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Evidence that patients with rheumatoid arthritis have asymptomatic ‘non-significant’ Proteus mirabilis bacteriuria more frequently than healthy controls
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(I/ Irr/~‘c~llorr I1 YYY) 3x. YY-1 Oh
i
Evidence that Patients with Rheumatoid Arthritis have Asymptomatic ‘Non-significant’ Proteus mirabilis Bacteriuria more Frequently than Healthy Controls B.W. Senior*‘,
G.A. Anderson’,
K.D. Morley2 and M.A. Kerr’
OhjrctivPs:
patients with rheumatoid arthritis (RA) are reported to have in their sera raised levels of antibodv specific to The aim of the study was to verify this and to determine an explanation for it by investigating the frequency of F! rJrirrrbi!is urinary tract infection in RA patients and matched controls. Md~ods: freshly voided urine was examined for the presence, number and identity of infecting bacteria. The levels of antibody in blood and in urine of the IgM, IgA and IgG classes to the common 0 serotypes of l? mirrrbilis and the antigens to which they reacted were determined by enzyme-linked immunosorbent assay (ELBA) and immunoblotting. R~srrlts: analysis of urine from 76 patients with RA and 18 age- and gender-matched healthy controls showed that only two (4%) of the control urines but 2 5 (33%) of those from the RA patients were infected. The commonest infecting organism in the RA patients’ urine was Protms JJlirrrbilis which occurred twice as frequently as Esclwrid~irr coli. Prohws mirrrbilis was found in 52% of the infected urines of the RA patients and was always detected as a pure growth and usually in insignificant (< I W/ml) numbers. It is highly improbable that this finding was the outcome of differences in age, physical ability or medication between the RA and control patient groups. Comparison of antibody levels to t? mirnhilis by EIJSA showed RA patients had raised (P < 0.000 1, P = 0.001, P = 0.006 3) levels of IgA, IgG and IgM respectively in their sera and raised (P < 0.0001, P C 0.0001, P = 0.0001) levels of I@, IgM and IgA respectively in their urine compared with the control group. It was not possible to detect an antibody reacting to a l? mirnhilis antigen that was specific to the RA patients. Corrclfrsion: the results confirm that RA patients have raised levels of antibody to I! r?hddis not only in blood but also in urine and suggest that this arises because RA patients have an asymptomatic. non-significant I! rrrirctbilis bacteriuria more frequently or more prolonged than control patients. This may be the trigger for their RA condition. Protcrrs
rnirabilis.
Introduction Rheumatoid arthritis (KA) is a chronic inflammatory disorder particularly of peripheral small joints which may also involve other joints and organs. It is the commonest inllammatory arthritis affecting middle-aged and elderly people and is more frequent in women than men. In most patients, rheumatoid factors (RF)-antibodies to the Fc region of immunoglobulin G (&G-can be found in the strum and synol+al fluid. The cause and pathogenesis of the disease are complex. It is known that there is a strong genetic predisposition to KA that is associated with some HLA-DR4 and DRl molecules.‘~ i Approximately 90% of patients with severe RA have been found to have HLA-DR haplotypes that contain the five amino acid motif O(K/R)KAA (glutamine, Iysine/arginine. arginine. lysine. lysine) in the third
hypervariable region of their p chains.4,i It has been proposed that T cells, positively selected in the thymus by lowaffinity interactions with self-major histocompatability complex (MHC) peptides. may be later triggered in the periphery upon exposure to similar but foreign crossreacting peptides which could be deri\,ed from infecting micro-organisnis.fl,T The QKKAA sequence has been found in such di\rerse micro-organisms as Epstein-Barr virus (KBV), Lr7ctocw~cus lactis, Hnu~~llr7 m?s and the dnaJ heat-shock protein of /isthel-ichirr dic3 Sm ’ I and KA patients with Hl,A-L)K haplotypes containing the QKRAA sequence have been shown to respond more strongly to this antigen on these organisms than normal control patients.12 It is likely that the QKRAA sequence will be found in antigens of other micro-organisms. The association known to exist between RA and the elderly, particularly women, is suggestive that a micro-organism associated with urinary tract infection may bc involved as a trigger for KA because this type of infection is most frequently found in elderly women. tl strong contender ( 1 YYYThc
British
Inkclion
Societ\
B.W. Senior et for this role is PIYUNLS r7hhilis because it is frequentI! found in urinary tract infections of the elderly. and particularly so in women of 60 years or more. ’ : Bbi-inger and colleagueslA first showed that RA patients had raised antibody levels specific for E minrbilis and that antisera to HI,&I>R4 lymphocytes reacted only against PJ-O~WS and not against many other bacteria tested.‘+ Other groups working indcpendcntly have subsequently confirmed these findings.‘im’; Raised antibody levels to P~~tc~s appear to be specific for KA for thcsc arc not found in patients with other autoimmunc conditions such as systemic lupus erythcmatosus. or the inflammator~~ arthritis of sarcoidosisli or ankylosin:: spondylitis.lq Is All these things point to 1-1 r~7il-rhilis being an aetiological agent of RA. The purpose of this study was to imwtigate the hypothesis by determining whether or not I? rttird~ilis M’S found more frequently in the urine of RA patients than controls, and comparing the levels and type of antibod!, responses to Pr-ok~rs in serum and urine of the RA patients bvith those of the control group.
Materials
a/.
overlayered to preiwt growth at the agar surface. All plates were incubated at 3 7°C overnight. The presence of 2 10’ colonies/ml of urine of one colony type was recorded as significant bacteriuria; 2 10’ colonies/ ml of urine of more thari one colony type as mixed growth; and < 10-l colonies/ ml of urine as no significant bacteriuria or, if appropriate. as no growth. I lrinc inoculated I IalS-clindamycin plates that remained yellow wcrc recorded as negative. i.e.. no ureadegrading organisms were present in I ml urine. Those that turned pink were recorded as positi\Te. i.e. urea degrading, Gram-ncgativc. clindamqicin-resistant organisms wcrc present in 1 ml urine. ‘l’hc pink colonies on such plates were suspected as being of I’JV~~ILSspp. and they were counted. The presence and number of suspect I’r0t~76 colonies (translucent. blue, i.e.. lactose nonfermenters) on CI,EI> plates \v;is also recorded. Representatic’es of all these types of colony M’ere isolated in purity and their identity determined by standard biochemical tests.“’ The identit!, of lactose-~ermentiti~ organisms on CLW was made by application of other standard biochemical tests. was
and Methods
The RA patients \h:ere known local patients attending the Rheumatology outpatient clinic who satisfied the diagnostic criteria for RA”’ and in whom clinical evidence ol urinary tract infection was not present. The control group comprised healthy people \vithout RA whose gender and age range was similar to that of the Rti patients. A fresh midstream specimen of urine was collected in a sterile container from each patient and stored at 4°C. After bacteriological analysis. sodium axide was added to the remainder to 0.04% w/v. A sample of \‘enous blood was taken from some patients. f\fter it had clotted and been centrifuged at 2OOOg foi- 5 min. the clear seruni was removed aseptically and stored at - 20°C’.
