Vo. 74, No. 3, March, pp. 252-256, 1995 Anti-ribosomal P Antibodies in Systemi¢ Lupus Erythematosus: A Case-Control Study Correlating Hepatic and Renal Disease" ‘Mans Huisey, Rose Goiosrein,* Linpa ScuLLy,* Wintiam SURBECK, AND Morris REICH iN Rhewnatology Section, University of Oblehoma Medical Center, Arthritis and Immunelogy Program, Ollahoma Medical Research Foundation, Ollahoma City, Oklahoma 73104; and *Deparimeni of Medicine, Oftewa General Heepial and Ottawe Civic Hospi, Uniersty of Ottawo, Ottewa, Ontario, Canada We report @ case-control study of the occurrence of liver and kidney disease in 20 systemic lupus erythematosi (SLE) patients with anti-ribosomal P antibodies and 20 age sex-, and race-matched (contro! group) SLE patients with- out anti-P antibodies. In the group with antisP antibodies, 7 patients were found to have had liver disease, compared with only 1 in the control group (P = 0.03), and 14 anti-P (+) patients have had kidney disease, compared with 4 in the control group (P = 0.01). A major serological difference be- tween the groups was an increased prevalence of anti- sDNA in the anti-P positive group (12120) vs the control group (4/20), P = 0.02, These statistically significant differ- ences suggest that antibodies to ribosomal P identify a sub- set of SLE patients at higher risk for liver and kidiey in- volvement, in addition to the previously recognized risk for neuropsychiatric disease. © 185 seems: res te INTRODUCTION Autoantibodies to ribosomal P protein have been rec- ognized since 1985 (1). ‘Their association with neuro- psychiatric disease has been demonstrated in three in- dependent reports (2-4), but not: in a fourth study (8). In addition, antibodies to ribosomal P protein in sys- temic lupus erythematosus (SLE) have also been a5s0- ciated with general disease activity (6, 7) and, in one report, with skin disease (7). ‘We have reported a case of a SLE patient in whom antibodies to ribosomal P proteins appeared in associ- ation with the development of abnormal liver function tests and chronic active hepatitis on liver biopsy (8). No other etiology for the liver disease was found. We pos- tulated that: antibodies to ribosomal P proteins might, be involved in this ease since we had found a form of ribosomal P protein on the surface of cultured human hepatoma cells (9) and had found that anti-ribosomal P * Supported by grants from the National Institute of Health (RO ‘AWL3L183, ROL AR 82244, and POL AT 121568) to Dr. Reichin and a {rent from Physicians’ Services Inc. Foundation to Drs. Goldstein fand Sealy, Dr. Goldstein isa Research Scholar of the Arthritis So ciety antibodies could bind to, penetrate, and injure hepato- ma cells in culture (10). ‘We now report a case-control study designed to in- vestigate the clinical relevance of anti-ribosomal P an- ibodies in a population of SLE patients in whom the prevalence of hepatic and renal disease was deter- mined. PATIENTS AND METHODS Anti-P Sera Sera of 260 patients with a physician diagnosis of SLE were identified from the database at the Rheu- matology Clinical Laboratory at the Oklahoma Medi- cal Research Foundation. These patients were from the referral area of the University of Oklahoma Medical Center. Each patient's earliest submitted sample, usu- ally coinciding with onset of SLE symptoms, was tested for the presence of anti-ribosomal P antibodies by Ouchterlony double-diffusion precipitation, using calf thymus extract as antigen. Twelve sera formed precip itates by double diffusion, and another eight sera were found to contain anti-P antibodies by combined reac- tivity in immunoblot and ELISA assays. Those sera with a 38-kDa “Po” band on immunoblot, using puri- fiod rabbit ribosomes as substrate, were assayed by an ELISA method using a 22-amino acid synthetic peptide glutaraldehyde rabbit serum albumin conjugate (P- RSA) as antigen. Control wells with glutaraldehyde- RSA carrier were run simultancously, and net. absor- bance greater than 0.156 (two standard deviations greater