang Schonfeld, Pharma Forschungszentrum, Wuppertal, Bayer AG, Institute for Chemotherapy, 5600 Wuppertal, Germany.
297
increased production of cytokines such as tumour necrosis factor-alpha (TNF-cx), IL-I and IL-6 (Mathison, Wolfson & Ulevitch, 1988; Beutler & Cerami, 1989; Akira et al., 1990). However, the precise mechanisms by which LPS acts on the immune system in vivo are not fully understood. In vitro effects are observed at much higher concentrations of LPS than might occur in septic patients. Moreover, in serum LPS is bound to lipoproteins (high-density lipoproteins) that partially inactivate the biological activity of LPS (Ulevitch, Johnston & Weinstein, 1979). However, LPS binding proteins may have opposite effects; in this regard Tobias et al. (1983, 1989) identified proteins that interact with LPS and cells mediating the effects of LPS on cellular activation. Very recently, the CD 14 molecule of macrophages and monocytes was recognized as binding site for the LPS-LPS-binding protein complex (Schumann et al., 1990; Wright et al., 1990); reaction of CD 14 with this complex triggers macrophages for TNF-ox production at a concentration below 100 pg/ml LPS. The CD14 molecule is primarily found on monocytes and macrophages as well as myelomonocytic leukaemia cells (Scott et al., 1990). Our own studies, and those of Bazil et al. (1986) have shown that the CD 14 can be released from the cells and may be found within biological fluids and cell supernatants of stimulated cells.
298
C. Kruger et al.
We analysed the concentration of soluble CD14 (sCD14) in serum from patients with polytrauma and severe burns and compared the data with levels from healthy volunteers. Both patient groups have a high risk of developing septicaemia and multi-organ failure, probably due to elevated serum levels of LPS and subsequent production of TNF-ac and IL-6 (Michie et al., 1988; Marks et al., 1990). In addition, we correlated sCD14 levels with immunological and clinical parameters of the patients.
Table 1. Serum levels of sCD14 in healthy volunteers (pg/ml)
Group
n
sCD14 level
50
10 30 9
3-25+0-17 3 68 + 0-05 424+0 17
Male Female
49 53
3 70+0 03 4-24 +0 05
SUBJECTS AND METHODS Materials All materials used were obtained from Merck (Darmstadt, Germany). Fine chemicals were obtained from Sigma (Munich, Germany). Patients
Polytraumatized patients (n = 16) from the Universititsklinikum Essen, who met the following criteria were examined: all patients were aged 15-65 years (mean 36 + 17) and had had no acute disease process prior to trauma. Patients with heart and renal failure as well as vascular disease processes were excluded. An injury severity score based on the scoring system of Baker et al. (1974) was used to describe the trauma. All patients had multiple fractures of the extremities, blunt trauma, and a hypovolaemic shock. The Glasgow coma score, which describes the awakeness of the patients, ranged above 8 points within 6 h post trauma, i.e. no clinical evidence for extensive brain trauma could be verified. The patients were divided into two groups, according to the severity of their injuries: the high-score group exhibited > 45 points, and in the low-level group the score was < 45 points. The therapeutical regimen was standardized and included i.v. volume substitution and administration of erythrocyte concentrates if the haemoglobulin levels fell below 110 g/l. Ventilatory support was performed as soon as possible within 60 min post trauma. Repeated surgery within the first days post trauma was necessary to stabilize the patients' condition; 700 o of the surgical interventions were performed within the first 24 h. Antibiotics were given according to the clinical signs of systemic infection. Corticosteroids, colloidal solutions, barbiturates as well as protease inhibitors were omitted. Serum samples were obtained every 6 h within the first 48 h and every 24 h in the following time period of 14 days. Five severely burned patients from the Department of Accident and Plastic Surgery (Kliniken Bergmannsheil, Ruhr University Bochum) were studied. All patients had severe burn trauma; total burned body area was 35-72% as described previously (Koller et al., 1989; Schdnfeld et al., 1990). The therapeutical regimen included early necrectomy, intensive care and local and systemic antibiotic treatment if signs for a postburn sepsis were evident. Pseudomonas aeruginosa and Staphylococcus aureus were regularly indentified on the burned area. Blood serum was obtained on the indicated time points post trauma. Within the second week post trauma, signs of septicaemia were evident in all patients such as increased number of bacterial specimen on the burned area, neutrophilia/neutropenia, thrombocytopenia as well as cardiovascular deterioration.