Within 2 h of collection. a standard loopful (0.004 ml) of each urine specimen was streaked out over a CLEL) agar (Oxoid CM 301 ) plate. In addition. to detect bacteria prcsent in small numbers. 1 ml of urine was pipetted into a sterile petri dish and mixed with 12 ml molten cool ITrea agnr Base (IJAB) medium (Oxoid CM 45) containing clindamycin ( 5 mg/l). C’lindamycin was used to prevent growth of (kam-positi\re bacteria and the detection ol urease forming staphylococci in urine. When the agar had set. a further 3 ml of llAK containing clindamycin
The 1 1 commonest 0 serotypes of I-I r7~irrrllili,s associated with urinary tract infection” (Table I) \izrere grown overnight on CLEII) agar at 3 7°C’. The grw:th was harvested and suspended in phosphate buffered saline (PBSI (pH i.2) containing sodium aide (0.04% w/v) (PBSAz). The absorption of the suspensions WIS read at 550 nm and adjusted ulth PBSAx to 1 .O. Equal volumes of all 1 1 suspensions were mixed togcthcr and stored at 4°C. This standard pooled suspension was diluted 1 in 10 in I’BSAx and 100 ~1 amounts added to each well of a microtitration plate (Nunc Maxisorp Fti. Life Technologies. Paisley, IJ.I<.). r\fter the co\wed plate had been left overnight at room temperature. the contents of the wells were removed and discarded. The wells were ti:ashed three times in PBSAx containingT~~,een 20 (0.05% X,/v) (PKS’I’). Two hundred microlitres of I’HS’I containing bovine serum albumin (0.05%) were added to each knell to block non-specific binding of any protein subsequently added, and the plate was incubated for 1 h at room temperature. The contents of the \l,elts were discarded and the wells washed three times with PBST. One hundred microlitrcs of tither neat urine or serum diluted 1 in 100 with I’KS’I’ were added in duplicate to each well and the plate was further incubated for 2 h at room tempcraturc. ‘l’hc well contents were then discarded and the wells \vashcd three times with I’RS’I’. ‘1’wo hundred microtitrcs of goat alltalinc phosphatase-conjugated IgM anti-human
P. mirabilis
and Rheumatoid
0 serotype 1’13h 1’111 I’13 I 5
0 3 0 6
F 0; 1’134x
0 10 0 11 0 I 3
SW 39 SW 19 I’H i
0 23 0 21 0 1;
1’13 1 3 S\‘l’ 74 S\‘17 79
0 2s 0 29 0 30
(u chain specific-Sigma X 32 75) or IgG (y chain specific-sigma A31 50) or IgA (a chain specific-Sigma A3300) diluted 1 in 1000 in PBST were added to each well as appropriate. The plate was then incubated at room temperature for 2 h. The well contents were then discarded and the wells washed three times with PBST. Two hundred microlitres of p-nitrophenyl phosphate solution (!I nitrophenyl phosphate tablet-Sigma X2765 and Tris buffer tabletSigma ‘I8790 suspended in 20 ml of distilled water) were added to each well and the plate incubated at 3 7”C, for 1 5 min when analysing sera but for 30 min when analysing urine. The absorption at 405 nm of each well was then immediately, read by a Dynatech 5000 plate reader.
101
Arthritis
a stacking gel of acrylamide 4% in 0.12 5 M Tris-HCI, pH 6.X. SDS 0.1% over a resolving gradient gel of acrylamide 5515% in 0.375 M’I’ris-HCl, pH X.X. SDS 0.2%. The upper tank buffer was 0.053 M Tris, 0.052 M glycine. SDS 0.1%. pH 8.9. amd the lower tank buffer was 0.1 M TrisHCl. SDS 0.1%. pH X.1. Electrophoresis was performed at 2 5 mA in the cold until the dye front reached the bottom of the gel. The separated proteins were transferred to a nitrocellulose membrane by electrophoresis in methanol 10% containing 2 5 mM Tris and 190 mM glycine at 10 V for 16 h at 4°C. The membrane was stained with Ponceau S dye (0.2% in trichloroacetic acid 3%) and the position of the reference protein standards marked. After washing the membrane in PBS, it was agitated for 2 h in dried milk powder (Marvel) 5% in PBS. The membrane was then cut into strips and each agitated in PBS-marvel containing a 1 in 50 dilution of the patients serum or urine for 2 h at room temperature. The strips were washed in several changes of PBS and then agitated for 2 h at room temperature with PBS-Marvel containing a 1 in 500 dilution of the appropriate class of human antibody labelled with alkaline phosphatase. After thorough washing in PBS. the strips were developed in 30 ml buffer (100 mM NaCl. 5 mM MgCl,, 100 mM Tris-HCl. pH 9.5) containing 100 ul bromochloro-indolyl phosphate (50 mg/ml in dimethyl formamide) and 200 yl nitroblue tetrazolium ( 50 mg/ml in dimethyl formamide 70% in water).
Stntistifs
Fifty microlitres of serum diluted 20-fold in glycine-saline buffer pH 8.2 were mixed with 50 pl of neat RF latex reagent (Biostat Diagnostics, Stockport. LJ.I<.) on a tile which was rocked for 2 min and then examined for agglutination of the IgG-coated latex particles. Positive and negative sera were included in each batch of tests. Agglutination indicated that the serum had an RF content of 2 20 ILl/ml. Doubling dilutions of such positive sera were re-tested. The RF concentration was equal to the highest dilution giving visible agglutination of the latex particles.
Washed cells fom the suspension of the pool of I? mimbilis strains of different 0 scrotype were mixed with an equal volume of sample buffer (0.12 5 M Tris-HCl pH 6.X. SDS 4%. glycerol 20%. mercaptoethanol 10% and a trace of bromophenol blue dye), boiled for 5 min and applied along with standard protein molecular weight markers to
The significance of ELISA results was examined by determination of Mann-Whitney and two-tailed I’ values with a Graphpad Instat programme.
Results The RA patient group of 76 from whom 3 5 blood samples were obtained were of age range 24&N years and comprised 65 females and 11 males. The healthy control group of 48 from whom 2 7 blood samples were obtained were of age range 2 l-79 years and comprised 42 females and 6 males. The age and sex distribution of both groups of patients was similar and is presented in Figure 1. The average age of the female and male RA patients was 58 and 62 years respectively whereas that of the female and male control patients was 59 and 61 respectively.
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Rheumatoid factor (RF) was measurable in 29 of the 3 5 sera from the RA patients with levels of 50-l 140 lLJ/ml.