than the mean absorbance of 10 normal sera) ‘was called positive. A total of 20 sera were found to be anti-P (+) by double difusion or immunoblot/ELISA assays. ‘As controls for the anti-ribosomal P antibody assays, 59 sera from patients with various liver diseases diag- nosed at the University of Ottawa were assayed for the presence of anti-P by double diffusion, Western blot, and the P-specific ELISA. Of these sera, 28 were from patients with chronic active hepatitis, 23 with primary 282 990.122996 $5.00 ‘Copyright © 1988 by Aesdcmse Prox, oe ‘A ighta a reproduction in any for ereSLE HEPATIC AND RENAL DISEASE CORRELATION biliary cirrhosis, and 8 with sclerosing cholangitis. Ad- ditionally, 20 normal human sera were run in these assays for anti-ribosomal P antibodies. Control Group The 20 anti-P patients were matched for age, sex, and race with 20 SLE patients whose sera tested neg- ative on immunoblot. Excluded were patients not meeting the 1982 revised criteria for SLE (11), patients with an identified liver disease not attributable to SLE (including ethanol abuse, identified toxin exposure, vi- ral hepatitis, and anti-mitochondrial antibodies), and patients with preeclampsia. All anti-P positive pa- tients were found to meet criteria for SLE, One anti-P pationt was excluded from the liver disease study due to the presence of anti-mitochondrial antibodies. Chart Review ‘The patient’s medical records were reviewed for ev- idence of autoimmune or otherwise unexplained liver and renal disease. Evidence for liver disease was taken as simultaneous elevations of three or more liver en- zymes (ic., SGOT, SGPT, LDH, alkaline phosphatase, total bilirubin, or GGTP), at any point in the disease course, as reported in the patient's medical chart. Ev- idence for kidney disease was taken as persistent pro- teinuria >0.5 g/24 hr or >3++ on dipstick urinalysis, persistent plasma creatinine of 2.0 mg % or greater, ot the presence of cellular casts. Disease outcomes for liver and kidney disease were compared between the anti-P positive and the control group using McNemar’s statistic for correlated proportions. RESULTS Patient Characteristics Table 1 compares the mean current age and age of onset of liver and renal disease, sex ratio, and racial breakdown of SLE pationts with and without anti-P. Female patients with anti-P exceed males with anti-P 5.6 to 1. The same ratio in the anti-P control group is by construct of the study to control for sex. Black pa- tients comprise the largest anti-P SLE group in our population. Almost as many white patients with anti-P exist in our study sample, but there is a much higher frequency of white SLE ‘sera submitted to the lab. Anti-P is apparently enriched in our black patient pop- ulation compared to the white patient population. ‘Comparable ages of onset of SLE, as well as renal and hepatic disease onset, are found in anti-P positive and negative SLE groups. Table 2 presents the list of patients in the anti-P + and control groups, summarizes the results of the study, and lists the serological findings in ali the pa- tients, 253 TABLE 1 Patient Characteristics of SLE Anti-P (+) Group and SLE ‘Anti-P (—] Control Group as to Sex, Age, and Race SLE ontiP (+) SLB anti-P (-) Femaleimale 8 178 Mean current age in years (range) 38.3(06-66) 33.8 (22-58) ‘Mean age onset SLE (range) 29.7 13-51) Mean age onset renal (range) 2887-39) Mean age onset hepatic (range) ra White 1 Black 10 Notive American 1 1 Hispanic 1 1 Asian ° 1 Note, Agecf onset defined as age at which diagnosis is made andor ‘treatment Is beyue. One patient in this group. Liver Disease ‘Table 3 summarizes the occurrence of liver disease in the study. Seven anti-P patients had simultaneous el- evations of three or more liver enzymes as evidence of at least one episode of liver disease apparently attrib- utable to SLE. Another patient (#15) with liver disease TABLE 2 Autoantibody Profile of Anti-P SLE and Control SLE. AntiP (row ‘anti? aro atone Paine Hep Ren Hep Ren Serclogy o ‘ax Ro RNP