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Molecular Cascade of Local Immune Response in Mice with Escherichia coli-Induced Ascending Pyelonephritis
Molecular Cascade of Local Immune Response in Mice with Escherichia coil-Induced Ascending Pyelonephritis Takaoki Hirose,* A k i f u m i Yokoo, Hiroshi Hotta, Yasuharu Kunishima, a n d Taiji Tsukamoto Department of Urology, 5apporo Medical University School of Medicine, Sapporo, Japan
We investigated the time course of gene expression of cytokines in mice with bacterial pyelonephritis. Murine kidneys were harvested at 2 and 6 hours, and 1, 3, 7, and 14 days after infection. Transcripts for murine cytokines (tumor necrosis factor (TNF)-tx, interferon (IFN)-7, interleukin (IL)-I ct, and IL-6) were measured in kidney homogenates by cDNA-equalized reverse transcription polymerase chain reaction (RT-PCR). There was no signal seen for any cytokines in the non-infected kidney. We found mRNA for TNF-c~ and IFN7in infected kidneys at 2 hours after infection, though no infiltrating immune-response cells were observed. It is possible that TNF-t~ and IFN-/,:were produced by intraepithelial lymphocytes in mucosa-associated lymphoid tissue in the urinary tract. IL-6 mRNA was detected at 6 hours after infection, concomitantly with infiltration of lgA-positive B ceils. As seen in the intestinal mucosa, IL-6 may stimulate the maturation of IgA-positive B cells in the urinary mucosa. IL-1 ct mRNA was detectable at 1 day after infection. IL-1 may help direct the neutrophils to the site of infection by inducing expression of intercellular adhesion molecule-1 expression. Our observations suggested that the inflammatory cytokines were of local origin and that urinary mucosa was activated directly by bacteria to produce cytokines. J Infect Chemother 1998;4:71-75 Key words: local cytokine response, urinary tract infection, RT-PCR, intraepithelial lymphocyte
INTRODUCTION
MATERIALS A N D METHODS
Inflammatory cytokines play an important role in the immune response to bacterial infection, 1 especially in inflammatory intestinal diseases 2 but little is known about inflammatory cytokines in urinary tract infections. Recently, interleuldn 01.)-8 has been detected in urine but not in serum after urinary tract infection, suggesting local production of this cytokine in the urinary tract. 3 In addition, uroepithelial cell lines have been shown to produce inflammatory cytokines in response to bacterial challenge. 4 T h e inflammatory cytokines, IL-1, IL6, IL-8, interferon ( I F N ) - 5 and t u m o r necrosis factor ( T N F ) - a may have important functions in the immune response to urinary tract infection. Because many studies reporting cytokines in urinary tract infections focus on only urinary or serum cytokines 5 little information is available a b o u t gene e x p r e s s i o n o f i n f l a m m a t o r y cytokines in bacterial urinary tract infections. In a time-course study of the local production of inflammatory cytokines in a murine model of ascending Escherichia coli-induced pyelonephritis we detected amplified cytokine-specific m R N A transcripts. This study presents a detailed analysis of the local cytokine response to pyelonephritis.
Animals
Received Aug. 14, 1997; accepted for publication in revised form Feb. 16, 1998.*Correspondence and requests for reprints to: Departmentof Urology, SapporoMedical University School of Medicine, S- 1 W- 16 Chuo-ku, Sapporo 060-8543, Japan.
1341-321X/98/O402-OO71/US$3.009 JSC/CLJ 1998
Five-week-old BAI.R/c female mice (Charles River Japan, Atsugi, Japan) weighing 18 to 20 g were used throughout the experiments. T h e mice were caged in groups of 5 and given food and water ad libitum.
Bacteria Escherichia coli (E. coh), a type 0 6 clinical isolate from urine, was cultured overnight in tryptic soy broth (Difco, Detroit, MI, USA) at 37~ suspended in p H 7.3 phosphate-buffered saline (PBS) at a cell density of 106 colony forming units (CFLI)/mL.
Experimentalascendingpyelonephritis T h e mice were infected according to a previously described method. 6 Briefly, a polyethylene catheter was inserted into the urethra under light ether anesthesia, and 0.25 m L of a bacterial suspension was stowly inoculated into the bladder. After the inoculation, the uretiara was obstructed with a clamp for 2 hours. Mice were sacrificed and the kidneys isolated before infection, 2 and 6 hours, and 1, 3, 7, and 14 days after infection. T h e kidneys were sliced in half and one-half was stored at - 7 0 ~ for experiments. T h e other half was homogenized in sterilized saline and serially diluted before being plated onto heart infusion agar (Eiken, Tokyo, Japan) for colony counts. For evaluation of local production of cytoldnes and cellular infiltration in infected kidneys under the same conditions, we used the frozen half kidneys,
ZI
J Infect Chemother 1998;4:71-75
which d e m o n s t r a t e d bacterial colonies of C F U / g renal tissues.