B.W. Senior
”
21-30
31-40
41-50 51-60 Aee i "ears )
61-70
71-80
RF was not detectable in any ol’ the L 7 sera Ii-om the cwtrol patients. llrine samples of 6 RA patients with high (i.e. > 875 TlJ/ml) serum RF levels were also examined but RI: was not detectable in any ol’ them.
‘fhe results ol’ the bacteriological analysis of the urine specimens oi’ the ~M’Ogroups arc‘ presented in Table II. I:rom this it can be seen that whereas Ih (96%) of the 48 specimens from the control group were sterile only 5 1 ((77%) of the ih urines li-om the RA group were stcrilc.
IOOI 3 il
4s IIOOI 1i-t)
Ii
0 IO1
1-I)
II21
9 I
1121
II
0 lOi
II
0 lOi
31
0 IOI
et al.
‘I’he organism found most l’requently in the inkted RA group urines was I-I r~~imbilis.‘I’his organism was l’ound in purity in 1 3 (SL’%) of the inkcted urine specimens of the RX patients and nearly twice as li-equently as the next common organism, I:. cdi. I-1r,tii-r&i/is was found in pm-it) in significant numbers in two ( 3%) and in ‘insignificant’ (i.e.. < IO-’ cfdml) numb-s in I 1 ( 14%) 01’ lhe if, urine specimens fi-om the R;\ patients. ‘I’his organism was not 1‘ound in significant numbers in any of the -I-S urines 01 the control group and was present in onI!, one (2%) 01’ them in ‘irisignilicant numbers. ‘fhe proportion of the inlkted urine female Iii\ patients M’ith I! rrlirrfhilis increased from 1 1Y, I’or those aged 5 l-C70 years lo 17.4% years to 35% for those aged Ibr those aged h I-/OY, 7 l-80 years. I: cdi was I’ound in urine in sigiiificant nunbcrs in seven (9%) of the RX patients hut in only one (Z’X,) urine of the control group.
01’ the 3 5 sera wailahlc from the RA group. there were only 11 that matched closely those of the control group with regard to patient gender and age. These were’ selected la- 13,ISA studies. ‘rhe ELIStI results of Igbl. IgC; and TgA antibody levels to the common 0 scrotypes 01 I-1~~irdilis in these sera are presented in Figure .? and ‘I’able III. ‘I’he RX patients sew were l’ound to have extremely significantly (I’ < 0.000 1 I or wry signilkmtl~ (P = 0.0010. I’ = 0.006 i) higher Igr\. Ig(: and IgbI antibody levels respect i\rely to II rrrirdlilis over thosc of 1he control group.
Antibody le\~els to I! ~rtiurbilis in the urine Ii-om the SIIIIIL’ 11 matched Rii patients and 2 1 control subjects kverc also determined by ELISA. ‘I’he results arc presented in b’igure 3 and Table 11’. The IgM. IgG and IgX Icvels to j-1rtlir-dilis in the urine of the KX patients were I’ound to be extremely significantly (I’ < 0.000 1, I’ < 0.0001, P = 0.0001 I raised respectively o\w those of’ the control group.
An analysis of the immunoblots of w-a ol’ RA and control group patients with high antibody Icvels to I? rrrir-~7bilis against lysntes ol’ E r~~irrrhilisshowed that all classes 01 ant ibodies against many I? ~nirnbilis antigens were present in both RA and control patients’ set-a. IM’ercnces in banding patterns with regard to the number. position and
P. mirabilis 1.00.
and Rheumatoid
Arthritis
103 1.00 Id
IgG
0.75.
0.75
..
. . 0.25
* . ::,A ..
.
8
0.25-
. Ar\
l:
l .* . ..* $1
A:A AA .:.
..:A
::*
. .
.:& .
.!$l .* :
0.00
Controls RA patients Serum samples
o.oc
Controls RA patients Serum samples
IL\ naticnts r\o. of samples ‘1 21 21
No. of samples
0.2x f 0.1 7 0.2 5 * 0.11 0.25*0.13
21 II
ent as determined by ELISA and the number or intensity of the bands given by it for some sera with low ELBA values gave many bands whereas some sera with high ELBA values gave few bands. It was not possible to detect an IgG. IgM or IgA antibody reacting to a given antigen that was exclusive to the RA patients. Analysis of immunoblots of nine I? rJCrnbilis infected urines from KA patients and one uninfected urine from the control group which had high I& levels to P mir-dCs as determined by ELI&l, against lysates of I? nGdGlis showed there were fewer IgG antibodies to I? mirabilis antigens in the urines than in the sera. IgG antibodies reacting to 97- and 7X-Ida antigens and an IgA antibody reacting to a 30-klk1 antigen were detected in several, but not all. of the infected urines from the RA
Controls RA patients Serum samples
C‘ontrol patients
CLISA * SD
intensity OS bands were observed for IgM. IgG and IgA and for both groups of patients. Sometimes there appeared to be no correlation between the amount of antibody pres-
patients.
0.25
21
ELISA i- ST) 0.1 ; i 0.04 0.14i0.09 0.08 k O.Oh
I’-value O.OOh j 0.0010 < 0.000 1
Discussion Rheumatoid arthritis (KA), like other autoimmune diseases, is strongly linked with women. Those who are middle-aged or elderly are three times more likely than men to develop the disease. KA patients characteristically have periods of remission alternating with periods of exacerbation of inflammatory arthritis. This suggests there may be factors, possibly microbial, that trigger the condition.
Women also have urinary tract infection (IJTI) more frequently than men. In middle-aged and cldcrly women the infecting organism is often I? mir-rrhilis.’ 1 It has been suggested that RA is a Proteus-reactive arthritis triggered by recurrent or asymptomatic urinary tract infections through antibodies to Prvtrrrs spp. cross-reacting with cells expressing HLA-DR4 antigens? This study attempted to examine whether or not RA patients had I? rnilahilis infected urine more frequently than control patients
and the immunological
consequences
of this.
B.W. Senior
104 1.00
et al. 1554
IgM 1.00
.
. 0.76
2 $ 2
0.50
3
.
2
0.25
0:.
l*
0.00
Controls IJrine
lgll I$; IL!,\
RA patients samples
II .?I II
..*..