a ‘ANA + 4 & 2 ANA + 3 kN a ANA. Sm, ‘BNP oO + Pola pNp 2 = ANA oe + RLSn ENP aya BNP +f RSMRNP + ANA m oy ta a ANA os a as +k ry + RNP 2 Re m+ a0 RNP. mot os 3 RNP. Sn 2 3 Ro, RNE as 2 + RP Moe + Pr ANA wet 38 + ee 6 a 36 a " + dfos 4, BNP ‘ins BNP 18 6 ANS 8 RNP 13 ANA 3 Ro, RNP. 2 + aS RNP 2 ANA ‘Note. Cumulative preipitin and antids DNA histories, All pa tients except 12 are ANA positive >1:160, Patients 1-12 have ant-P precipitin, and 13-20 have aut by Western blot and ELISA. de, fanti-native DNA antibodies: Ro, anti-Ro; La, anti-Le; RNP, anti ‘BRNP: Sm, anti-Sm. Omitted are ants DNA and unidentified pre cipitns.254 TABLE 3 Liver Disease in SLE Patients with and without ‘Anti-ribosomal P Antibodies = Hepatitie Be 9 Se Now Ae 2% Neg Bx CAH Tote Hey RIBA eg 1 New Mo Neg € 6 a c ec 4 A ‘Note. Excluded are cases with hepatitis due to infection (e.., HBV of HCV), drug intoxication (eg., ethanol abuse), oF enti mitochondrial antibodies. GGT, serum gamma glutamyl a notransferse level; CAB, chronic active hepatitis; US, ultrasound maging; A, acute liver disease; C, chronic liver disease had anti-mitochondrial antibody (by indirect immuno- fluorescence on HEp-2 cells) noted a few years after her initial hepatitis and was not included in this part of the study. Patient 10 had biopsy evidence of chronic active hepatitis and serum anti-HCV activity by ELISA, but stored serum from the time of the biopsy tested nega- tive on recombinant immunoblot for five HCV anti- gens. Autoimmune hepatitis is associated with a high frequency of false-positives on HOV ELISA, and since her transaminases improved with immunosuppres- sion, patient 10 was included. Two other anti-P pa- tients with liver enzyme elevation had liver biopsy ev- idence of chronic active hepatitis with negative viral hepatitis studies, another had demonstrated hepato- splenomegaly by ultrasound (and gallstones with no ductal stones or alkaline phosphatase elevation), and two others had elevated GGT to corroborate liver dam- age. ‘A sixth patient developed lupus 4 years after diag- nosis of Philadelphia chromosome positive chronic my- elogenous leukemia, which had been treated sequen- tially with busulfan, DOAP, and interferon a, and was in hematologic and eytogenetic remission, on inter- feron, at the time he developed tenderness over the liver, proteinuria, malar rash, 1:3240 titer of anti- native DNA, and was diagnosed with SLE. He had not shown abnormal elevations of three simultaneous liver enzymes (SGOT, LDH, and AP) until after 4 years of ‘management with the above regimen, and these eleva- tions coincided with the development of SLE. Serum from this date showed anti-P by Western blot and ELISA. Immunosuppressive treatment and discontin- ‘uanee of interferon « improved his condition and nor- malized the liver enzymes. Subsequent to SLE remi sion, he was restarted on interferon « and did not re- HULSEY ET AL, ‘peat the pattern of liver enzyme elevation nor did other features of his SLE reappear. This case was not ex- cluded because the occurrence of liver damage was temporally associated with the onset of his SLE and anti-P antibodies and was not clearly attributable to & factor other than Tupus. Of the control patients, 1 had liver disease not at- tributable to other causes and had a biopsy consistent with autoimmune hepatitis. Using McNemar’s test to compare the 19 evaluable anti-P patients with their matched controls for prevalence of liver disease, a P value of 0.03 was obtained. Renal Disease Fourteen anti-P positive SLE patients met criteria for renal disease in SLE, as shown in Table 2. Of these, 3 had renal biopsy; I showed diffuse proliferative glo- merulonephritis, 1 had mesangial proliferative glo- merulonephritis, and 1 had a biopsy specified only as glomerulonephritis. Bight other anti-P positive SLE patients had persistent proteinuria, Of the 4 nephritic patients in the anti-P negative group, 1 had DPGN and another focal sclerosing glomerulonephritis by biopsy, and 2 patients had proteinuria, The