10 6 to
10 7
Immunohistochemical staining T h e remaining specimens were sliced on a cryostat into 6 # m sections and fixed in acetone for 10 minutes at r o o m t e m p e r a t u r e . T h e sections were stained by the avidin-biotin-peroxidase complex (ABC) m e t h o d as described by Hjelm. 7 T h e antibodies used were Mac-1 ( M 1 / 7 0 . 1 5 ) reactive with m a c r o p h a g e s ; B 2 2 0 / L y - 5 (DNL-1.9) reactive with all B cells; anti-mouse IgA (R5135), I g M (R6-71.1), IgG1 (G1-6.5) reactive with their appropriately positive B cells; Ly-1 (53-7.8), reactive with all T cells. All the antibodies were obtained f r o m P h a r M i n g e n (San Diego, CA, USA). T h e kidney sections were washed in PBS and incubated overnight at 4~ with the different p r i m a r y monoclonal antibodies. After incubation the sections were washed several times with PBS. T h e sections were then incubated with biotinylated goat anti-mouse immunoglobulin (PharMingen) for 1 h o u r at r o o m temperature. After extensive washes with PBS, the sections were incubated with mouse ABC (Vector, Burlingame, CA, USA) for 30 minutes at r o o m temperature. T h e color reaction was developed by incubating the sections with a diaminobenzidine tetrahydrochloride solution containing 0.01% H 2 0 2 . T h e sections were counter-stained with hematoxylin, dehydrated and mounted. For observation ofneutrophils, the sections were stained with hematoxylin-eosin. U n d e r a light microscope the positively labeled cells were identified by their dark b r o w n m e m b r a n e - r e l a t e d staining. In every case the degree of cells reacting with any individual monoclonal antibody was d e t e r m i n e d in the pyelonephritic lesion where the greatest infiltration of reactive cells had oc-
curred. T h e degree of cellular infiltration was graded into 5 categories: 1) no cell infiltration; 2) the n u m b e r of reactive cells was < 10 using light microscopy at x 200 magnification; 3) the n u m b e r of reactive cells was in double figures; 4) the n u m b e r of reactive cells was in 3 figures; and 5) the n u m b e r o f reactive cells was in 4 figures (Fig. 1).
Cytokine cDNA amplification Transcripts for murine cytokines and fl-actin were measured in the kidney h o m o g e n a t e by cDNA-equalized reverse transcription polymerase chain reaction (RT-PCR).S Briefly, kidney h o m o g e n a t e was suspended i n T R I Z O L reagent (Gibco G R L , G r a n d Island, NY, USA), and total R N A was extracted by an adaptation of the acid guanidine phenol chloroform method. 9 R N A concentrations were estimated by s p e c t r o p h o t o m e t r y at 260 n m and the absence of R N A degradation was confirmed by electrophoresis on 1% agarose gels. One/~g aliquots of total R N A were reverse-transcribed into c D N A in 20 /,tL of 1 x P C R buffer containing of 2.5 m m o l / L r a n d o m h e x a m e r as a primer, 1 m m o l / L (each) dATP, d G T P , d T T P , a n d d C T P , 5 m m o l / L MgCI2, 1 U of R N a s e inhibitor and 2.5 U of murine LV reverse transcriptase (all p u r c h a s e d f r o m C e t u s - P e r k i n - E l m e r , Branchburg, NJ, USA). T h e reaction mixtures were incubated for 15 minutes at 42~ and for 5 minutes at 99~ and chilled for 5 minutes at 5~ After reverse transcription, the resulting c D N A was amplified using P C R primers specific for m u r i n e cytokines and fl-actin shown in Table 1. Murine IL-8 was excluded f r o m the analysis because its sequence has not yet been reported. P C R was done in 100//L reactions of 2 m m o F L MgC12, 0.15 m m o l / L primers, 0.0125 U of Taq D N A p o l y m e r ase and 1 x P C R buffer. T h e a u t o m a t e d t e m p e r a t u r e
Fig. 1. The degree of cellular infiltration. Cryostat sections (6/~m) were stained by the avadinbiotin-peroxidase complex method for macrophages using Mac-1 (M1/70.15) in murine renal tissue. Macrophages were identified by their dark brown membrane-related staining. Original magnification, x 170. +, number of reactive cells was < 10 using light microscopy at x 200 magnification; +, number of reactive cells was in double figures; ++, number of reactive cells was in 3 figures; i p I, number of reactive cells was in 4 figures.
