653
Comparison of Urine and Serum Concentrations of Interleukin-6 in Women with Acute Pyelonephritis or Asymtomatic Bacteriuria S. Hedges, K. Stenqvist, G. Lidin-Janson, J. Martinell, T. Sandberg, and C. Svanborg
Department of Medical Microbiology. Division of Clinical Immunology. Lund University. and Departments ofInfectious Diseases and Pediatrics. Goteborg University. Sweden
Urinary tract infections (UTI) can remain localized to the kidneys or bladder or can involve tissues outside the urinary tract. In most cases, the systemic host response is activated without bacterial invasion of the bloodstream [1, 2]. The mechanisms whereby a mucosal bacterial stimulus can give rise to a systemic host response have not been investigated. Interleukin-6 (IL-6) has a spectrum of biologic activities that corresponds to symptoms ofpatients with acute pyelonephritis. IL-6 acts as an endogenous pyrogen [3] and induces the synthesis of acute-phase reactants, such as C-reactive protein, in the liver [4]. Spread ofIL-6 from the site of infection in the urinary tract via the bloodstream to the liver and temperature regulatory center might induce or enhance the systemic host response in patients with UTI. IL-6 production is activated by experimental bacterial stimulation of the urinary tract mucosa. The urinary IL-6 levels ofhuman volunteers increased within minutes ofdeliberate colonization of the urinary tract with Escherichia coli [5]. In these patients, IL-6 was secreted into the urine intermittently for as long as bacteriuria remained but was not secreted into serum. However, the IL-6 response to natural UTI has not been described. The aim of this study was to analyze whether IL-6 is activated in patients with natural UTI and to quantitate the local and systemic IL-6 response to acute pyelonephritis and asymptomatic bacteriuria.
Received 5 August 1991; revised 6 April 1992. Grant support: Swedish Medical Research Council (07934-0 I). "Forenade Liv" Mutual Group Life Insurance Company. Stockholm. Medical Faculties at the Universities of Lund and Goteborg, and the Tesdorpf Osterlund. and Crawford Foundations. Reprints or correspondence: Dr. Spencer Hedges. Department of Medical Microbiology. Division of Clinical Immunology. Solvegatan 23. S-223 62 Lund. Sweden. The Journal of Infectious Diseases 1992;166:653-6 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0035$01.00
Materials and Methods Patients with asymptomatic bacteriuria. Twenty-two nonpregnant women with asymptomatic bacteriuria contributed samples from 42 episodes. They were enrolled in the prospective UTI follow-up program in Goteborg in connection with their first episode of childhood bacteriuria at a median age of 7.5 years (range, 2-16). The initial UTI episode was treated. Subsequent episodes of bacteriuria were detected in connection with scheduled control visits and were left untreated. The age of the patients at the time of sampling for this study was 22-38 years [6]. Patients with acute pyelonephritis. The patients were enrolled at the Department of Infectious Diseases and followed according to the standardized protocol [7]. The pyelonephritis patients represented a sample of29 women with a median age of 33 years (range, 19-67). Healthy controls. Serum and urine samples were obtained from healthy pregnant and nonpregnant volunteers. The urine samples were semiquantitatively cultured and found to be free of bacteria. Diagnostic criteria. Asymptomatic bacteriuria was diagnosed at screening if two consecutive midstream urine samples showed significant growth (~105 cfu/ml.) of the same bacterial strain. The clinical diagnosis of acute pyelonephritis was based on fever ~38.0°C and flank pain or costovertebral angle tenderness (or both) in patients with a midstream urine culture yielding a single strain at ~ 105 cfu/rnl., The clinical diagnosis was confirmed with laboratory values including C-reactive protein > 20 mg/L, erythrocyte sedimentation rate >25 rnrn/h, and a renal concentrating capacity 20 units/mL occurred in 25 of 29 women with acute pyelonephritis and in 36 of 42 women with asymptomatic bacteriuria. Elevated serum IL-6 levels were found mainly in patients with acute pyelonephritis: Levels > 20 units/mL occurred in 14 of 28 women with acute pyelonephritis compared with 0 of 28 women with asymptomatic bacteriuria. These results suggest that bacteriuria is accompanied by elevated urinary IL-6 levels and that this IL-6 is locally produced. The spread of IL-6 to the circulation in patients with acute pyelonephritis may contribute to the elevation of fever and C-reactive protein characteristic of the disease.