difference in prev- alence of renal disease between these two group is sta- tistically significant (P = 0.012), The odds ratio for renal disease given anti-P (+) is 6 (95% CI, 1.5-30.8). Anti-ribosomal P antibody was detected in samples rior to or concomitantly with development of liver dis- ease in all cases despite the fact that the study was conducted retrospectively. It is quite interesting that all the patients with liver disease also had renal dis- ease sometime in their course, and in this sense, the pationts with liver disease are a subset of the larger group of anti-P (+) patients with nephritis. Sera from 59 Ottawa General Hospital patients with autoimmune-associated liver disease, but not SLE, ‘were assayed for presence of anti-ribosomal P antibod- ies and none of the sera were positive. In addition, 20 normal sera were assayed for anti-P antibodies and were negative for anti-P antibodies. Autoantibody Profile ‘Table 2 also compares the autoantibody profiles of the two groups; any identified precipitin or positive anti-native DNA antibody (Crithidia) result in the pa- tient’s record is included, Enrichment for anti-dsDNA was found in the anti-P positive group compared to the anti-P negative group. Anti-dsDNA antibody was found in 12 anti-P positive patients compared with 4 in the control group (P = 0.02). The odds ratio for pres- ence of anti-dsDNA given anti-P (+) is 9 (95% CI, 1.1— 71.0). There were no statistically significant differ- ‘ences in the frequency of antibodies to Ro, La, U,RNP, ‘or Sm between the two groups.‘SLE HEPATIC AND RENAL DISEASE CORRELATION Medication Toxicity Care was taken to evaluate for liver disease or renal disease caused by medication toxicity. Essentially all patients in the anti-P and control groups used NSAID's and corticosteroids, and several required the use of ey- totoxic agents. Review in each case of the physician notes, changes in medications, and pattern of liver ot kidney disease did not implicate medication toxicity as cause of the organ involvement in question. DISCUSSION Autoantibodies reacting to ribosomal phosphopro- teins identify on Western blot three distinct, bands, P02, Pi, and P2, corresponding to MW sizes of 38, 19, and 17 kDa, respectively (1). These autoantibodies hhave been reported to occur in sera of about 10% of patients with SLE (2). In agreement with these results is the finding that 20 of 260 sera submitted with the diagnosis SLE to the clinical immunology lab at the Oklahoma Medical Research Foundation had anti- ribosomal P autoantibodies detectable by Ouchterlony double diffusion or Western bloELISA. A pathogenic role for anti-P antibodies has been sug- gested by studies showing an association between the presence of these antibodies in SLE patients and nou- ropsychiatric disease (2, 4) and photosensitive rash (7). Beyond this, clinical studies have not established a link between anti-P antibodies and other organ in- volvement. A positive correlation has also been found in SLE patients between anti-DNA antibodies and anti-P antibodies (7). However, despite the well- established correlation between anti-DNA antibodies and renal disease, in the cited study only 4 of 33 anti-P positive SLE patients had renal disease, compared with 5 of 26 anti-P negative SLE patients. Individual cases from our clinic of SLE patients who developed anti-P precipitins concomitantly with symp- toms of either hepatic or renal disease argued for the possibility of such a pathogenic association and led us to formally evaluate SLE patients with and without anti-P for liver or kidney disease. This case-control study of 20 SLE patients with anti-ribosomal P anti- bodies and their 20 age, sex-, and race-matched con- trols provides evidence of an association between the presence of anti-P and the prevalence of liver and renal disease. ‘Comparing the anti-P (+) and control groups, it was possible to exclude other known causes of liver disease, yet still find a positive association between the pres- tence of anti-P and