1425

BRIEF REPORT

AGALACTOSYL IgG: AN AID TO DIFFERENTIAL DIAGNOSIS IN EARLY SYNOVITIS

ADAM YOUNG, NAZIRA SUMAR. KATHERINE BODMAN, SNEH GOYAL, HILARY SINCLAIR, IVAN ROITT, and DAVID ISENBERG

Sixty consecutive patients presenting with early- onset synovitis were studied by measuring rheumatoid factor (RF) titers and the percentage of oligosaccharide chains attached to the Cy2 domain of IgG that lack galactose (GAL[OI). After 2 years of followup, 39 pa- tients (65%) had developed rheumatoid arthritis (RA), and 21 had developed a variety of other inflammatory joint diseases. A combination of RF positivity and GAL(0) levels above the age-corrected mean gave a positive predictive value for a diagnosis of RA in 94% of these patients. These observations may well have clinical utility.

High levels of agalactosyl IgG (GAL[O]) have been reported in adult-onset and juvenile-onset rheu- matoid arthritis (RA), particularly in patients with

From the Department of Rheumatology, St. Alban’s City Hospital and the Department of Rheumatotogy, Queen Elizabeth I1 Hospital, Welwyn Garden City, and the Department of Rheumatol- OW Research and Immunology, University College and the Middle- sex Hospital School of Medicine, London, England.

Adam Young, FRCP: Consultant Rheumatologist, Queen Elizabeth I1 Hospital; Nazira Sumar, PhD: Associate Research Fellow, University College and the Middlesex Hospital School of Medicine; Katherine Bodman, BSc: Associate Research Assistant, University College and the Middlesex Hospital School of Medicine; Sneh Goyal, BSc: Research Student, University College and the Middlesex Hospital School of Medicine; Hilary Sinclair, MRCP: Research Associate, University College and the Middlesex Hospital School of Medicine; Ivan Roitt, FRS: Director, Department of Rheumatology Research and Immunology, University College and the Middlesex Hospital School of Medicine; David Isenberg, FRCP Consultant Rheumatologist and Honorary Senior Lecturer, Univer- sity College and the Middlesex Hospital School of Medicine.

Address reprint requests to David Isenberg, FRCP, Bloomsbury Rheumatology Unit, Arthur Stanley House, 40-50 Tottenham Street, London WlP 9PG, England.

Submitted for publication October 15, 1990; accepted in revised form May 16, 1991.

active disease (1,2), Thus, when the RA is active, these patients invariably have an increased percentage of oligosaccharide chains attached to the Cy2 domain of IgG that lack galactose and terminate in N-acetyl- glucosamine (2,3). These agalactosylated immuno- globulins may be functionally deficient in their ability to bind monocyte and macrophage receptors or to induce cellular cytotoxicity (4-6). Furthermore, im- mune complexes formed by these immunoglobulins with antigen may not be eliminated rapidly from the circulation (4). In studies using a precise but complex chemical method (1) to analyze several hundred sera from patients with over 25 different diseases, the defect in glycosylation of IgG was shown to be largely confined to individuals with RA, Crohn’s disease, tuberculosis, or systemic lupus erythematosus (SLE) complicated by Sjogren’s syndrome (7). The disorders in which normal GAL(0) levels were found included osteoarthritis, with or without a synovid effusion, and various non-RA conditions associated with rheuma- toid factor (RF) (e.g., Klebsiellu infections and malar- ia). It has also become evident that the degree of glycosylation of IgG varies with age and that it is important to relate every value to a reference age curve, which has now been published (8). Recently, the differential binding properties of certain lectins to particular sugars have been utilized in a novel method in which equivalent results can be obtained faster (9). Using this technique, we have confirmed our original results showing elevated GAL(0) levels in both adult- onset and juvenile-onset RA (10).