Controls llrine
0.2 1 k 0. IO 0.2; i 0. I : 0.1 ; * 0.05
1111the patients in the KA group were known to have rheumatoid arthritis and most of them had detectable RF in their scra whereas the patients of the control group were free from KA and Kl: was not detected in any of them. In other respects regarding age and gender. the two groups of patients were very similar. Because of this it was surprising to find that whereas. as was expected, virtually all (96%) of the urines of the control group were sterile, 2 5 ( 3 3%) of the RA patients. who otherwise were healthy and free of clinical symptoms of urinary tract infection. had infected urine. This could not he explained on the basis ol differences in number in the two groups of patients in the age range most likely to get urinary tract infections (i.e.. those above 50 pears) because the proportions of the males and females in both groups were sery similar and almost identical. namely. 8 1.8% female and 80% male in the KA group and X0.9’%,fcmak and 8 3% male in the control group. Nor is it likely to bc an outcome of the greatel difficulty the KA patients may have had in maintaining
RA patients samples
.?I 11 21
rrf.r
Controls Urinr
0. I I) * 0.05 0, I I IL (l.Oh (1. I1 * 0.01
RA patients samples
< 0.000 I < 0.0001 0.000I
personal hygiene because none of the infected urines of the RA patients gave rise to a mixed gro\vth of faecal types of bacteria such as is found when there is faecal soiling around the area where the urine is collected. Instead each infected urine was a pure growth of one organism. The organism most frequently encountered in the RA patients’ urine was I? rlrimhilis. It was dctcctcd t\s+e as frequently as I:‘. rdi which. in normal people, is by far the commonest urinary tract pathogen. I? Itlirdlilis was found in 1 3 ( 1 7%) of the KX patients’ urine (and in 5.2% of those with infected urine) but in on1y one (.?‘!Y,J of the COP trol patients’ urine. Information was not available of medication which any of the individuals of either the KA OI the control group might have been receiving. That such medication might be an explanation for the more frequently infected, particularly with /? ~~jr~~l~ili.s. urines in the R/1 group is thought to bc improbable because it would need to cause im increase in susceptibility to infcction specifically to I’r~olvrfso\rer that of I:. c~li.
P. mirabilis
and Rheumatoid
In most of the P rJlirnhilis infected urines of the RA patients, the organism was found in numbers that would be regarded as being too low to constitute a significant bacteriuria. Hence it was not surprising that none of them had clinical symptoms of IJTI. The presence of these smaller numbers of I-1rJJir-&lis was revealed through the sampling of a larger volume ( 1 ml) of urine than is normal in the analysis of urine for significant bacteriuria. If the urine had been examined by conventional methods in which only a small volume C. 4 1.11 of urine was examined. in many instances bacteria would not have been detected. Although the results above which indicate a more frequent carriage of I? mirrrbilis (often in low numbers) in the urinary tract by RA patients than controls are at variance with those of McDonagh it OS. they are in agreement with the work of others2’,2i and they give support to the hypothesis that KA may have developed in them as a result of a subclinical ITT1 with a small number of I! mimbilis bacteria providing a frequent or prolonged exposure to I? mitnhilis antigens. Moreover. an earlier comparative analysis”’ of the types of 1-Irnirrrhilis strains found in the urine of RA and control patients which showed that the types of strains found in RA patients are often different from those found in controls in being of types that are either more virulent or persistent or both, substantiates this idea. Although Deighton and colleagues’; were unable to find antibody to 1-1rJGrnhilismore frequently in the urine of KA patients than controls by indirect immunofluorescence with an unclassified immunoglobulin to one serotype of 1: mirddis. the ELISA results in this study of the levels of the different classes of antibody in urine to a pool of l? rnirddis strains of different common 0 serotypes showed unequivocally that the levels of IgM. IgG and also IgA for P r~~ilabiliswere raised extremely significantly in the RA group compared with the control group. Furthermore they support the interpretation of the results of the bacteriological analysis of the urines and suggest that in RA patients. the P utirrrhilis antigenic stimulus is both frequent and prolonged. If this is true it would explain the finding of significantly raised IgM levels to I? rJk&ilis in the sera of RA patients reported previously I ; and again here. It was not possible from the immunoblot analyses in this study to find a F! rnir-c&ilisantigen that was reactive in all the KA sera and unreactive in the control sera. However, two antigens which may be important are the haemolysin-associated protein Hpm B and the urease of P JJJinrhilis.These proteins contain respectively the polypeptide sequences ESRRAL and IRRET and are similar in molecular structure to the amino acid motifs QRRAA and LRREI that are associated respectively with RA and type XI collagen. Although in a previous study’; we were
Arthritis
105
unable to detect antibody in RA sera that reacted with HpmB, others have reported that sera from RA patients. but not those from patients with ankylosing spondylitis or controls, had raised antibody levels to the ESRRAL polypeptide and Pmteus haemolysin and urease.‘”
References 1 Stastny I? Mixed Ivmphocpte culture typing cells kom patients lvith rheumatoid arthrk. Tissw :\r~tiqer~s 1 YT4: 4: 5/l-Sic). 2 Lanchbury JSS. hIolecular genetics of the IILrl-I) region component ol’ inherited susceptibility to rheumatoid arthritis. fjr 1 Klw~r~~~rrlol 19X8:27: 1;1-175. 3 Harris CL). Kheumatoid arthritis. Pathophysiology and implications lirr therapy. S f:ryl \ .Lfwl 1 YYO: 322: 11 iT-I ‘Xc). 4 Oilier 1\: Thomson \k! Population genetics 01‘ rheum:~toitl arthritis. KIIPUIII Ilk C‘lifl s A!,! 199.2: 1x: r41-761. 5 ;\lhani S. Carson DA. Ruudier J. Genetic and enrironmrntal l’actors in the immune p;lthogenesis 01 rheumatoid arthritis. I
X Kwdicr J. Petersen J. Khodcs (;[I. Luka J. C’arson DA. Suscrptibility to rheumatoid arthritis maps to a ‘I-cc‘ll epitope shared by the Ill,;\I1w-l I)K p- 1 chain and the Epstein-Barr virus glycoprotrin gp I 10. PI-W Ytrtl ,hrl Sri USA I YXY: 86: 5 101-5 1 OX. Y ;Ubani S. ‘I’ucltwell JE. Esparza I,. Carson DA. Koudier J. The susceptibility sequence to rheumatoid arthritis is a cwss-rcactivc B cell epitape shared by I:sc~/wr-ic,flitr coli heat shock protein dnaJ and the hiatocompatihilit~ leulwcytr antigen UKBl *O-I01 molecule. / C‘lirr Irl~wt lYY1:89: 327-311.
17 Senior l<\L’. McBride l’L)E Morley I<. krr hlA. The detection 01‘ raised levels of Ig’ll to I’rotcws Itlit-trhilia in sew l’rom patients \vith rhrum+ toid arthritis. 1 Mrtl Aficwbiol lYY5: 43: 1 ih-1 X1. 18 Ebringer A. Cox NL. ,\huljadayel I ef (II. lif~~l~.\i~~llnantibodies in anlqlosing apondylitis and I’rotcws altihodies in rheumatoid arthritis. ET-/ Khr~rr~ttrf~l 198X: 27 Suppl II: iL-X 5. I Y Arnett AK. Mworthy FC. Bloch IM CT (11.The ,\merican Rheumatism Association 1 YXi revised criteria for the classilication of rhcum;ltoid arthritis. Ar-llrritis Klw~rrtr 1 YXX: 31: 3 1 T-114.