Determination of sCDJ4 sCD14 levels were determined by enzyme immunoassay (EIA) using specific monoclonal antibodies (MEM 18 and Romo- 1) in a sandwich assay as described previously (Schitt et al., 1988). Briefly, serum samples were diluted 1/41 in phosphate-buffered saline (PBS) with 0- I % Tween (PBS-T). The EIA was performed in 96-well microtitre plates precoated with the monoclonal antiCD14 antibody (MEM 18) (Bazil et al., 1986) overnight, washed with PBS-T and incubated for 3 h with the samples in duplicates at room temperature. The wells were washed with PBS-T and further incubated for 2 h with anti-CD 14 peroxidase-conjugated antibody (Romo-1) (Schitt et al., 1988). OPD (Merck) was added (0-1 M citrate buffer, pH 5-0) and the absorbance was detected after 15 min in an ELISA reader at 492 nm. CD 14 was prepared by affinity chromatography from urine of patients with proteinuria, as described previously (Bazil et al., 1986). sCD14 concentration in the biological fluids was determined by external standardization with the chromatographically purified CD14 in each assay. Intra-assay and inter-assay variation coefficients were below 10%. Neopterin and elastase levels were kindly determined by Prof. M. Jochum (Universitiit Munchen). C-reactive protein (CRP) was determined by Prof. E. Kreuzfelder (Universititsklinikum Essen). High-density lipoprotein (HDL) levels were determined using a conventional assay (HDL-cholesterin) from Boehringer (Mannheim, Germany). C3a levels were kindly determined by Dr G. Zilow (University of Heidelberg, Germany) by a specific radioimmunoassay.
Statistical analysis The statistic evaluation was performed using the Complete Statistic Software (CSS; Statsoft, New England Software, Release 2.1, 1988). Student's t-test for independent means was used; P < 0-05 was considered significant, unless otherwise stated. The data are given as mean + s.e.m. Correlation coefficients were calculated using correlation matrices; the significance level for each correlation coefficient was calculated. RESULTS CD14 is found in its soluble form, sCD14, in human blood serum. Table 1 summarizes the data of 102 healthy donors. Mean levels were 368+0005 jg/mI. A slight age-dependent increase of sCD14 levels was observed, whereas no significant differences were obtained between female and male donors. Since we suggested a relationship between sCD14 levels and activation of inflammatory cascades, we analysed the levels of
299
sCDJ4 after polytrauma and burns 6 *
.*
5
E
E
031
4
co
-L
01
10 0
5) 3 I*
L
I
I I
0
12
24
&:21 0
1
24
1
1
1
.
.V
"M' -I
36 48h 4 5 6 7 8 9 10 11 121314 Time post trauma (days)
Fig. 3. sCD14 and C-reactive protein (CRP) levels after polytrauma in 16 subjects. Data are mean+ s.e.m. Inset: correlation between sCD14 versus CRP; individual data pairs are given, r=0-43, P 45 points; low-score group (-) < 45 points. * Significant differences between both groups (P < 0 05). Data are mean + s.e.m. 0
12
24
Table 2. Correlation between CD14 and laboratory findings Parameter
C-reactive protein Neopterin Elastase (free) Elastase complexed to a I -macroglobulin C3a levels Leucocyte count Platelet count Band cells Monocyte count Bilirubin Lactate-dehydrogenase Glutamic-oxaloacetic transaminase HDL-cholesterin
r
P
0-43 0-14 029 -0 19 0-29 0 099 -0-15 -0-31 0-23 014 0 13 -0-14 -0-06
< 000001 0-13 0002
HDL, high-density lipoprotein.
0 05
Clin. exp. Immunol. (1991) 85, 297-301
000991049100233Y
Serum CD14 levels in polytraumatized and severely burned patients C. KR(JGER*1, C. SCHUTT*, U. OBERTACKEt, T. JOKAt, F. E. MULLERt, J. KNOLLER, M. KOLLER, W. KONIG & W. SCHONFELD Medical Microbiology and Immunology, Ruhr University, Bochum, *Ernst Moritz Arndt University, Geifswald, tDepartment of Medical Immunology, University Clinic of Accident Surgery, Essen, and $University Clinic Bergmannsheil, Department of Plastic Surgery and Burn Unit, Bochum, Germany
(Acceptedfor publication 5 February 1991)
SUMMARY Recently it has been demonstrated that the CD14 molecule which is expressed on monocytes and macrophages serves as a receptor for lipopolysaccharide (LPS) bound to LPS-binding protein (LBP) and thus mediates LPS-induced tumour necrosis factor (TNF) production. Here we report that CD14 is found as a soluble (s) molecule in serum. In healthy volunteers sCD14 levels (mean+ s.e.m.) were 3-7 + 0 05 yg/ml (n = 30, 25-50 years of age) as determined by ELISA (detection limit 20 ng/ml serum) using two monoclonal antibodies in a sandwich technique. In polytraumatized patients (n = 16) significantly decreased levels (1 -7 + 0 3) were detected immediately after the trauma, which increased to 4-9+0 3 yg/ml within the first 6 days post trauma. sCD14 remained elevated during the first 14 days post trauma in patients with the most severe injuries (injury severity score > 45 points), whereas a return to normal levels was observed in patients with an injury score of < 45 points. In addition, the levels of the high-density lipoproteins that partially inactivate free endotoxin are significantly decreased post trauma. No correlation between parameters of inflammation (C3a and neopterin levels, leucocyte counts, amount of band cells), liver function and sCD14 levels was established. Comparable to polytraumatized patients, increased sCD14 serum levels were observed in five patients with burn trauma (burned area> 35%) within the second week post trauma when clinical signs of septicaemia were evident. Keywords polytrauma sepsis lipopolysaccharide CD14
