Vol. 31, No. 1INFECTION AND IMMUNITY, Jan. 1981, p. 17-200019-9567/81/010017-04$02.00/0

Local Immune Response to Escherichia coli Pili inExperimental Pyelonephritis

JAMES W. SMITH,'2* SCOTT WAGNER,3t AND ROBERT M. SWENSON3Veterans Administration Medical Center, Dallas, Texas 752161; The University of Texas Health ScienceCenter at Dallas, Southwestern Medical School, Dallas, Texas 752352; and Temple University Health

Sciences Center at Philadelphia, Philadelphia, Pennsylvania 191403

The local immune response to pili of Escherichia coli 06:K13:H1 was deter-mined in experimental hematogenous pyelonephritis in rabbits. Pili purified fromsheared cells by ammonium sulfate precipitation were found to be pure byelectron microscopy and negative for lipopolysaccharide by limulus lysate assay.Antipilus antibody was detected in serum and newly synthesized protein frominfected animals with enzyme-linked immunosorbent assay. Serum and local(intrarenal) antibodies were of the immunoglobulin G class, were detectable byday 20 of infection, and persisted through 250 days of infection. These datasuggest that pili are present on the organism at the site of infection, since theyinduce the local synthesis of antipilus antibody in experimental pyelonephritis.

Considerable interest has been focused re-cently on the significance of common pili in thepathogenesis of pyelonephritis (5, 6, 10). Pili ofEnterobacteriaceae have been postulated to beimportant in the in vitro attachment of orga-nisms to uroepithelhal cells. However, there arefew data relative to the question of whether pilipersist on the organisms during and after actualinvasion of the kidney itself. If these structuresremain on organisms during the infection, thenpili should induce a local immune response, ashas been shown for other bacterial somatic an-tigens (7, 9). Consequently, in experimental an-imals with hematogenous pyelonephritis, wemeasured local and serum antibody response topili with the enzyme immunosorbent assay(ELISA) (7, 9). In these studies, antibody to thepili of the infecting Escherichia coli strain wasdemonstrated both in serum and at the localsite.

MATERIALS AND METHODSExperimental pyelonephritis. Experimental

pyelonephritis was produced in male New Zealandwhite rabbits as described previously by injecting 108E. coli 06:K13:H1 organisms intravenously while theright kidney was transiently obstructed (2). Animalsthat became infected were observed through thecourse of infection and were bled and sacrificed onvarious days after infection. Local syntheses ofprotein,immunoglobin, and specific antibody were determinedon minced kidney slices incubated with "4C-labeledamino acids as previously described (2, 9).Antigen preparation. Pili were purfied from E.

coli 06:K13:H1 as previously described (1). This E.coli strain agglutinated guinea pig, but not human A

t Present address: 209 Fairway Rd., Ambler, PA 19002.

17

or B, erythrocytes, and the agglutination was inhibitedby D-mannose. The pili were shown to be present byelectron microscopic examination. In brief, the E. coliwere grown in 10 liters of Trypticase soy broth (BBLMicrobiology Systems), and the cells were centrifugedat 4,000 x g for 20 min. The cell pellet was washedwith 100 ml of 0.01 M tris(hydroxymethyl)amino-methane-hydrochloride and recentrifuged. The cellswere suspended in 80 ml of 0.01 M tris(hydroxy-methyl)aminomethane-hydrochloride and sheared for2 min at high speed in a blender. The sheared cellswere centrifuged at 20,000 x g for 30 min to removecells and debris. The resulting supernatant was cen-trifuged at 150,000 x g for 16 h. Pellets were sus-pended, and the protein was precipitated with 45%ammonium sulfate. The resulting flocculent pellet wassuspended again in buffer and dialyzed to remove theammonium sulfate. The samples were lyophilized, andthere was a yield of 11 mg of pili from the incubationof approximately 10'3 bacteria. The material wasshown by the diphenylamine assay to be protein freeof DNA and by the Limulus radioimmunoassay to beprotein free of lipopolysaccharide (3). (The Limuluslysate immunoassay showed less than 0.1 jLg of lipo-polysaccharide per ml in a sample of pili at a concen-tration of 500 1g/ml, or less than 1 part per 5,000.)Sodium dodecyl sulfate-polyacrylamide slag gel elec-trophoresis showed a single band with a molecularweight of approximately 18,000. The dialyzed suspen-sion was fixed in 1% Formalin, placed on a copper grid,and negatively stained with saturated uranyl acetate.After drying, the specimens were examined in a Sie-mens Model 1A electron microscope. Electron micro-graphs revealed purified pili (1).Antiserum preparation. Normal adult male New