All the studies performed to date have used sera from patients with established disease. It seemed important to determine whether raised GAL(0) levels were of diagnostic or prognostic significance in the

Arthritis and Rheumatism, Vol. 34, No. 11 (November 1991)

1426 BRIEF REPORT

Table 1. Outcome (at 1 year) for 60 patients with inflammatory joint pains of recent origin

Number (%) Diagnosis of patients ~ _ _ _ _ _ ~ ~

Early rheumatoid arthritis Palindromic rheumatism Psoriatic arthritis Reactive arthritis Systemic lupus erythematosus Polymyalgia rheumatica Viral arthritis Transient synovitis

39 (65) 1 (1.7) l(1.7) 2 (2.4) 3 (5 ) l(1.7) 3 (5 )

10 (16.7)

early stages of adult-onset synovitis. To this end, we studied 60 consecutive patients attending early-onset synovitis clinics at 2 district general hospitals in Hert- fordshire (St. Albans City Hospital and Queen Eliza- beth I1 Hospital, Welwyn Garden City) and The Mid- dlesex Hospital. These patients had had synovitis for less than a year, and they have been followed up for a minimum of 2 years. This report details the eventual clinical diagnosis in these patients and demonstrates that abnormalities in glycosylation of IgG are present from a very early stage of the disease in those who were eventually diagnosed as having rheumatoid ar- thritis.

Patients and methods. Sixty patients (20 males, 40 females, mean age 51.1, age range 2!9-73) with synovitis of less than 1 year’s duration (mean 7.95 months) have had regular clinical and laboratory as- sessments for a minimum of 2 years. Detailed assess- ments of the time and type of onset of this synovitis, any prodromal iIIness, reIevant family history, and history of other diseases were recorded. Measure- ments of disease activity and functional capacity in- cluded assessments of the following features: duration of early morning stiffness, pain (by visual analog

scales), joint scores, and functional assessments (by grip strength and by Health Assessment Question- naire). Blood samples were taken and tested for the following values: complete blood cell count, blood urea nitrogen, electrolytes, liver function, erythrocyte sedimentation rate (ESR; Westergren), RF (by RA hemagglutination assay [RAHA], positive titer >1:40), antinuclear antibodies (ANA; on H E p 2 cells, positive titer >1:40), and GAL(0) (see below).

Over the course of the 2-3-year followup pe- riod, the diagnosis has become evident in each case. The majority of these patients (n = 39) were eventually diagnosed as having RA, according to the revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association [ARA]) (11). The remaining 21 patients were eventu- ally diagnosed as having other forms of inflammatory joint disease (Table 1).

The mean ages, age ranges, and sex ratios were similar in the 2 groups (Table 2). There was an expected overlap of the number of ARA clinical crite- ria met and the presence of RF between the 2 groups,

The method used to measure GAL(0) levels is described in detail elsewhere (9). Briefly, purified IgG samples (1-2 pg) and IgG standards containing known percentages of GAL(0) were dot-blotted onto nitrocel- lulose using a dot-blotter from Bio-Rad (Richmond, CA). Two identical blots of each sample were pre- pared. The blots were boiled in phosphate buffered saline (PBS) at 90°C for 15 minutes, followed by blocking in PBS-Tween (0.01%) containing 1 .O% bo- vine serum albumin (PBS-Tween-BSA). The following steps were carried out at room temperature, and the blots were shaken on a horizontal shaker. One of the blocked blots was treated with biotinylated Ricinus communis agglutinin (RCA-I; 1 : 250 dilution) and the

Table 2. Demographic data of the study population*

Duration Diagnostic Sex Age (mean ARA criteria RF GAL(0) GAL(0) group (n) (no. malelfemale) (mean) months) (%with >2) (% positive) (% positive) (mean value)

Non-RA (21) 5/16 49.1 7.11 44.4 4.8 14.3 -0.57 Early RA (39) 15/24 52.2 8.33 81.6t 64.1$ 76.98 1.06 Total (60) 20140 51.1 7.95 70.6 43.3 55.0 0.49

See ref. I I for the American Rheumatism Association (ARA) criteria. Rheumatoid factor (RF) was determined by rheumatoid arthritis hemagglutination assay (positive >1:40). Levels of agalactosylated (GAL[Ol) IgG were calculated as described in Patients and Methods, and represent standard deviations from (above or below) the age-corrected mean for healthy controls (see ref. 9). The non-rheumatoid arthritis (non-RA) group had other inflammatory joint diseases. t 2 = 6.3. P < 0.05, versus non-RA group. t 2 = 17.2, P < O.OOO1, versus non-RA group. 8 J = 19.1, P < 0.0001. versus non-RA group.