72
Local cytokine response in pyelonephritis
cycling p r o g r a m was set for 35 cycles, each cycle with the following conditions: 94~ for 1 minute, 55~ for 1 minute and 72~ for 1 minute. Final extension was done at 72~ for 10 minutes. T h e P C R products were analyzed in 3% agarose gels with ethidium bromide stain.
after. I g M - and IgG-positive B cells were observed at 1 day after infection and gradually increased in n u m b e r thereafter (Table 2).
RESULTS
T h e r e was no signal seen for any cytokines in non-infected kidney.TNF-ct and I F N - T m R N A were detected in infected kidney at 2 hours after infection, though no infiltrating immune-response cells were observed.These cytokine m R N A were thereafter present at all t i m e points in infected kidney. IL-6 m R N A was detected at 6 hours after infection, concomitantly with infiltration o f IgA-positive B cells. This cytokine m R N A was seen in infected kidney at 1, 3, 7, and 14 days after infection. I L - l a m R N A was detected at 1 day after infection and at 3 and 14 days after infection. I L - l a m R N A in the infected kidney correlated with increased infiltration of i m m u n e response cells (Fig. 3).
Expression of cytokine mRNA in kidney tissue Cellular infiltration in the pyelonephritic lesion In n o n - i n f e c t e d kidneys, a few Ly-1 reactive cells (intraepithelial lymphocytes) 2 were visible in renal pelvic m u c o s a (Fig. 2). Mac-1 reactive cells (histiocytes) were also found in the submucosa. At 2 hours after infection, infiltration of i m m u n e response cells was not detected. At 6 hours after infection, neutrophils, macrophages a n d T cells were observed in the pyelonephritic lesion. F r o m 1 day after infection, the degree of infiltration of these cells increased. IgA-positive B cells were observed at 6 hours after infection and increased there-
Table 1. The sequences of primers for inflammatory cytokines. mRNA species IL-la
Sequence upstream 5'- CTCTAGAGCACCATGCTACAGAC-3' downstream 5'-TGGAATCCAGGGGAAACACTG-3'
IL-6
upstream 5'-TGGAGTCACAGAAGGAGTGGCTAAG-3' downstream 5'-TCTGACCACAGTGAGGAATGTCCAC-3'
IFN-~,
upstream 5'- AGCGCCTGACTGAACTCAGATTGTAG-3' downstream 5'-GTCACAGTTTTCAGCTGTATAGGG-3'
TNF-tz
upstream 5'- GGCAGGTCTACTTTGGAGTCATTGC-3' downstream 5'-ACATTCGAGGCTCCAGTGAATTCGG-3'
fl-actin
upstream 5'-TGGAATCCTGTGGCATCCATGAAAC-3' downstream 5'-TAAAACGCAGCTCAGT~AACAGTCCG-3'
Length (bp) 309 155 243 307 349
IL, interleukin; IFN, interferon; TNF, tumor necrosis factor.
Fig. 2. A histologic section in the murine renal pelvis of noninfected kidney. A cryostat section was stained by the ABC method for T cells using Ly-1 (53-7.8). A few Ly-1 positive cells were detectable in renal mucosa. Original magnification, x 85.
73
J Infect Chemother 1998;4:71-75 Table 2. Cellular infiltration a in infected kidney. After infection Non-infection Neutrophils Macrophages T cells IgA(+) B cells IgM(+) B cells IgG(+) B cells
+b +c -
2 hours
6 hours
1 day
3 days
7 days
14 days
+ + -
+ + + + -
+ -H+ + + +
+ -H-H++ + +
-H-H~ ++ + +
++ i it tit
++ + +
aCellular infiltration was graded as follows: - , no cell infiltration; +, n u m b e r of reactive cells was < 10 using light microscopy at x 200 magnification; +, number of reactive cells was in d o u b l e figures; -H-, n u m b e r of reactive cells was in 3 figures; I t t, n u m b e r of reactive cells was in 4 figures, bMac-1 reactive cells in submucosa (histiocytes). eLy-1 reactive cells in pelvic mucosa (intraepithelial lymphocytes).
Fig. 3. Reverse transcription-polymerase chain reaction detection of inflammatory cytokine mRNAs in murine kidneys. (A) T N F - a mRNA, (B) IFN-TmRNA, (C) IL-6 mRNA, (D) IL-|o~ mRNA, (E) fl-actin mRNA. T N F - a and I F N - T m R N A w e r e detectable at all time points in infected kidney. IL-6 m R N A was detectable at 6 hours and seen at 1, 3, 7, and 14 days after infection. IL-a m R N A was detectable at 1 day after infection and present at 3 and 14 days after infection.