Concise Communications
654
>250 J 250
line abolished the IL-6-dependent growth ofthe B9 cells in both the standard and random patient samples. Statistical methods. Differences between groups were evaluated by using the Mann-Whitney U test and the X2 test with Yates's correction factor. Pairwise comparisons were made by Wilcoxon's rank sum test.
Results Healthy controls. The IL-6 activity in urine and sera from the healthy controls is shown in figure 1. The urine samples did not contain IL-6 activity detectable by the B9 assay. The serum samples induced B9 cell proliferation equivalent to 0-20 units/ml, of the recombinant IL-6 standard. IL-6 activity ~20 units/rnl. was subsequently used to define a positive response. IL-6 response in women with UTI. A urinary IL-6 response occurred at the time of diagnosis in most women with significant bacteriuria. The mean urinary IL-6 levels were 81 units/rnl, for women with acute pyelonephritis and 86 units/ mL for those with asymptomatic bacteriuria (nonsignificant) (table I). Urinary IL-6 levels >20 units/nil. occurred in 25 of29 women with acute pyelonephritis and in 36 of 42 sam-
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PYELONEPHRITIS ASYMPTOMATIC NON-INFECTED BACTERIURIA
PYELONEPHRITIS ASYMPTOMATIC NON-INFECTED BACTERIURIA
URINE
SERUM
Figure 1. IL-6 in urine and serum of women with urinary tract infections and noninfected controls.
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 13, 2015
Sampling of blood and urine. Blood and urine specimens were collected before beginning treatment and 1 month after ending treatment. Some samples were used for other purposes, and in a few cases, samples were not obtained at the time of infection. Analyses of IL-6 activity were done on 29 urine and 28 serum samples from women with acute pyelonephritis and on 42 urine and 38 serum samples from women with asymptomatic bacteriuria. IL-6 assay. The cell line BI3.29, which is dependent on IL-6 for its growth, has been described [8]. For IL-6 determinations, the more sensitive subclone B9 was used [3]. The B9 cells were harvested from the tissue culture flasks, and 5000 cells/ well were seeded into microtiter plates (Nunc, Roskilde, Denmark) containing dilutions of sample or recombinant human IL-6 (as standard) and Iscove's modified Dulbecco's medium supplemented with 50 mM 2-mercaptoethanol, 5% fetal calf serum (SeraLab, Crawley Down, UK), and gentamicin (0.1 mg/ mL) in a total volume of 0.2 mL. eH]thymidine was added between 68 and 72 h ofculture, and the incorporated radioactivity was measured and compared with a standard curve. The standard curve was prepared using stock recombinant human IL-6 (8000 units/rnl.; 1 unit = concentration of IL-6 required for half-maximal thymidine incorporation). The specificity of the assay was tested on random samples with monoclonal antiIL-6 antibody 6B4 (IgG 1) [9]. Supernatants from the 6B4 cell
1ID 1992; 166 (September)
JID 1992; 166 (September)
Concise Communications
Discussion In the present study, we analyzed the local and systemic IL-6 response to UTI. The urinary IL-6 levels were elevated in most patients with asymptomatic bacteriuria or acute pyelonephritis, suggesting that bacterial stimulation of the urinary tract mucosa elicited a local IL-6 response. In contrast, elevated serum IL-61evels were found mainly in women with acute pyelonephritis. We propose that this spread of IL-6 to the circulation contributes to the systemic host response in acute pyelonephritis. Bacteriuria was accompanied by elevated urinary IL-6 levels in 86%of the women. The urinary IL-6 disappeared after treatment, and no IL-6 was detected in urine samples from healthy controls. Therefore, it appeared that the infection caused the IL-6 response. The sensitivity of IL-6 to indicate significant bacteriuria in this group was 86%, the specificity 100%. At present there is a major controversy regarding the definition of significant bacteriuria. While the classic studies proposed a cutoff of --10 5 cfu/ml, [10], several groups have