106
B.W. Senior
et al.
(I/ Irr/~‘c~llorr I1 YYY) 3x. YY-1 Oh
i
Evidence that Patients with Rheumatoid Arthritis have Asymptomatic ‘Non-significant’ Proteus mirabilis Bacteriuria more Frequently than Healthy Controls B.W. Senior*‘,
G.A. Anderson’,
K.D. Morley2 and M.A. Kerr’
OhjrctivPs:
patients with rheumatoid arthritis (RA) are reported to have in their sera raised levels of antibodv specific to The aim of the study was to verify this and to determine an explanation for it by investigating the frequency of F! rJrirrrbi!is urinary tract infection in RA patients and matched controls. Md~ods: freshly voided urine was examined for the presence, number and identity of infecting bacteria. The levels of antibody in blood and in urine of the IgM, IgA and IgG classes to the common 0 serotypes of l? mirrrbilis and the antigens to which they reacted were determined by enzyme-linked immunosorbent assay (ELBA) and immunoblotting. R~srrlts: analysis of urine from 76 patients with RA and 18 age- and gender-matched healthy controls showed that only two (4%) of the control urines but 2 5 (33%) of those from the RA patients were infected. The commonest infecting organism in the RA patients’ urine was Protms JJlirrrbilis which occurred twice as frequently as Esclwrid~irr coli. Prohws mirrrbilis was found in 52% of the infected urines of the RA patients and was always detected as a pure growth and usually in insignificant (< I W/ml) numbers. It is highly improbable that this finding was the outcome of differences in age, physical ability or medication between the RA and control patient groups. Comparison of antibody levels to t? mirnhilis by EIJSA showed RA patients had raised (P < 0.000 1, P = 0.001, P = 0.006 3) levels of IgA, IgG and IgM respectively in their sera and raised (P < 0.0001, P C 0.0001, P = 0.0001) levels of I@, IgM and IgA respectively in their urine compared with the control group. It was not possible to detect an antibody reacting to a l? mirnhilis antigen that was specific to the RA patients. Corrclfrsion: the results confirm that RA patients have raised levels of antibody to I! r?hddis not only in blood but also in urine and suggest that this arises because RA patients have an asymptomatic. non-significant I! rrrirctbilis bacteriuria more frequently or more prolonged than control patients. This may be the trigger for their RA condition. Protcrrs
rnirabilis.
Introduction Rheumatoid arthritis (KA) is a chronic inflammatory disorder particularly of peripheral small joints which may also involve other joints and organs. It is the commonest inllammatory arthritis affecting middle-aged and elderly people and is more frequent in women than men. In most patients, rheumatoid factors (RF)-antibodies to the Fc region of immunoglobulin G (&G-can be found in the strum and synol+al fluid. The cause and pathogenesis of the disease are complex. It is known that there is a strong genetic predisposition to KA that is associated with some HLA-DR4 and DRl molecules.‘~ i Approximately 90% of patients with severe RA have been found to have HLA-DR haplotypes that contain the five amino acid motif O(K/R)KAA (glutamine, Iysine/arginine. arginine. lysine. lysine) in the third
hypervariable region of their p chains.4,i It has been proposed that T cells, positively selected in the thymus by lowaffinity interactions with self-major histocompatability complex (MHC) peptides. may be later triggered in the periphery upon exposure to similar but foreign crossreacting peptides which could be deri\,ed from infecting micro-organisnis.fl,T The QKKAA sequence has been found in such di\rerse micro-organisms as Epstein-Barr virus (KBV), Lr7ctocw~cus lactis, Hnu~~llr7 m?s and the dnaJ heat-shock protein of /isthel-ichirr dic3 Sm ’ I and KA patients with Hl,A-L)K haplotypes containing the QKRAA sequence have been shown to respond more strongly to this antigen on these organisms than normal control patients.12 It is likely that the QKRAA sequence will be found in antigens of other micro-organisms. The association known to exist between RA and the elderly, particularly women, is suggestive that a micro-organism associated with urinary tract infection may bc involved as a trigger for KA because this type of infection is most frequently found in elderly women. tl strong contender ( 1 YYYThc
British
Inkclion
Societ\
B.W. Senior et for this role is PIYUNLS r7hhilis because it is frequentI! found in urinary tract infections of the elderly. and particularly so in women of 60 years or more. ’ : Bbi-inger and colleagueslA first showed that RA patients had raised antibody levels specific for E minrbilis and that antisera to HI,&I>R4 lymphocytes reacted only against PJ-O~WS and not against many other bacteria tested.‘+ Other groups working indcpendcntly have subsequently confirmed these findings.‘im’; Raised antibody levels to P~~tc~s appear to be specific for KA for thcsc arc not found in patients with other autoimmunc conditions such as systemic lupus erythcmatosus. or the inflammator~~ arthritis of sarcoidosisli or ankylosin:: spondylitis.lq Is All these things point to 1-1 r~7il-rhilis being an aetiological agent of RA. The purpose of this study was to imwtigate the hypothesis by determining whether or not I? rttird~ilis M’S found more frequently in the urine of RA patients than controls, and comparing the levels and type of antibod!, responses to Pr-ok~rs in serum and urine of the RA patients bvith those of the control group.
Materials
a/.
overlayered to preiwt growth at the agar surface. All plates were incubated at 3 7°C overnight. The presence of 2 10’ colonies/ml of urine of one colony type was recorded as significant bacteriuria; 2 10’ colonies/ ml of urine of more thari one colony type as mixed growth; and < 10-l colonies/ ml of urine as no significant bacteriuria or, if appropriate. as no growth. I lrinc inoculated I IalS-clindamycin plates that remained yellow wcrc recorded as negative. i.e.. no ureadegrading organisms were present in I ml urine. Those that turned pink were recorded as positi\Te. i.e. urea degrading, Gram-ncgativc. clindamqicin-resistant organisms wcrc present in 1 ml urine. ‘l’hc pink colonies on such plates were suspected as being of I’JV~~ILSspp. and they were counted. The presence and number of suspect I’r0t~76 colonies (translucent. blue, i.e.. lactose nonfermenters) on CI,EI> plates \v;is also recorded. Representatic’es of all these types of colony M’ere isolated in purity and their identity determined by standard biochemical tests.“’ The identit!, of lactose-~ermentiti~ organisms on CLW was made by application of other standard biochemical tests. was
and Methods
The RA patients \h:ere known local patients attending the Rheumatology outpatient clinic who satisfied the diagnostic criteria for RA”’ and in whom clinical evidence ol urinary tract infection was not present. The control group comprised healthy people \vithout RA whose gender and age range was similar to that of the Rti patients. A fresh midstream specimen of urine was collected in a sterile container from each patient and stored at 4°C. After bacteriological analysis. sodium axide was added to the remainder to 0.04% w/v. A sample of \‘enous blood was taken from some patients. f\fter it had clotted and been centrifuged at 2OOOg foi- 5 min. the clear seruni was removed aseptically and stored at - 20°C’.