DISCUSSION
To evaluate the local cytokine response to ascending bacterial pyelonephritis, we analyzed cytokine-specific m R N A in the murine kidney. Amplification of murine kidney c D N A revealed a reproducible time course of gene expression for IL-1, IL-6, TNF-c~ and IFN- 7. On the other hand, spleen homogenate from infected mice revealed no signal for these cytokines.These results suggested that the cytokines were produced in local response to bacterial pyelonephritis.
74
T N F - a and I F N - y m R N A were detected in infected kidney at 2 hours after infection, although no immuneresponse cells infiltrated the pyelonephritic lesion. Immunohistochemical study revealed that Ly-1 positive cells were present in urinary mucosa of non-infected kidney (Fig. 2). It is known that normal intestinal mucosa contains intraepithelial lymphocytes. 2 Thus, it is assumed that Ly-1 positive cells in non-infected urinary mucosa represent intraepithelial lymphocytes. Recent studies have d e m o n s t r a t e d that intraepithelial lymphocytes are capable of producing cytokines such as
Local cytokine response in pyelonephritis
I F N - y i n response to bacterial adhesion at the mucosal surface. 1~ On the other hand, there are some reports that renal tubular epithelia can produce T N F - a in response to lipopolysaccharide.11 Though the source of T N F - a and I F N - y m R N A was not determined, the mucosal surface was the primary site of bacteria-host encounter. Ly-1 positive cells, intraepithelial lymphocytes, may e x p r e s s T N F - a and I F N - y m R N A in response to urinary mucosal challenge with bacteria before immune-response cells infiltrate. Forget al. 12reported thatTNF-tz and I F N ywere regarded as obligate precursors of further cytokine responses, including that oflL-6.Thus, Ly-1 positive cells represent intraepithelial lymphocytes that may further regulate mucosal local immune responses to bacterial pyelonephritis by p r o d u c i n g T N F - a and IFN-y. IL-6 m R N A was detected at 6 hours after infection, concomitantly with infiltration of IgA-positive B cells. In the intestinal mucosa, IL-6 stimulates the maturation of IgA-positive B cells, which secrete lgA. 13 According to our results, IL-6 has the same role as seen in the intestinal mucosa. IL-1 m R N A was detected at 1 day after infection. T h o u g h the source of IL- 1 m R N A was not clear in our experiment, IL-1 m R N A has been detected in uroepithelial cells after exposure to E. coll. 4 Thus, II_,-1 m R N A seemed to originate from uroepithelial cells. In addition, IL-1 has been reported to enhance endothelial cell adhesiveness for p o l y m o r p h o n u c l e a r leukocytes and to increase intercellular adhesion molecule1 expression. 14 IL-1 may help direct the polymorphonuclear leukocytes to the site of infection. In conclusion, our results demonstrated that through cytokine production the urinary mucosa plays an active immunoregulatory role against bacterial infections. Our results suggested that prior to infiltration o f i m m u n e response cells, bacteria-activated intraepithelial lymphocytes and histiocytes in the urinary mucosa produce T N F - a and I F N - y a s a primarycytokineresponse. These cytokines may influence adjacent uroepithelial cells and induce the migration of immune-response cells to the infected sites. IL-6 may be important for the IgA response as seen in the intestinal mucosa. IL-1 seemed to originate from adjacent uroepithelial cells and may stimulate more neutrophil infiltration by inducing intercellular adhesion molecule-1 expression.