Table 1. Urine and serum IL-6 levels in women with urinary tract infection. Group. n Asymptomatic bacteriuria. 42 Pyelonephritis. 29 p
Urine
Serum*
86.2 (0-190) 81.2 (0-512) Nonsignificant
10.9 (0-24) 49.6 (0-400) 38.5°C, C-reactive protein >25 rng/L, and elevated erythrocyte sedimentation rate. The pyelonephritis patients in this study were enrolled on the basis ofbacteriuria and fever but also had elevated C-reactive protein levels. Second, serum IL-6 levels were significantly higher in the febrile patients than in nonfebrile patients, although both groups had a urinary IL-6 response. Locally produced IL-6 that enters the bloodstream may determine the host response to the infection. However there is no information about how the systemic spread of IL-6 occurs. Febrile UTI is often caused by E. coli strains with P fimbriae and other virulenceassociated traits that are lacking in the strains causing asymptomatic bacteriuria [15]. It is possible that bacterial properties can influence both the magnitude of the IL-6 response and the cell type that is activated to produce IL-6 and thus the host response to the infection. The patients who acquire significant bacteriuria may develop a severe systemic infection, as in acute pyelonephritis, or may carry the bacteria without apparent discomfort, as in asymptomatic bacteriuria [I, 2]. The difference between these conditions is not attributable to whether bacteria remain localized to the urinary tract or invade the bloodstream. Positive blood cultures are rare in children with febrile UTI and occur in only ,...., 30% of adults with acute pyelonephritis [I]. For example, only 2 ofthe pyelonephritis patients in the present study had concomitant septicemia. It has previously been unclear how a local bacterial stimulus can give rise to a systemic host response in the absence of bacterial invasion. The IL-6 response to UTI demonstrated in the present study is the first step toward the elucidation of such a mechanism.
JID 1992; 166 (September)
Comparison of Urine and Serum Concentrations of Interleukin-6 in Women with Acute Pyelonephritis or Asymtomatic Bacteriuria S. Hedges, K. Stenqvist, G. Lidin-Janson, J. Martinell, T. Sandberg, and C. Svanborg
Department of Medical Microbiology. Division of Clinical Immunology. Lund University. and Departments ofInfectious Diseases and Pediatrics. Goteborg University. Sweden
Urinary tract infections (UTI) can remain localized to the kidneys or bladder or can involve tissues outside the urinary tract. In most cases, the systemic host response is activated without bacterial invasion of the bloodstream [1, 2]. The mechanisms whereby a mucosal bacterial stimulus can give rise to a systemic host response have not been investigated. Interleukin-6 (IL-6) has a spectrum of biologic activities that corresponds to symptoms ofpatients with acute pyelonephritis. IL-6 acts as an endogenous pyrogen [3] and induces the synthesis of acute-phase reactants, such as C-reactive protein, in the liver [4]. Spread ofIL-6 from the site of infection in the urinary tract via the bloodstream to the liver and temperature regulatory center might induce or enhance the systemic host response in patients with UTI. IL-6 production is activated by experimental bacterial stimulation of the urinary tract mucosa. The urinary IL-6 levels ofhuman volunteers increased within minutes ofdeliberate colonization of the urinary tract with Escherichia coli [5]. In these patients, IL-6 was secreted into the urine intermittently for as long as bacteriuria remained but was not secreted into serum. However, the IL-6 response to natural UTI has not been described. The aim of this study was to analyze whether IL-6 is activated in patients with natural UTI and to quantitate the local and systemic IL-6 response to acute pyelonephritis and asymptomatic bacteriuria.