Zealand white rabbits were injected intravenouslywith increasing quantities of pili (10 to 100 ,Lg sus-pended in phosphate-buffered saline) at 5-day inter-vals for a total of eight doses. Other rabbits wereimmunized with one of the following materials, which

18 SMITH, WAGNER, AND SWENSON

were derived from the same lyophilized samples pre-pared from E. coli 06:K13:H1 as was the pill prepa-ration: (i) E. coli, which had been heated at 100°C for2 h to destroy the pili, in 1 ml injected intravenouslyevery 5 days for five doses (9); (ii) purified 06 lipo-polysaccharide, prepared by phenol-water prepara-tion, every 7 days for six doses (9); or (iii) murein-lipoprotein, given intravenously at 250 fig/ml every 5days for four doses (7). Animals given pili, heat-killed06 organisms, purified 06 lipopolysaccharide, or mu-rein-lipoprotein were bled at weekly intervals, andserum was separated and frozen until antibody deter-minations were performed.Antibody determination. Serum and local (intra-

renal) antibodies to 0 antigen and pili were deter-mined with the ELISA technique (7, 9). This methodhas been shown to detect local synthesis of antibodytoO antigen in fractions from diethylaminoethyl chro-matography of soluble protein after the incubation ofkidney slices with '4C-labeled amino acids (9). Polysty-rene plastic tubes were coated with 1 ml of pili at aconcentration of 10 Ag/ml (the concentration of pilishown to give optimal activity in ELISA with hyper-immune sera). Unattached antigen was removed bywashing the tubes. Tenfold dilutions of serum or kid-ney fractions were then added to the tubes. Thesewere mixed on a shaker at 22°C for 6 h. Unattachedprotein was then removed by repeated washing withphosphate-buffered normal saline (pH 7.4) containingTween 20. The antigen-antibody complexes were thenincubated with alkaline phosphatase conjugates ofgoat anti-rabbit immunoglobulin G (IgG), IgM, or IgA,after which the enzyme activity of the bound conju-gates was measured spectrophotometrically by addingthe substrate p-nitrophenyl phosphate. In ELISA, thecalculated titer was the logio of the reciprocal of theserum or kidney fraction which had an extinctionvalue 0.15 above the base-line level.

RESULTSImmunization of rabbits with purified pili led

to a significant antibody response after at least20 days. Serum antibody was demonstrated inthe IgG class at titers of 4.8 to 5.0 logio and wasdetectable for up to 4 months after immuniza-tion with eight injections. No antibody responseto purified pili was detected in animals immu-nized with heat-killed E. coli 06 or with purified06 lipopolysaccharide. However, these rabbitsproduced a significant antibody response to the0 antigen of E. coli (Table 1). Animals immu-nized with murein-lipoprotein of the outer mem-brane of E. coli had a brisk antibody responseto this antigen but had no antibody activity inELISA with pilus antigen. Also, two animalsgiven a single injection of 10i viable organismswithout ureter ligation produced no antibody topii.A serum antibody response in the IgG class to

the 0 antigen of the infecting organism wasdetectable by day 6 of infection (Fig. 1). Anti-body could be detected in both the IgG and the

TABLE 1. Antibody response to various antigens ofE. coli

Titer of serum antibody toa:Animal immunized

with: Pili 0 antigen Murein-lipo-protein

Purified pili 4.8 0 0Heat-killed E. coli 06 0 5.0 0Purified 06 antigen 0 4.0 0Murein-lipoprotein 0 0 5.2

a Antibody in ELISA is expressed as the log10 of the recip-rocal of the dilution of serum from rabbits after repeatedimmunizations. Antibody is detectable only if the extinctionvalue is 0.15 above the base line at 400 nm.