REFERENCES 1. Fowrie F, Coffman RL. Cytokine regulation ofT-cell function: potential for therapeutic intervention. Immunol Today 1993;14:270-274. 2. KatoT, Owen RT. Structure and function of intestinal mucosal epithelium. In: Ogra PL (ed) Handbook of mucosal immunology. San Diego: Academic Press, 1994:11-26. 3. AgeceWW, Hedges SR, Ceska M, Svanborg C. Interleukin8 and neutrophil response to mucosal gram-negative infection. J Clin Invest 1993;92:780-785. 4. Heges SR, Agace WW, Svensson M, Sjogren AN, Ceska M, Svanborg C. Uroepithelial cells are part of a mucosal cytokine network. Infect Immun 1994;62:2315 2321. 5. Roberts JA, Kaack MB, Martin LN. Cytokine and lymphocyte activation during experimental acute pyelonephritis. Urol Res 1995;23:33-38. 6. HiroseT, KumamotoY,Tanaka N,Yoshioka M,Tsukamoto T. Study on pathogenesis of Enterococcus faecalis in urinary tract. Urol Res 1989; 17:125-129. 7. Hjelm EM. Local cellular immune response in ascending urinary tract infection: occurrence of T-cells, immunoglobulin-producing cells, and Ia-expressing cells in rat urinary tract tissue. Infect Immun1984;44:627-632. 8. Kolls J, Deininger P, Cohen J, Larson JL. cDNA equalization for reverse transcription-polymerase chain reaction. Anal Biochem 1993;208:264-269. 9. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. 10. Yamamoto S, Russ F, Teixeira HC, Conradt P, Kaufmann SHE. Listeria monocywgenes-induced gamma interferon secretion by intestinal intraepithelial g/d T lymphocytes. Infect Immun 1993;61:2154-2161. 11. Jevnikar AM, Brennan DC, Singer GG, Herg JE, Maslinski W, Wuthrich R,P, et al. Stimulated kidney tubular epithelial cells express membrane associated and secretedTNFa. Kidney Int 1991;40:203-211. 12. ForgY,Tracey KJ, Moldawer LL, Hesse DG, Manogue KB, Kenney JS, et al. Antibodies to cachecfin/tumor necrosis factor reduce interleukin lfl and interleukin 6 appearance during lethal bacteremia. J Exp Med 1989; 170:1627-1633. 13. Beagley KW, Eldridge JH, Lee F, Kuyono H, Everson MP, Koopman WJ, et al. Interleukins and IgA synthesis. Human and murine interleukin-6 induce high rate of IgA secretion in IgA committed B cells. J Exp Med 1989;169: 2133-2148. 14. Pober JS, Gimbrone MA, Lapierre LA, Mendrick DL, FiersW, Rothlein R, et al. Overlapping patterns of activation of human endothelial cells by interleukin-1, tumor necrosis factor, and immune interferon. J Immunol 1986; 137:1893-1896.
75
We investigated the time course of gene expression of cytokines in mice with bacterial pyelonephritis. Murine kidneys were harvested at 2 and 6 hours, and 1, 3, 7, and 14 days after infection. Transcripts for murine cytokines (tumor necrosis factor (TNF)-tx, interferon (IFN)-7, interleukin (IL)-I ct, and IL-6) were measured in kidney homogenates by cDNA-equalized reverse transcription polymerase chain reaction (RT-PCR). There was no signal seen for any cytokines in the non-infected kidney. We found mRNA for TNF-c~ and IFN7in infected kidneys at 2 hours after infection, though no infiltrating immune-response cells were observed. It is possible that TNF-t~ and IFN-/,:were produced by intraepithelial lymphocytes in mucosa-associated lymphoid tissue in the urinary tract. IL-6 mRNA was detected at 6 hours after infection, concomitantly with infiltration of lgA-positive B ceils. As seen in the intestinal mucosa, IL-6 may stimulate the maturation of IgA-positive B cells in the urinary mucosa. IL-1 ct mRNA was detectable at 1 day after infection. IL-1 may help direct the neutrophils to the site of infection by inducing expression of intercellular adhesion molecule-1 expression. Our observations suggested that the inflammatory cytokines were of local origin and that urinary mucosa was activated directly by bacteria to produce cytokines. J Infect Chemother 1998;4:71-75 Key words: local cytokine response, urinary tract infection, RT-PCR, intraepithelial lymphocyte
INTRODUCTION
MATERIALS A N D METHODS
Inflammatory cytokines play an important role in the immune response to bacterial infection, 1 especially in inflammatory intestinal diseases 2 but little is known about inflammatory cytokines in urinary tract infections. Recently, interleuldn 01.)-8 has been detected in urine but not in serum after urinary tract infection, suggesting local production of this cytokine in the urinary tract. 3 In addition, uroepithelial cell lines have been shown to produce inflammatory cytokines in response to bacterial challenge. 4 T h e inflammatory cytokines, IL-1, IL6, IL-8, interferon ( I F N ) - 5 and t u m o r necrosis factor ( T N F ) - a may have important functions in the immune response to urinary tract infection. Because many studies reporting cytokines in urinary tract infections focus on only urinary or serum cytokines 5 little information is available a b o u t gene e x p r e s s i o n o f i n f l a m m a t o r y cytokines in bacterial urinary tract infections. In a time-course study of the local production of inflammatory cytokines in a murine model of ascending Escherichia coli-induced pyelonephritis we detected amplified cytokine-specific m R N A transcripts. This study presents a detailed analysis of the local cytokine response to pyelonephritis.
Animals
Received Aug. 14, 1997; accepted for publication in revised form Feb. 16, 1998.*Correspondence and requests for reprints to: Departmentof Urology, SapporoMedical University School of Medicine, S- 1 W- 16 Chuo-ku, Sapporo 060-8543, Japan.