Received 5 August 1991; revised 6 April 1992. Grant support: Swedish Medical Research Council (07934-0 I). "Forenade Liv" Mutual Group Life Insurance Company. Stockholm. Medical Faculties at the Universities of Lund and Goteborg, and the Tesdorpf Osterlund. and Crawford Foundations. Reprints or correspondence: Dr. Spencer Hedges. Department of Medical Microbiology. Division of Clinical Immunology. Solvegatan 23. S-223 62 Lund. Sweden. The Journal of Infectious Diseases 1992;166:653-6 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0035$01.00
Materials and Methods Patients with asymptomatic bacteriuria. Twenty-two nonpregnant women with asymptomatic bacteriuria contributed samples from 42 episodes. They were enrolled in the prospective UTI follow-up program in Goteborg in connection with their first episode of childhood bacteriuria at a median age of 7.5 years (range, 2-16). The initial UTI episode was treated. Subsequent episodes of bacteriuria were detected in connection with scheduled control visits and were left untreated. The age of the patients at the time of sampling for this study was 22-38 years [6]. Patients with acute pyelonephritis. The patients were enrolled at the Department of Infectious Diseases and followed according to the standardized protocol [7]. The pyelonephritis patients represented a sample of29 women with a median age of 33 years (range, 19-67). Healthy controls. Serum and urine samples were obtained from healthy pregnant and nonpregnant volunteers. The urine samples were semiquantitatively cultured and found to be free of bacteria. Diagnostic criteria. Asymptomatic bacteriuria was diagnosed at screening if two consecutive midstream urine samples showed significant growth (~105 cfu/ml.) of the same bacterial strain. The clinical diagnosis of acute pyelonephritis was based on fever ~38.0°C and flank pain or costovertebral angle tenderness (or both) in patients with a midstream urine culture yielding a single strain at ~ 105 cfu/rnl., The clinical diagnosis was confirmed with laboratory values including C-reactive protein > 20 mg/L, erythrocyte sedimentation rate >25 rnrn/h, and a renal concentrating capacity 20 units/mL occurred in 25 of 29 women with acute pyelonephritis and in 36 of 42 women with asymptomatic bacteriuria. Elevated serum IL-6 levels were found mainly in patients with acute pyelonephritis: Levels > 20 units/mL occurred in 14 of 28 women with acute pyelonephritis compared with 0 of 28 women with asymptomatic bacteriuria. These results suggest that bacteriuria is accompanied by elevated urinary IL-6 levels and that this IL-6 is locally produced. The spread of IL-6 to the circulation in patients with acute pyelonephritis may contribute to the elevation of fever and C-reactive protein characteristic of the disease.
Concise Communications
654
>250 J 250
line abolished the IL-6-dependent growth ofthe B9 cells in both the standard and random patient samples. Statistical methods. Differences between groups were evaluated by using the Mann-Whitney U test and the X2 test with Yates's correction factor. Pairwise comparisons were made by Wilcoxon's rank sum test.
Results Healthy controls. The IL-6 activity in urine and sera from the healthy controls is shown in figure 1. The urine samples did not contain IL-6 activity detectable by the B9 assay. The serum samples induced B9 cell proliferation equivalent to 0-20 units/ml, of the recombinant IL-6 standard. IL-6 activity ~20 units/rnl. was subsequently used to define a positive response. IL-6 response in women with UTI. A urinary IL-6 response occurred at the time of diagnosis in most women with significant bacteriuria. The mean urinary IL-6 levels were 81 units/rnl, for women with acute pyelonephritis and 86 units/ mL for those with asymptomatic bacteriuria (nonsignificant) (table I). Urinary IL-6 levels >20 units/nil. occurred in 25 of29 women with acute pyelonephritis and in 36 of 42 sam-
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PYELONEPHRITIS ASYMPTOMATIC NON-INFECTED BACTERIURIA
PYELONEPHRITIS ASYMPTOMATIC NON-INFECTED BACTERIURIA
URINE
SERUM
Figure 1. IL-6 in urine and serum of women with urinary tract infections and noninfected controls.