° 5-01

-JI.-4-

crw

I3-

cn- 2w

oO ANTIGENA Pili

8T 8 8 0 0

0

0

I

0

-A10 20 30 >40

DAYSFIG. 1. Serum antibody in the IgG class to 0 an-

tigen and pili measured by ELISA over the course ofexperimental pyelonephritis. The antibody titer wasexpressed as the log1o ofthe reciprocal ofthe dilutionof serum that had an extinction value 0.15 above thebase line.

IgM classes. The peak IgG level was at 12 days,and synthesis persisted for up to 250 days afterinfection. The peak IgM level was at 20 days,and synthesis persisted for up to 250 days afterinfection. No serum antibody to the pill couldbe detected through day 13 of infection, butserum antibody was measurable (in the IgG classalone) by day 30 (Fig. 1). The peak IgG level ofpilus antibody was achieved at 27 days, andantibody was detectable in animals through 250days of infection at 3 to 4 log1o. No antibody topili was detectable in the IgA or IgM class.

Synthesis of local antibody in the IgG class tothe 0 antigen occurred in these studies by day11 of infection, and levels reached a peak of 4logio by 13 days (Fig. 2). Synthesis of local IgGantibody to 0 antigen was detected in animalsthrough 250 days of infection, but at lower levels(2.2 to 2.7 logio). IgA and IgM antibodies to 0antigen was detectable in animals by day 13.Local synthesis of IgG antibody to pilus antigenwas detected by day 20 of infection (9 days afterdetection to antibody to 0 antigen). Synthesisof antipilus antibody by kidneys reached its peakby 27 days and persisted through day 240 at

INFECT. IMMUN.

IMMUNE RESPONSE TO PILI IN PYELONEPHRITIS 19

0 5° 5.

0

-j.

4.w

I~-

P 3.

jn

_j P.

* 0 ANTIGENA Pili

S

0

0

0

a

AA I

0

A

10 20 30 >40DAYS

FIG. 2. Local antibody in the IgG class to 0 anti-gen and pili measured by ELISA over the course ofexperimental pyelonephritis. The antibody titer withthe ELISA method was expressed as the log1o of thereciprocal of the dilution of kidney fraction that hadan extinction value 0.15 above the base line.

levels of 3.0 logio. No IgA or IgM local antibodywas detectable throughout the course of infec-tion.

DISCUSSIONPrevious studies which have evaluated the

importance of pili in the pathogenesis of infec-tion with Enterobacteriaceae have comparedthe in vitro attachment of piliated organismsand nonpiliated organisms to uroepithelial cells(4, 10). However, direct evidence of the in vivoimportance of pili on E. coli has been difficult toobtain. Thus, an indirect means for evaluatingthe presence and synthesis of pili by the invadingorganism is presented in these studies. Rabbitsinfected with an E. coli strain which possessedpili were shown to develop a serum antibodyresponse to pili of the infecting organism. Inaddition, tissue slices from infected kidneys alsosynthesized specific antibody (local) to the piliof the infecting organism.The onset of this local and systemic immune

response to the pili was delayed, compared withthe antibody responses to the 0 antigen and themurein-lipoprotein of the infecting organism (7,9). A serum response to these antigens was de-tected 2 weeks before response to pili and 7 daysbefore local synthesis ofantibody could be meas-ured with pilus antigen in ELISA. One expla-nation for this delay could be that a small quan-tity of pili is present in vivo, and thus a longertime is required for a local anitbody response.This seems possible, since 100 ,ug of purified pili(derived from 1011 organisms) was required toimmunize normal rabbits, whereas infected rab-bits were given only 10' organisms intrave-nously. Another possibility is that early in infec-tion the organisms are in log-phase growth,which is not conducive to pili synthesis by or-