1341-321X/98/O402-OO71/US$3.009 JSC/CLJ 1998
Five-week-old BAI.R/c female mice (Charles River Japan, Atsugi, Japan) weighing 18 to 20 g were used throughout the experiments. T h e mice were caged in groups of 5 and given food and water ad libitum.
Bacteria Escherichia coli (E. coh), a type 0 6 clinical isolate from urine, was cultured overnight in tryptic soy broth (Difco, Detroit, MI, USA) at 37~ suspended in p H 7.3 phosphate-buffered saline (PBS) at a cell density of 106 colony forming units (CFLI)/mL.
Experimentalascendingpyelonephritis T h e mice were infected according to a previously described method. 6 Briefly, a polyethylene catheter was inserted into the urethra under light ether anesthesia, and 0.25 m L of a bacterial suspension was stowly inoculated into the bladder. After the inoculation, the uretiara was obstructed with a clamp for 2 hours. Mice were sacrificed and the kidneys isolated before infection, 2 and 6 hours, and 1, 3, 7, and 14 days after infection. T h e kidneys were sliced in half and one-half was stored at - 7 0 ~ for experiments. T h e other half was homogenized in sterilized saline and serially diluted before being plated onto heart infusion agar (Eiken, Tokyo, Japan) for colony counts. For evaluation of local production of cytoldnes and cellular infiltration in infected kidneys under the same conditions, we used the frozen half kidneys,
ZI
J Infect Chemother 1998;4:71-75
which d e m o n s t r a t e d bacterial colonies of C F U / g renal tissues.
10 6 to
10 7
Immunohistochemical staining T h e remaining specimens were sliced on a cryostat into 6 # m sections and fixed in acetone for 10 minutes at r o o m t e m p e r a t u r e . T h e sections were stained by the avidin-biotin-peroxidase complex (ABC) m e t h o d as described by Hjelm. 7 T h e antibodies used were Mac-1 ( M 1 / 7 0 . 1 5 ) reactive with m a c r o p h a g e s ; B 2 2 0 / L y - 5 (DNL-1.9) reactive with all B cells; anti-mouse IgA (R5135), I g M (R6-71.1), IgG1 (G1-6.5) reactive with their appropriately positive B cells; Ly-1 (53-7.8), reactive with all T cells. All the antibodies were obtained f r o m P h a r M i n g e n (San Diego, CA, USA). T h e kidney sections were washed in PBS and incubated overnight at 4~ with the different p r i m a r y monoclonal antibodies. After incubation the sections were washed several times with PBS. T h e sections were then incubated with biotinylated goat anti-mouse immunoglobulin (PharMingen) for 1 h o u r at r o o m temperature. After extensive washes with PBS, the sections were incubated with mouse ABC (Vector, Burlingame, CA, USA) for 30 minutes at r o o m temperature. T h e color reaction was developed by incubating the sections with a diaminobenzidine tetrahydrochloride solution containing 0.01% H 2 0 2 . T h e sections were counter-stained with hematoxylin, dehydrated and mounted. For observation ofneutrophils, the sections were stained with hematoxylin-eosin. U n d e r a light microscope the positively labeled cells were identified by their dark b r o w n m e m b r a n e - r e l a t e d staining. In every case the degree of cells reacting with any individual monoclonal antibody was d e t e r m i n e d in the pyelonephritic lesion where the greatest infiltration of reactive cells had oc-
curred. T h e degree of cellular infiltration was graded into 5 categories: 1) no cell infiltration; 2) the n u m b e r of reactive cells was < 10 using light microscopy at x 200 magnification; 3) the n u m b e r of reactive cells was in double figures; 4) the n u m b e r of reactive cells was in 3 figures; and 5) the n u m b e r o f reactive cells was in 4 figures (Fig. 1).