Within 2 h of collection. a standard loopful (0.004 ml) of each urine specimen was streaked out over a CLEL) agar (Oxoid CM 301 ) plate. In addition. to detect bacteria prcsent in small numbers. 1 ml of urine was pipetted into a sterile petri dish and mixed with 12 ml molten cool ITrea agnr Base (IJAB) medium (Oxoid CM 45) containing clindamycin ( 5 mg/l). C’lindamycin was used to prevent growth of (kam-positi\re bacteria and the detection ol urease forming staphylococci in urine. When the agar had set. a further 3 ml of llAK containing clindamycin
The 1 1 commonest 0 serotypes of I-I r7~irrrllili,s associated with urinary tract infection” (Table I) \izrere grown overnight on CLEII) agar at 3 7°C’. The grw:th was harvested and suspended in phosphate buffered saline (PBSI (pH i.2) containing sodium aide (0.04% w/v) (PBSAz). The absorption of the suspensions WIS read at 550 nm and adjusted ulth PBSAx to 1 .O. Equal volumes of all 1 1 suspensions were mixed togcthcr and stored at 4°C. This standard pooled suspension was diluted 1 in 10 in I’BSAx and 100 ~1 amounts added to each well of a microtitration plate (Nunc Maxisorp Fti. Life Technologies. Paisley, IJ.I<.). r\fter the co\wed plate had been left overnight at room temperature. the contents of the wells were removed and discarded. The wells were ti:ashed three times in PBSAx containingT~~,een 20 (0.05% X,/v) (PKS’I’). Two hundred microlitres of I’HS’I containing bovine serum albumin (0.05%) were added to each knell to block non-specific binding of any protein subsequently added, and the plate was incubated for 1 h at room temperature. The contents of the \l,elts were discarded and the wells washed three times with PBST. One hundred microlitrcs of tither neat urine or serum diluted 1 in 100 with I’KS’I’ were added in duplicate to each well and the plate was further incubated for 2 h at room tempcraturc. ‘l’hc well contents were then discarded and the wells \vashcd three times with I’RS’I’. ‘1’wo hundred microtitrcs of goat alltalinc phosphatase-conjugated IgM anti-human
P. mirabilis
and Rheumatoid
0 serotype 1’13h 1’111 I’13 I 5
0 3 0 6
F 0; 1’134x
0 10 0 11 0 I 3
SW 39 SW 19 I’H i
0 23 0 21 0 1;
1’13 1 3 S\‘l’ 74 S\‘17 79
0 2s 0 29 0 30
(u chain specific-Sigma X 32 75) or IgG (y chain specific-sigma A31 50) or IgA (a chain specific-Sigma A3300) diluted 1 in 1000 in PBST were added to each well as appropriate. The plate was then incubated at room temperature for 2 h. The well contents were then discarded and the wells washed three times with PBST. Two hundred microlitres of p-nitrophenyl phosphate solution (!I nitrophenyl phosphate tablet-Sigma X2765 and Tris buffer tabletSigma ‘I8790 suspended in 20 ml of distilled water) were added to each well and the plate incubated at 3 7”C, for 1 5 min when analysing sera but for 30 min when analysing urine. The absorption at 405 nm of each well was then immediately, read by a Dynatech 5000 plate reader.
101
Arthritis
a stacking gel of acrylamide 4% in 0.12 5 M Tris-HCI, pH 6.X. SDS 0.1% over a resolving gradient gel of acrylamide 5515% in 0.375 M’I’ris-HCl, pH X.X. SDS 0.2%. The upper tank buffer was 0.053 M Tris, 0.052 M glycine. SDS 0.1%. pH 8.9. amd the lower tank buffer was 0.1 M TrisHCl. SDS 0.1%. pH X.1. Electrophoresis was performed at 2 5 mA in the cold until the dye front reached the bottom of the gel. The separated proteins were transferred to a nitrocellulose membrane by electrophoresis in methanol 10% containing 2 5 mM Tris and 190 mM glycine at 10 V for 16 h at 4°C. The membrane was stained with Ponceau S dye (0.2% in trichloroacetic acid 3%) and the position of the reference protein standards marked. After washing the membrane in PBS, it was agitated for 2 h in dried milk powder (Marvel) 5% in PBS. The membrane was then cut into strips and each agitated in PBS-marvel containing a 1 in 50 dilution of the patients serum or urine for 2 h at room temperature. The strips were washed in several changes of PBS and then agitated for 2 h at room temperature with PBS-Marvel containing a 1 in 500 dilution of the appropriate class of human antibody labelled with alkaline phosphatase. After thorough washing in PBS. the strips were developed in 30 ml buffer (100 mM NaCl. 5 mM MgCl,, 100 mM Tris-HCl. pH 9.5) containing 100 ul bromochloro-indolyl phosphate (50 mg/ml in dimethyl formamide) and 200 yl nitroblue tetrazolium ( 50 mg/ml in dimethyl formamide 70% in water).
Stntistifs
Fifty microlitres of serum diluted 20-fold in glycine-saline buffer pH 8.2 were mixed with 50 pl of neat RF latex reagent (Biostat Diagnostics, Stockport. LJ.I<.) on a tile which was rocked for 2 min and then examined for agglutination of the IgG-coated latex particles. Positive and negative sera were included in each batch of tests. Agglutination indicated that the serum had an RF content of 2 20 ILl/ml. Doubling dilutions of such positive sera were re-tested. The RF concentration was equal to the highest dilution giving visible agglutination of the latex particles.
Washed cells fom the suspension of the pool of I? mimbilis strains of different 0 scrotype were mixed with an equal volume of sample buffer (0.12 5 M Tris-HCl pH 6.X. SDS 4%. glycerol 20%. mercaptoethanol 10% and a trace of bromophenol blue dye), boiled for 5 min and applied along with standard protein molecular weight markers to
The significance of ELISA results was examined by determination of Mann-Whitney and two-tailed I’ values with a Graphpad Instat programme.
Results The RA patient group of 76 from whom 3 5 blood samples were obtained were of age range 24&N years and comprised 65 females and 11 males. The healthy control group of 48 from whom 2 7 blood samples were obtained were of age range 2 l-79 years and comprised 42 females and 6 males. The age and sex distribution of both groups of patients was similar and is presented in Figure 1. The average age of the female and male RA patients was 58 and 62 years respectively whereas that of the female and male control patients was 59 and 61 respectively.
Rhcurrzrftoid~irctou
1m3~swfw~fv1f
Rheumatoid factor (RF) was measurable in 29 of the 3 5 sera from the RA patients with levels of 50-l 140 lLJ/ml.