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 13, 2015
Sampling of blood and urine. Blood and urine specimens were collected before beginning treatment and 1 month after ending treatment. Some samples were used for other purposes, and in a few cases, samples were not obtained at the time of infection. Analyses of IL-6 activity were done on 29 urine and 28 serum samples from women with acute pyelonephritis and on 42 urine and 38 serum samples from women with asymptomatic bacteriuria. IL-6 assay. The cell line BI3.29, which is dependent on IL-6 for its growth, has been described [8]. For IL-6 determinations, the more sensitive subclone B9 was used [3]. The B9 cells were harvested from the tissue culture flasks, and 5000 cells/ well were seeded into microtiter plates (Nunc, Roskilde, Denmark) containing dilutions of sample or recombinant human IL-6 (as standard) and Iscove's modified Dulbecco's medium supplemented with 50 mM 2-mercaptoethanol, 5% fetal calf serum (SeraLab, Crawley Down, UK), and gentamicin (0.1 mg/ mL) in a total volume of 0.2 mL. eH]thymidine was added between 68 and 72 h ofculture, and the incorporated radioactivity was measured and compared with a standard curve. The standard curve was prepared using stock recombinant human IL-6 (8000 units/rnl.; 1 unit = concentration of IL-6 required for half-maximal thymidine incorporation). The specificity of the assay was tested on random samples with monoclonal antiIL-6 antibody 6B4 (IgG 1) [9]. Supernatants from the 6B4 cell
1ID 1992; 166 (September)
JID 1992; 166 (September)
Concise Communications
Discussion In the present study, we analyzed the local and systemic IL-6 response to UTI. The urinary IL-6 levels were elevated in most patients with asymptomatic bacteriuria or acute pyelonephritis, suggesting that bacterial stimulation of the urinary tract mucosa elicited a local IL-6 response. In contrast, elevated serum IL-61evels were found mainly in women with acute pyelonephritis. We propose that this spread of IL-6 to the circulation contributes to the systemic host response in acute pyelonephritis. Bacteriuria was accompanied by elevated urinary IL-6 levels in 86%of the women. The urinary IL-6 disappeared after treatment, and no IL-6 was detected in urine samples from healthy controls. Therefore, it appeared that the infection caused the IL-6 response. The sensitivity of IL-6 to indicate significant bacteriuria in this group was 86%, the specificity 100%. At present there is a major controversy regarding the definition of significant bacteriuria. While the classic studies proposed a cutoff of --10 5 cfu/ml, [10], several groups have
Table 1. Urine and serum IL-6 levels in women with urinary tract infection. Group. n Asymptomatic bacteriuria. 42 Pyelonephritis. 29 p
Urine
Serum*
86.2 (0-190) 81.2 (0-512) Nonsignificant
10.9 (0-24) 49.6 (0-400) 38.5°C, C-reactive protein >25 rng/L, and elevated erythrocyte sedimentation rate. The pyelonephritis patients in this study were enrolled on the basis ofbacteriuria and fever but also had elevated C-reactive protein levels. Second, serum IL-6 levels were significantly higher in the febrile patients than in nonfebrile patients, although both groups had a urinary IL-6 response. Locally produced IL-6 that enters the bloodstream may determine the host response to the infection. However there is no information about how the systemic spread of IL-6 occurs. Febrile UTI is often caused by E. coli strains with P fimbriae and other virulenceassociated traits that are lacking in the strains causing asymptomatic bacteriuria [15]. It is possible that bacterial properties can influence both the magnitude of the IL-6 response and the cell type that is activated to produce IL-6 and thus the host response to the infection. The patients who acquire significant bacteriuria may develop a severe systemic infection, as in acute pyelonephritis, or may carry the bacteria without apparent discomfort, as in asymptomatic bacteriuria [I, 2]. The difference between these conditions is not attributable to whether bacteria remain localized to the urinary tract or invade the bloodstream. Positive blood cultures are rare in children with febrile UTI and occur in only ,...., 30% of adults with acute pyelonephritis [I]. For example, only 2 ofthe pyelonephritis patients in the present study had concomitant septicemia. It has previously been unclear how a local bacterial stimulus can give rise to a systemic host response in the absence of bacterial invasion. The IL-6 response to UTI demonstrated in the present study is the first step toward the elucidation of such a mechanism.
JID 1992; 166 (September)