Cytokine cDNA amplification Transcripts for murine cytokines and fl-actin were measured in the kidney h o m o g e n a t e by cDNA-equalized reverse transcription polymerase chain reaction (RT-PCR).S Briefly, kidney h o m o g e n a t e was suspended i n T R I Z O L reagent (Gibco G R L , G r a n d Island, NY, USA), and total R N A was extracted by an adaptation of the acid guanidine phenol chloroform method. 9 R N A concentrations were estimated by s p e c t r o p h o t o m e t r y at 260 n m and the absence of R N A degradation was confirmed by electrophoresis on 1% agarose gels. One/~g aliquots of total R N A were reverse-transcribed into c D N A in 20 /,tL of 1 x P C R buffer containing of 2.5 m m o l / L r a n d o m h e x a m e r as a primer, 1 m m o l / L (each) dATP, d G T P , d T T P , a n d d C T P , 5 m m o l / L MgCI2, 1 U of R N a s e inhibitor and 2.5 U of murine LV reverse transcriptase (all p u r c h a s e d f r o m C e t u s - P e r k i n - E l m e r , Branchburg, NJ, USA). T h e reaction mixtures were incubated for 15 minutes at 42~ and for 5 minutes at 99~ and chilled for 5 minutes at 5~ After reverse transcription, the resulting c D N A was amplified using P C R primers specific for m u r i n e cytokines and fl-actin shown in Table 1. Murine IL-8 was excluded f r o m the analysis because its sequence has not yet been reported. P C R was done in 100//L reactions of 2 m m o F L MgC12, 0.15 m m o l / L primers, 0.0125 U of Taq D N A p o l y m e r ase and 1 x P C R buffer. T h e a u t o m a t e d t e m p e r a t u r e
Fig. 1. The degree of cellular infiltration. Cryostat sections (6/~m) were stained by the avadinbiotin-peroxidase complex method for macrophages using Mac-1 (M1/70.15) in murine renal tissue. Macrophages were identified by their dark brown membrane-related staining. Original magnification, x 170. +, number of reactive cells was < 10 using light microscopy at x 200 magnification; +, number of reactive cells was in double figures; ++, number of reactive cells was in 3 figures; i p I, number of reactive cells was in 4 figures.
72
Local cytokine response in pyelonephritis
cycling p r o g r a m was set for 35 cycles, each cycle with the following conditions: 94~ for 1 minute, 55~ for 1 minute and 72~ for 1 minute. Final extension was done at 72~ for 10 minutes. T h e P C R products were analyzed in 3% agarose gels with ethidium bromide stain.
after. I g M - and IgG-positive B cells were observed at 1 day after infection and gradually increased in n u m b e r thereafter (Table 2).
RESULTS
T h e r e was no signal seen for any cytokines in non-infected kidney.TNF-ct and I F N - T m R N A were detected in infected kidney at 2 hours after infection, though no infiltrating immune-response cells were observed.These cytokine m R N A were thereafter present at all t i m e points in infected kidney. IL-6 m R N A was detected at 6 hours after infection, concomitantly with infiltration o f IgA-positive B cells. This cytokine m R N A was seen in infected kidney at 1, 3, 7, and 14 days after infection. I L - l a m R N A was detected at 1 day after infection and at 3 and 14 days after infection. I L - l a m R N A in the infected kidney correlated with increased infiltration of i m m u n e response cells (Fig. 3).
Expression of cytokine mRNA in kidney tissue Cellular infiltration in the pyelonephritic lesion In n o n - i n f e c t e d kidneys, a few Ly-1 reactive cells (intraepithelial lymphocytes) 2 were visible in renal pelvic m u c o s a (Fig. 2). Mac-1 reactive cells (histiocytes) were also found in the submucosa. At 2 hours after infection, infiltration of i m m u n e response cells was not detected. At 6 hours after infection, neutrophils, macrophages a n d T cells were observed in the pyelonephritic lesion. F r o m 1 day after infection, the degree of infiltration of these cells increased. IgA-positive B cells were observed at 6 hours after infection and increased there-
Table 1. The sequences of primers for inflammatory cytokines. mRNA species IL-la
Sequence upstream 5'- CTCTAGAGCACCATGCTACAGAC-3' downstream 5'-TGGAATCCAGGGGAAACACTG-3'
IL-6
upstream 5'-TGGAGTCACAGAAGGAGTGGCTAAG-3' downstream 5'-TCTGACCACAGTGAGGAATGTCCAC-3'
IFN-~,
upstream 5'- AGCGCCTGACTGAACTCAGATTGTAG-3' downstream 5'-GTCACAGTTTTCAGCTGTATAGGG-3'
TNF-tz
upstream 5'- GGCAGGTCTACTTTGGAGTCATTGC-3' downstream 5'-ACATTCGAGGCTCCAGTGAATTCGG-3'
fl-actin
upstream 5'-TGGAATCCTGTGGCATCCATGAAAC-3' downstream 5'-TAAAACGCAGCTCAGT~AACAGTCCG-3'
Length (bp) 309 155 243 307 349
IL, interleukin; IFN, interferon; TNF, tumor necrosis factor.
Fig. 2. A histologic section in the murine renal pelvis of noninfected kidney. A cryostat section was stained by the ABC method for T cells using Ly-1 (53-7.8). A few Ly-1 positive cells were detectable in renal mucosa. Original magnification, x 85.