INTRODUCTION Infectious processes determine the fate of patients with polytrauma and severe burns (Antonacci, 1986; DeCamp & Demling, 1988). Two general principles are recognized which essentially influence the outcome of the disease: the mechanisms of the specific and non-specific host defence system, and the pathogenicity of the bacterial pathogen (Arturson, 1985; Green & Faist, 1988; Finlay & Falkow, 1989; Konig et al., 1990). In this regard, the lipopolysaccharide (LPS), a component of Gram-negative bacteria, is recognized as one major pathogenicity factor (Westphal, 1975; Morrison & Ulevitch, 1978). In vitro and in vivo experiments indicate clearly that LPS triggers inflammatory cells, such as monocytes, macrophages, granulocytes and lymphocytes, for the release and production of inflammatory mediators and cytokines (Morrison & Ryan, 1987; Movat et al., 1987). In vivo application of LPS leads to typical symptoms of Gram-negative septicaemia such as fever, shock and neutrophilia, which are supposed to be induced by Correspondence: Dr med. Wolfgang Schonfeld, Pharma Forschungszentrum, Wuppertal, Bayer AG, Institute for Chemotherapy, 5600 Wuppertal, Germany.
297
increased production of cytokines such as tumour necrosis factor-alpha (TNF-cx), IL-I and IL-6 (Mathison, Wolfson & Ulevitch, 1988; Beutler & Cerami, 1989; Akira et al., 1990). However, the precise mechanisms by which LPS acts on the immune system in vivo are not fully understood. In vitro effects are observed at much higher concentrations of LPS than might occur in septic patients. Moreover, in serum LPS is bound to lipoproteins (high-density lipoproteins) that partially inactivate the biological activity of LPS (Ulevitch, Johnston & Weinstein, 1979). However, LPS binding proteins may have opposite effects; in this regard Tobias et al. (1983, 1989) identified proteins that interact with LPS and cells mediating the effects of LPS on cellular activation. Very recently, the CD 14 molecule of macrophages and monocytes was recognized as binding site for the LPS-LPS-binding protein complex (Schumann et al., 1990; Wright et al., 1990); reaction of CD 14 with this complex triggers macrophages for TNF-ox production at a concentration below 100 pg/ml LPS. The CD14 molecule is primarily found on monocytes and macrophages as well as myelomonocytic leukaemia cells (Scott et al., 1990). Our own studies, and those of Bazil et al. (1986) have shown that the CD 14 can be released from the cells and may be found within biological fluids and cell supernatants of stimulated cells.
298
C. Kruger et al.
We analysed the concentration of soluble CD14 (sCD14) in serum from patients with polytrauma and severe burns and compared the data with levels from healthy volunteers. Both patient groups have a high risk of developing septicaemia and multi-organ failure, probably due to elevated serum levels of LPS and subsequent production of TNF-ac and IL-6 (Michie et al., 1988; Marks et al., 1990). In addition, we correlated sCD14 levels with immunological and clinical parameters of the patients.
Table 1. Serum levels of sCD14 in healthy volunteers (pg/ml)
Group
n
sCD14 level
50
10 30 9
3-25+0-17 3 68 + 0-05 424+0 17
Male Female
49 53
3 70+0 03 4-24 +0 05
SUBJECTS AND METHODS Materials All materials used were obtained from Merck (Darmstadt, Germany). Fine chemicals were obtained from Sigma (Munich, Germany). Patients
Polytraumatized patients (n = 16) from the Universititsklinikum Essen, who met the following criteria were examined: all patients were aged 15-65 years (mean 36 + 17) and had had no acute disease process prior to trauma. Patients with heart and renal failure as well as vascular disease processes were excluded. An injury severity score based on the scoring system of Baker et al. (1974) was used to describe the trauma. All patients had multiple fractures of the extremities, blunt trauma, and a hypovolaemic shock. The Glasgow coma score, which describes the awakeness of the patients, ranged above 8 points within 6 h post trauma, i.e. no clinical evidence for extensive brain trauma could be verified. The patients were divided into two groups, according to the severity of their injuries: the high-score group exhibited > 45 points, and in the low-level group the score was < 45 points. The therapeutical regimen was standardized and included i.v. volume substitution and administration of erythrocyte concentrates if the haemoglobulin levels fell below 110 g/l. Ventilatory support was performed as soon as possible within 60 min post trauma. Repeated surgery within the first days post trauma was necessary to stabilize the patients' condition; 700 o of the surgical interventions were performed within the first 24 h. Antibiotics were given according to the clinical signs of systemic infection. Corticosteroids, colloidal solutions, barbiturates as well as protease inhibitors were omitted. Serum samples were obtained every 6 h within the first 48 h and every 24 h in the following time period of 14 days. Five severely burned patients from the Department of Accident and Plastic Surgery (Kliniken Bergmannsheil, Ruhr University Bochum) were studied. All patients had severe burn trauma; total burned body area was 35-72% as described previously (Koller et al., 1989; Schdnfeld et al., 1990). The therapeutical regimen included early necrectomy, intensive care and local and systemic antibiotic treatment if signs for a postburn sepsis were evident. Pseudomonas aeruginosa and Staphylococcus aureus were regularly indentified on the burned area. Blood serum was obtained on the indicated time points post trauma. Within the second week post trauma, signs of septicaemia were evident in all patients such as increased number of bacterial specimen on the burned area, neutrophilia/neutropenia, thrombocytopenia as well as cardiovascular deterioration.