BRIEF REPORT 1427

other with biotinylated Bandeiraea simplicifolia agglu- tinin (BS-11; 1 : 100 dilution) for 2.5 hours. After wash- ing the blots with PBS-Tween-BSA (5 times for 10 minutes each), streptavidin-biotin-horseradish peroxidase conjugate was added (1 : 1,000 dilution), and the blots were incubated for 2 hours.

The blots were then washed with PBS-Tween, developed with chloronaphthol, and scanned using a Bio-Rad video densitometer. The ratios of BS-I1 bind- ing to RCA-I binding for the standards were plotted against the %GAL(O) values, and the values for test samples were read against this curve. TLe values were scored as standard deviations above or below the age-corrected mean for a healthy population (9), now consisting of nearly 100 individuals between ages 20 and 70. The test standards, measured by the original hydrazinolysis method (l), were kindly provided by Dr. Rademacher and colleagues from the Oxford Gly- cobiology Group.

Data were entered on the UCL Amdahl com- puter and analyzed with the SPSS” package (12), employing chi-square contingency tables and Stu- dent’s t-test where appropriate. Discriminant function analysis used mean values of the chosen variables to obtain Rao’s V to separate outcome groups, as previ- ously described (12).

Results. There was a significant difference be- tween the GAL(0) levels corrected for age-at-first-visit in the group that developed RA and the group that did not (Student’s t = -4.86, P = 0.0001) (Figure 1) . Only 3 patients from the non-RA group had GAL(0) levels above the age-corrected mean (only 1 above the mean + 1 SD), 2 of whom had SLE (1 also had Sjogren’s syndrome). The third patient had a transient synovitis with a high ESR but no detectable RF or ANA and negative viral serology. This patient’s symptoms sub- sided spontaneously.

Nine patients with early RA (23%) had low GAL(0) levels (Le., below the age-corrected mean) at study entry. Six of these patients had serial samples of blood drawn for GAL(0) levels, 5 of whom subse- quently had raised GAL(0) levels, and only 1 had persistently low levels (data not shown). Thirteen patients had a transient synovitis; 3 of them had positive findings on viral serology for parvovirus (2 patients) and for rubella ( I patient), but 1 of them had raised GAL(0) levels.

Discriminant analysis of the ARA clinical crite- ria present at study entry predicted the eventual outcome in 41 of the 60 patients (68%), whereas the RF titer successfully distinguished 50 of the 60 (83%) and

G A L

( 0 )

L E V E L S *

*

*** **

* *

* * * ** *

****** ** - - - -

** *** ** ** c

**** * ** ** *** *** ** *

**** ** ** *

- - - -

No* RA Figure 1. Levels of agalactosylated [GAL(O)] IgG in patients with early-onset synovitis ( < I year’s duration) who, after 2 years of followup, developed rheumatoid arthritis (RA; n = 39) or other forms of inflammatory joint disease (Non-RA; n = 21). Values are standard deviations from the age-corrected mean (@point, solid line) for healthy controls (see ref. 9). Broken lines show group means.

the GAL(0) levels 47 (78%). Combining RF and GAL(0) improved this discrimination to 91% (sensitivity 90%, specificity 95%, positive predictive value 94%).

As Table 3 indicates, of 18 patients in whom RF

Table 3. Correlations of outcome with RF positivity and GAL(0) level*

Non-RA patients RA patients (n = 21) (n = 39)

RF negative and GAL(0) normal 16 2 RF negative, GAL(@ raised 2 3 RF positive, GAL(0) normal 2 7 RF positive and GAL(0) raised 1 27

* Rheumatoid factor (RF) was determined by rheumatoid arthritis hemagglutination assay (positive > 1:40). Levels of agalactosylated (GAL[O]) IgG were compared with the mean age-corrected level in healthy controls, as described in Patients and Methods (see also ref. 9). The non-rheumatoid arthritis (non-RA) group had other inflam- matory joint diseases.