B.W. Senior
”
21-30
31-40
41-50 51-60 Aee i "ears )
61-70
71-80
RF was not detectable in any ol’ the L 7 sera Ii-om the cwtrol patients. llrine samples of 6 RA patients with high (i.e. > 875 TlJ/ml) serum RF levels were also examined but RI: was not detectable in any ol’ them.
‘fhe results ol’ the bacteriological analysis of the urine specimens oi’ the ~M’Ogroups arc‘ presented in Table II. I:rom this it can be seen that whereas Ih (96%) of the 48 specimens from the control group were sterile only 5 1 ((77%) of the ih urines li-om the RA group were stcrilc.
IOOI 3 il
4s IIOOI 1i-t)
Ii
0 IO1
1-I)
II21
9 I
1121
II
0 lOi
II
0 lOi
31
0 IOI
et al.
‘I’he organism found most l’requently in the inkted RA group urines was I-I r~~imbilis.‘I’his organism was l’ound in purity in 1 3 (SL’%) of the inkcted urine specimens of the RX patients and nearly twice as li-equently as the next common organism, I:. cdi. I-1r,tii-r&i/is was found in pm-it) in significant numbers in two ( 3%) and in ‘insignificant’ (i.e.. < IO-’ cfdml) numb-s in I 1 ( 14%) 01’ lhe if, urine specimens fi-om the R;\ patients. ‘I’his organism was not 1‘ound in significant numbers in any of the -I-S urines 01 the control group and was present in onI!, one (2%) 01’ them in ‘irisignilicant numbers. ‘fhe proportion of the inlkted urine female Iii\ patients M’ith I! rrlirrfhilis increased from 1 1Y, I’or those aged 5 l-C70 years lo 17.4% years to 35% for those aged Ibr those aged h I-/OY, 7 l-80 years. I: cdi was I’ound in urine in sigiiificant nunbcrs in seven (9%) of the RX patients hut in only one (Z’X,) urine of the control group.
01’ the 3 5 sera wailahlc from the RA group. there were only 11 that matched closely those of the control group with regard to patient gender and age. These were’ selected la- 13,ISA studies. ‘rhe ELIStI results of Igbl. IgC; and TgA antibody levels to the common 0 scrotypes 01 I-1~~irdilis in these sera are presented in Figure .? and ‘I’able III. ‘I’he RX patients sew were l’ound to have extremely significantly (I’ < 0.000 1 I or wry signilkmtl~ (P = 0.0010. I’ = 0.006 i) higher Igr\. Ig(: and IgbI antibody levels respect i\rely to II rrrirdlilis over thosc of 1he control group.
Antibody le\~els to I! ~rtiurbilis in the urine Ii-om the SIIIIIL’ 11 matched Rii patients and 2 1 control subjects kverc also determined by ELISA. ‘I’he results arc presented in b’igure 3 and Table 11’. The IgM. IgG and IgX Icvels to j-1rtlir-dilis in the urine of the KX patients were I’ound to be extremely significantly (I’ < 0.000 1, I’ < 0.0001, P = 0.0001 I raised respectively o\w those of’ the control group.
An analysis of the immunoblots of w-a ol’ RA and control group patients with high antibody Icvels to I? rrrir-~7bilis against lysntes ol’ E r~~irrrhilisshowed that all classes 01 ant ibodies against many I? ~nirnbilis antigens were present in both RA and control patients’ set-a. IM’ercnces in banding patterns with regard to the number. position and
P. mirabilis 1.00.
and Rheumatoid
Arthritis
103 1.00 Id
IgG
0.75.
0.75
..
. . 0.25
* . ::,A ..
.
8
0.25-
. Ar\
l:
l .* . ..* $1
A:A AA .:.
..:A
::*
. .
.:& .
.!$l .* :
0.00
Controls RA patients Serum samples
o.oc
Controls RA patients Serum samples
IL\ naticnts r\o. of samples ‘1 21 21
No. of samples
0.2x f 0.1 7 0.2 5 * 0.11 0.25*0.13
21 II
ent as determined by ELISA and the number or intensity of the bands given by it for some sera with low ELBA values gave many bands whereas some sera with high ELBA values gave few bands. It was not possible to detect an IgG. IgM or IgA antibody reacting to a given antigen that was exclusive to the RA patients. Analysis of immunoblots of nine I? rJCrnbilis infected urines from KA patients and one uninfected urine from the control group which had high I& levels to P mir-dCs as determined by ELI&l, against lysates of I? nGdGlis showed there were fewer IgG antibodies to I? mirabilis antigens in the urines than in the sera. IgG antibodies reacting to 97- and 7X-Ida antigens and an IgA antibody reacting to a 30-klk1 antigen were detected in several, but not all. of the infected urines from the RA
Controls RA patients Serum samples
C‘ontrol patients
CLISA * SD
intensity OS bands were observed for IgM. IgG and IgA and for both groups of patients. Sometimes there appeared to be no correlation between the amount of antibody pres-
patients.
0.25
21
ELISA i- ST) 0.1 ; i 0.04 0.14i0.09 0.08 k O.Oh
I’-value O.OOh j 0.0010 < 0.000 1
Discussion Rheumatoid arthritis (KA), like other autoimmune diseases, is strongly linked with women. Those who are middle-aged or elderly are three times more likely than men to develop the disease. KA patients characteristically have periods of remission alternating with periods of exacerbation of inflammatory arthritis. This suggests there may be factors, possibly microbial, that trigger the condition.
Women also have urinary tract infection (IJTI) more frequently than men. In middle-aged and cldcrly women the infecting organism is often I? mir-rrhilis.’ 1 It has been suggested that RA is a Proteus-reactive arthritis triggered by recurrent or asymptomatic urinary tract infections through antibodies to Prvtrrrs spp. cross-reacting with cells expressing HLA-DR4 antigens? This study attempted to examine whether or not RA patients had I? rnilahilis infected urine more frequently than control patients
and the immunological
consequences
of this.
B.W. Senior
104 1.00
et al. 1554
IgM 1.00
.
. 0.76
2 $ 2
0.50
3
.
2
0.25
0:.
l*
0.00
Controls IJrine
lgll I$; IL!,\
RA patients samples
II .?I II
..*..