ganisms (6, 10). Perhaps pili are synthesized only

later in the kidney, during the stationary phase.It is unlikely that synthesis of antibody to othercell surface components would bind to pilusantigen and block the appearance of anti-pilus antibody, since antisera from animals giventhese other antigens did not cross-react inELISA with pili. The antibody detected in in-fected animals was specific antipilus antibodyand not antibody to another cell surface com-ponent which cross-reacts with pili.The functional importance of the local and

serum antibodies to pili remains unknown. Pilihave been shown to be an important determi-nant of attachment of E. coli to uroepithelialcells (4, 10). Antibodies synthesized locallymight prevent attachment of bacteria to theuroepithelial cells, but this would be a late phe-nomenon at the infected site. Although antibodyto pili was not demonstrated in urine in the fewinfected animals in which it was tested, previousstudies have shown that antibody to 0 antigenin present in the urine and can attach to bacteria,giving rise to antibody-coated bacteria (8). Stud-ies are in progress to determine whether theantibody which coats bacteria is also directedagainst pili. Further evaluation will also be re-quired to determine whether antibody directedagainst pili can inhibit the attachment of bac-teria to renal epithelium. Detection of antibodyfor up to 250 days after infection indicates thatpili are likely present as immunogens at theinfected site for a long period. Thus, these stud-ies provide substantial evidence that the hostprovides the proper milieu for E. coli to synthe-size pili at the site of infection in sufficientquantity to provoke a local and systemic im-mune response.

ACKNOWLEDGMENTSThis work was supported by program-directed research

funds from the U.S. Veterans Administration.We thank Robert Munford for performing the Linulus

radioimmunoassay. We also express our appreciation to Mar-tha Jane Adkins for her long-term involvement in these stud-ies.

LITERATURE CITED

1. Buchanan, T. M., J. Swanson, K. K. Holmes, S. J.Kraus, E. C. Gotschlich. 1973. Quantitative determi-nation of antibody to gonococcal pili. Changes in anti-body levels with gonococcal infection. J. Clin. Invest.52:2896-2909.

2. Lehmann, J. D., J. W. Smith, T. E. Miller, J. A.Barnett, and J. P. Sanford. 1968. Local immuneresponse in experimental pyelonephritis. J. Clin. Invest.47:2541-2550.

3. Munford, R. S. 1978. Quantitative limulus lysate assayfor endotoxin activity: aggregation of radioiodinatedcoagulogen monomers. Anal. Biochem. 91:509-515.

4. Orskov, I., F. Orskov, A. Birch-Andersen. 1980. Com-parison of Escherichia coli fimbrial antigen F7 withtype 1 fimbriae. Infect. Immun. 27:657-666.

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20 SMITH, WAGNER, AND SWENSON

5. Salit, I. E., E. C. Gotachlich. 1977. Type 1 Escherichiacoli pili: characterization of binding to monkey kidneycells. J. Exp. Med. 146:1182-1194.

6. Silverblatt, F. J. 1974. Host-parasite interaction in therat renal pelvis. A possible role for pili in the pathogen-esis of pyelonephritis. J. Exp. Med. 140:1696-1711.

7. Smith, J. W. 1977. Local immune response to lipoproteinof the outer membrane of Escherichia coli in experi-mental pyelonephritis. Infect. Immun. 17:366-370.

8. Smith, J. W., S. R. Jones, and B. Kaijser. 1977. Signif-

INFECT. IMMUN.

icance of antibody-coated bacteria in urinary sedimentin experimental pyelonephritis. J. Infect. Dis. 135:577-581.

9. Smith, J. W., and B. Kaijser. 1976. The local immuneresponse to Escherichia coli 0 and K antigen in exper-imental pyelonephritis. J. Clin. Invest. 58:276-281.

10. Svanborg, E. C. and H. A. Hansson. 1978. Escherichiacoli pili as possible mediators of attachment to humanurinary tract epithelial cells. Infect. Immun. 21:229-237.