1428 BRIEF REPORT

was negative (i,e., RAHA titer < I :40) and the GAL(0) value was at or below the age-corrected mean, 16 were in the non-RA group. Five patients with RA were seronegative for RF at study entry; 3 of them had raised GAL(0) levels. Three patients with other types of inflammatory joint disease were RF positive; 2 of these 3 patients had low GAL(0) levels. The patient with the highest GAL(0) level in the non-RA group was RF negative.

Discussion. The purpose of this study was to determine whether the finding of an increased level of GAL(0) might help guide the rheumatologist when presented with a patient who has clinical evidence of synovitis but no diagnosis has been made. The prac- ticing clinician seeks, in particular, a reliable diagnos- tic and prognostic marker for RA. The commonly used ARA diagnostic criteria were designed essentially for epidemiologic studies, and the practical problem of their application to individual patients is well recog- nized. The positive predictive value of 94%, obtained by combining the GAL(0) and RF results, is impres- sive. From these results it is clear that the impairment in normal glycosylation of IgG is present from a very early stage in RA. Our detailed earlier studies reported elsewhere (3,7) have confirmed that in other diseases in which RF has been reported (13), including osteoar- thritis with synovial effusion and Escherichia coli and Klebsiella infections, GAL(0) levels are not increased. We observed with interest, though have no simple explanation, that the individuals in the non-RA group tended to have lower GAL(0) levels than did the healthy controls.

It is possible that the patients we studied rep- resent some atypical group of individuals who are more likely to develop RA. We think this is unlikely for several reasons. The only clinical criterion was whether the patient had early-onset synovitis. Further- more, the study patients were drawn from 2 different populations, one at the Middlesex Hospital, a teaching hospital in the center of London, whose patients are drawn from a wide variety of sources, and the other at St. Albans City and Queen Elizabeth I1 hospitals, both 30 miles outside London, whose patients are drawn from a much more homogeneous population. Finally, over one-third of the patients (21 of 60) were eventu- ally diagnosed as having disorders other than RA.

Rheumatoid factor has been the best laboratory aid to diagnosis. It has, however, to be interpreted with great care in patients with early inflammatory joint conditions, because it may be unreliable in dif- ferentiating early RA from the overlapping clinical

features commonly seen in SLE and some forms of reactive or psoriatic arthropathies that affect the body symmetrically. It has recently been confirmed, for example, that up to 25% of lupus patients have a persistently positive RF (14). For the clinician, the only realistic approach, in many cases, is to provide supportive therapy while waiting for definitive diag- nostic symptoms to evolve. Given that GAL(0) levels higher than the age-corrected mean + 2 SD were confined in this study to patients who were eventually diagnosed as having RA, and that an increased level has been shown to be disease restricted (7), this new measurement appears to be useful in the diagnosis of RA. The relationship between RF activity and the extent of galactosylation of IgG remains uncertain, though recently published reports suggest that they are not associated (5,15).

The question of prognosis is important both to the patient and to the clinician who may favor early intervention. The adverse features identified in early studies as poor prognostic indicators included early erosions, high ESR, and seropositivity (16-18). How- ever, of these and other features analyzed in a study using discriminant function analysis, only a combina- tion of RF (by sheep cell agglutination and by latex fixation), hemoglobin level, and platelet count suc- cessfully predicted radiologic outcome in 72%. This was confirmed in 79% in a separate group (19). Im- provement in these values is essential to the clinician intending to use powerful second-line agents in early disease. Longer-term studies are needed to determine whether GAL(0) levels can usefully predict outcome in this potentially serious condition. However, the re- sults in the present study suggest that there is consid- erable clinical utility in measuring GAL(0) and RF as a way of helping the rheumatologist arrive at the correct diagnosis in a patient presenting with early-onset synovitis.

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