Controls llrine
0.2 1 k 0. IO 0.2; i 0. I : 0.1 ; * 0.05
1111the patients in the KA group were known to have rheumatoid arthritis and most of them had detectable RF in their scra whereas the patients of the control group were free from KA and Kl: was not detected in any of them. In other respects regarding age and gender. the two groups of patients were very similar. Because of this it was surprising to find that whereas. as was expected, virtually all (96%) of the urines of the control group were sterile, 2 5 ( 3 3%) of the RA patients. who otherwise were healthy and free of clinical symptoms of urinary tract infection. had infected urine. This could not he explained on the basis ol differences in number in the two groups of patients in the age range most likely to get urinary tract infections (i.e.. those above 50 pears) because the proportions of the males and females in both groups were sery similar and almost identical. namely. 8 1.8% female and 80% male in the KA group and X0.9’%,fcmak and 8 3% male in the control group. Nor is it likely to bc an outcome of the greatel difficulty the KA patients may have had in maintaining
RA patients samples
.?I 11 21
rrf.r
Controls Urinr
0. I I) * 0.05 0, I I IL (l.Oh (1. I1 * 0.01
RA patients samples
< 0.000 I < 0.0001 0.000I
personal hygiene because none of the infected urines of the RA patients gave rise to a mixed gro\vth of faecal types of bacteria such as is found when there is faecal soiling around the area where the urine is collected. Instead each infected urine was a pure growth of one organism. The organism most frequently encountered in the RA patients’ urine was I? rlrimhilis. It was dctcctcd t\s+e as frequently as I:‘. rdi which. in normal people, is by far the commonest urinary tract pathogen. I? Itlirdlilis was found in 1 3 ( 1 7%) of the KX patients’ urine (and in 5.2% of those with infected urine) but in on1y one (.?‘!Y,J of the COP trol patients’ urine. Information was not available of medication which any of the individuals of either the KA OI the control group might have been receiving. That such medication might be an explanation for the more frequently infected, particularly with /? ~~jr~~l~ili.s. urines in the R/1 group is thought to bc improbable because it would need to cause im increase in susceptibility to infcction specifically to I’r~olvrfso\rer that of I:. c~li.
P. mirabilis
and Rheumatoid
In most of the P rJlirnhilis infected urines of the RA patients, the organism was found in numbers that would be regarded as being too low to constitute a significant bacteriuria. Hence it was not surprising that none of them had clinical symptoms of IJTI. The presence of these smaller numbers of I-1rJJir-&lis was revealed through the sampling of a larger volume ( 1 ml) of urine than is normal in the analysis of urine for significant bacteriuria. If the urine had been examined by conventional methods in which only a small volume C. 4 1.11 of urine was examined. in many instances bacteria would not have been detected. Although the results above which indicate a more frequent carriage of I? mirrrbilis (often in low numbers) in the urinary tract by RA patients than controls are at variance with those of McDonagh it OS. they are in agreement with the work of others2’,2i and they give support to the hypothesis that KA may have developed in them as a result of a subclinical ITT1 with a small number of I! mimbilis bacteria providing a frequent or prolonged exposure to I? mitnhilis antigens. Moreover. an earlier comparative analysis”’ of the types of 1-Irnirrrhilis strains found in the urine of RA and control patients which showed that the types of strains found in RA patients are often different from those found in controls in being of types that are either more virulent or persistent or both, substantiates this idea. Although Deighton and colleagues’; were unable to find antibody to 1-1rJGrnhilismore frequently in the urine of KA patients than controls by indirect immunofluorescence with an unclassified immunoglobulin to one serotype of 1: mirddis. the ELISA results in this study of the levels of the different classes of antibody in urine to a pool of l? rnirddis strains of different common 0 serotypes showed unequivocally that the levels of IgM. IgG and also IgA for P r~~ilabiliswere raised extremely significantly in the RA group compared with the control group. Furthermore they support the interpretation of the results of the bacteriological analysis of the urines and suggest that in RA patients. the P utirrrhilis antigenic stimulus is both frequent and prolonged. If this is true it would explain the finding of significantly raised IgM levels to I? rJk&ilis in the sera of RA patients reported previously I ; and again here. It was not possible from the immunoblot analyses in this study to find a F! rnir-c&ilisantigen that was reactive in all the KA sera and unreactive in the control sera. However, two antigens which may be important are the haemolysin-associated protein Hpm B and the urease of P JJJinrhilis.These proteins contain respectively the polypeptide sequences ESRRAL and IRRET and are similar in molecular structure to the amino acid motifs QRRAA and LRREI that are associated respectively with RA and type XI collagen. Although in a previous study’; we were
Arthritis
105
unable to detect antibody in RA sera that reacted with HpmB, others have reported that sera from RA patients. but not those from patients with ankylosing spondylitis or controls, had raised antibody levels to the ESRRAL polypeptide and Pmteus haemolysin and urease.‘”
References 1 Stastny I? Mixed Ivmphocpte culture typing cells kom patients lvith rheumatoid arthrk. Tissw :\r~tiqer~s 1 YT4: 4: 5/l-Sic). 2 Lanchbury JSS. hIolecular genetics of the IILrl-I) region component ol’ inherited susceptibility to rheumatoid arthritis. fjr 1 Klw~r~~~rrlol 19X8:27: 1;1-175. 3 Harris CL). Kheumatoid arthritis. Pathophysiology and implications lirr therapy. S f:ryl \ .Lfwl 1 YYO: 322: 11 iT-I ‘Xc). 4 Oilier 1\: Thomson \k! Population genetics 01‘ rheum:~toitl arthritis. KIIPUIII Ilk C‘lifl s A!,! 199.2: 1x: r41-761. 5 ;\lhani S. Carson DA. Ruudier J. Genetic and enrironmrntal l’actors in the immune p;lthogenesis 01 rheumatoid arthritis. I
X Kwdicr J. Petersen J. Khodcs (;[I. Luka J. C’arson DA. Suscrptibility to rheumatoid arthritis maps to a ‘I-cc‘ll epitope shared by the Ill,;\I1w-l I)K p- 1 chain and the Epstein-Barr virus glycoprotrin gp I 10. PI-W Ytrtl ,hrl Sri USA I YXY: 86: 5 101-5 1 OX. Y ;Ubani S. ‘I’ucltwell JE. Esparza I,. Carson DA. Koudier J. The susceptibility sequence to rheumatoid arthritis is a cwss-rcactivc B cell epitape shared by I:sc~/wr-ic,flitr coli heat shock protein dnaJ and the hiatocompatihilit~ leulwcytr antigen UKBl *O-I01 molecule. / C‘lirr Irl~wt lYY1:89: 327-311.
17 Senior l<\L’. McBride l’L)E Morley I<. krr hlA. The detection 01‘ raised levels of Ig’ll to I’rotcws Itlit-trhilia in sew l’rom patients \vith rhrum+ toid arthritis. 1 Mrtl Aficwbiol lYY5: 43: 1 ih-1 X1. 18 Ebringer A. Cox NL. ,\huljadayel I ef (II. lif~~l~.\i~~llnantibodies in anlqlosing apondylitis and I’rotcws altihodies in rheumatoid arthritis. ET-/ Khr~rr~ttrf~l 198X: 27 Suppl II: iL-X 5. I Y Arnett AK. Mworthy FC. Bloch IM CT (11.The ,\merican Rheumatism Association 1 YXi revised criteria for the classilication of rhcum;ltoid arthritis. Ar-llrritis Klw~rrtr 1 YXX: 31: 3 1 T-114.
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et al.