Determination of sCDJ4 sCD14 levels were determined by enzyme immunoassay (EIA) using specific monoclonal antibodies (MEM 18 and Romo- 1) in a sandwich assay as described previously (Schitt et al., 1988). Briefly, serum samples were diluted 1/41 in phosphate-buffered saline (PBS) with 0- I % Tween (PBS-T). The EIA was performed in 96-well microtitre plates precoated with the monoclonal antiCD14 antibody (MEM 18) (Bazil et al., 1986) overnight, washed with PBS-T and incubated for 3 h with the samples in duplicates at room temperature. The wells were washed with PBS-T and further incubated for 2 h with anti-CD 14 peroxidase-conjugated antibody (Romo-1) (Schitt et al., 1988). OPD (Merck) was added (0-1 M citrate buffer, pH 5-0) and the absorbance was detected after 15 min in an ELISA reader at 492 nm. CD 14 was prepared by affinity chromatography from urine of patients with proteinuria, as described previously (Bazil et al., 1986). sCD14 concentration in the biological fluids was determined by external standardization with the chromatographically purified CD14 in each assay. Intra-assay and inter-assay variation coefficients were below 10%. Neopterin and elastase levels were kindly determined by Prof. M. Jochum (Universitiit Munchen). C-reactive protein (CRP) was determined by Prof. E. Kreuzfelder (Universititsklinikum Essen). High-density lipoprotein (HDL) levels were determined using a conventional assay (HDL-cholesterin) from Boehringer (Mannheim, Germany). C3a levels were kindly determined by Dr G. Zilow (University of Heidelberg, Germany) by a specific radioimmunoassay.
Statistical analysis The statistic evaluation was performed using the Complete Statistic Software (CSS; Statsoft, New England Software, Release 2.1, 1988). Student's t-test for independent means was used; P < 0-05 was considered significant, unless otherwise stated. The data are given as mean + s.e.m. Correlation coefficients were calculated using correlation matrices; the significance level for each correlation coefficient was calculated. RESULTS CD14 is found in its soluble form, sCD14, in human blood serum. Table 1 summarizes the data of 102 healthy donors. Mean levels were 368+0005 jg/mI. A slight age-dependent increase of sCD14 levels was observed, whereas no significant differences were obtained between female and male donors. Since we suggested a relationship between sCD14 levels and activation of inflammatory cascades, we analysed the levels of
299
sCDJ4 after polytrauma and burns 6 *
.*
5
E
E
031
4
co
-L
01
10 0
5) 3 I*
L
I
I I
0
12
24
&:21 0
1
24
1
1
1
.
.V
"M' -I
36 48h 4 5 6 7 8 9 10 11 121314 Time post trauma (days)
Fig. 3. sCD14 and C-reactive protein (CRP) levels after polytrauma in 16 subjects. Data are mean+ s.e.m. Inset: correlation between sCD14 versus CRP; individual data pairs are given, r=0-43, P 45 points; low-score group (-) < 45 points. * Significant differences between both groups (P < 0 05). Data are mean + s.e.m. 0
12
24
Table 2. Correlation between CD14 and laboratory findings Parameter
C-reactive protein Neopterin Elastase (free) Elastase complexed to a I -macroglobulin C3a levels Leucocyte count Platelet count Band cells Monocyte count Bilirubin Lactate-dehydrogenase Glutamic-oxaloacetic transaminase HDL-cholesterin
r
P
0-43 0-14 029 -0 19 0-29 0 099 -0-15 -0-31 0-23 014 0 13 -0-14 -0-06
< 000001 0-13 0002
HDL, high-density lipoprotein.
0 05