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J Infect Chemother 1998;4:71-75 Table 2. Cellular infiltration a in infected kidney. After infection Non-infection Neutrophils Macrophages T cells IgA(+) B cells IgM(+) B cells IgG(+) B cells
+b +c -
2 hours
6 hours
1 day
3 days
7 days
14 days
+ + -
+ + + + -
+ -H+ + + +
+ -H-H++ + +
-H-H~ ++ + +
++ i it tit
++ + +
aCellular infiltration was graded as follows: - , no cell infiltration; +, n u m b e r of reactive cells was < 10 using light microscopy at x 200 magnification; +, number of reactive cells was in d o u b l e figures; -H-, n u m b e r of reactive cells was in 3 figures; I t t, n u m b e r of reactive cells was in 4 figures, bMac-1 reactive cells in submucosa (histiocytes). eLy-1 reactive cells in pelvic mucosa (intraepithelial lymphocytes).
Fig. 3. Reverse transcription-polymerase chain reaction detection of inflammatory cytokine mRNAs in murine kidneys. (A) T N F - a mRNA, (B) IFN-TmRNA, (C) IL-6 mRNA, (D) IL-|o~ mRNA, (E) fl-actin mRNA. T N F - a and I F N - T m R N A w e r e detectable at all time points in infected kidney. IL-6 m R N A was detectable at 6 hours and seen at 1, 3, 7, and 14 days after infection. IL-a m R N A was detectable at 1 day after infection and present at 3 and 14 days after infection.
DISCUSSION
To evaluate the local cytokine response to ascending bacterial pyelonephritis, we analyzed cytokine-specific m R N A in the murine kidney. Amplification of murine kidney c D N A revealed a reproducible time course of gene expression for IL-1, IL-6, TNF-c~ and IFN- 7. On the other hand, spleen homogenate from infected mice revealed no signal for these cytokines.These results suggested that the cytokines were produced in local response to bacterial pyelonephritis.
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T N F - a and I F N - y m R N A were detected in infected kidney at 2 hours after infection, although no immuneresponse cells infiltrated the pyelonephritic lesion. Immunohistochemical study revealed that Ly-1 positive cells were present in urinary mucosa of non-infected kidney (Fig. 2). It is known that normal intestinal mucosa contains intraepithelial lymphocytes. 2 Thus, it is assumed that Ly-1 positive cells in non-infected urinary mucosa represent intraepithelial lymphocytes. Recent studies have d e m o n s t r a t e d that intraepithelial lymphocytes are capable of producing cytokines such as
Local cytokine response in pyelonephritis
I F N - y i n response to bacterial adhesion at the mucosal surface. 1~ On the other hand, there are some reports that renal tubular epithelia can produce T N F - a in response to lipopolysaccharide.11 Though the source of T N F - a and I F N - y m R N A was not determined, the mucosal surface was the primary site of bacteria-host encounter. Ly-1 positive cells, intraepithelial lymphocytes, may e x p r e s s T N F - a and I F N - y m R N A in response to urinary mucosal challenge with bacteria before immune-response cells infiltrate. Forget al. 12reported thatTNF-tz and I F N ywere regarded as obligate precursors of further cytokine responses, including that oflL-6.Thus, Ly-1 positive cells represent intraepithelial lymphocytes that may further regulate mucosal local immune responses to bacterial pyelonephritis by p r o d u c i n g T N F - a and IFN-y. IL-6 m R N A was detected at 6 hours after infection, concomitantly with infiltration of IgA-positive B cells. In the intestinal mucosa, IL-6 stimulates the maturation of IgA-positive B cells, which secrete lgA. 13 According to our results, IL-6 has the same role as seen in the intestinal mucosa. IL-1 m R N A was detected at 1 day after infection. T h o u g h the source of IL- 1 m R N A was not clear in our experiment, IL-1 m R N A has been detected in uroepithelial cells after exposure to E. coll. 4 Thus, II_,-1 m R N A seemed to originate from uroepithelial cells. In addition, IL-1 has been reported to enhance endothelial cell adhesiveness for p o l y m o r p h o n u c l e a r leukocytes and to increase intercellular adhesion molecule1 expression. 14 IL-1 may help direct the polymorphonuclear leukocytes to the site of infection. In conclusion, our results demonstrated that through cytokine production the urinary mucosa plays an active immunoregulatory role against bacterial infections. Our results suggested that prior to infiltration o f i m m u n e response cells, bacteria-activated intraepithelial lymphocytes and histiocytes in the urinary mucosa produce T N F - a and I F N - y a s a primarycytokineresponse. These cytokines may influence adjacent uroepithelial cells and induce the migration of immune-response cells to the infected sites. IL-6 may be important for the IgA response as seen in the intestinal mucosa. IL-1 seemed to originate from adjacent uroepithelial cells and may stimulate more neutrophil infiltration by inducing intercellular adhesion molecule-1 expression.
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