disease. The groups did not differ significantly in their profiles of medication, and after excluding patients with ethanol abuse, viral hepatitis, infection, and anti-mitochondrial antibodies, there was still a significant preponderance of patients in the anti-P group (7/19) with episodes of clinically sigmifi- cant liver damage than in the control group (1/20). Hal of these were cases of chronic active hepatitis proven by biopsy, a fourth patient had chronic liver disease not 255 characterized by a biopsy, and the other three were episodes that largely remitted with immunosuppres- sive therapy, without being subjected to biopsy. This heterogeneity of liver disease is not surprising given the reported spectrum of liver disease in SLE; one study of liver biopsies in 33 SLE patients with evidence of liver disease revealed 12 patients with ste- atosis, 4 with CAH, 4 with cirrhosis, 3 with granulo- matous disease, 2 with chronic persistent hepatitis, 2 with microabscesses, 1 with PBC, 1 with hemochroma- tosis, and 1 with “nonspecific reactive changes” (11). A similar spectrum of liver disease was seen in an au- topsy study of lupus patients from Japan (12), It is likely that more than one autoimmune mechanism is at play, even within the same individual. Many more ceases may be subclinical. The fact that anti-ribosomal P antibodies were not found in sera from patients with autoimmune chronic Tiver diseases (without SLE) indicates that the pres- ‘ence of anti-P antibodies is not simply secondary to hepatic damage. Many have assumed that hepatitis in SLE patients occurs by the same mechanism as idio- pathic autoimmune hepatitis, but our study suggests that, at least for the anti-P subset of SLE hepatitis patients, an alternate pathogenesis involving anti-P antibodies may be present. ‘That anti-P antibodies are pathogenic to liver tissue is possible since recent in vitro studies show binding of afinity-purified human anti-P antibodies to human hepatoma cells (HEpG2) (9). The mechanism for liver damage is to be determined, but recent in vitro studies show that anti-P antibodies can directly injure hepatic cells in a complement-independent fashion (10). Inter- ference with hepatic membrane function has also been shown (Koren, Reichlin, unpublished studies). For the ‘cases reported in this study, sera containing anti-P an- tibodies were identified which had been submitted ei- ther before or concomitantly with the episode(s) of liver enzyme elevation, suggesting that presence of the an- tibody predates the liver disease. With rogard to renal disease, it is not possible to separate the relationship of anti-P to nephritis from other known renal disease-associated autoantibodies, particularly anti-native DNA which occurred in 8 of the 14 anti-P positive cases with nephritis. Previous studies of anti-native DNA antibodies by the Crithidia assay have shown a 75% correlation with nephritis (13), Assuming that anti-P antibodies and anti-native DNA antibodies are independent variables for renal disease, this study suggests that the occurrence of anti-P antibodies in SLE patients may be a significant marker for renal disease in the 10% of SLE patients in which anti-P antibodies are seen. Since preparing this report, we have observed two SLE patients whose ne- phritic episodes were associated with the appearance of, antibodies to ribosomal “P” protein in the absence of antibodies to native DNA. Several factors should be kept in mind in assessing256 the significance of the results in this study. The sample group is small, increasing the margin for type I (and type Il) error. It is a retrospective study with the prob- lems inherent in the interpretations of charted histor- ical events. The study group was culled from a larger group of SLE patients by testing only the earliest se- rum from each patient, and it is possible that some patients initially testing negative for anti-P may have developed the antibody in later, untested sera. 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