GALACTOSYLATION OF IgG ASSOCIATED OLIGOSACCHARIDES: REDUCTION IN PATIENTS WITH ADULT AND JUVENILE ONSET RHEUMATOID ARTHRITIS AND RELATION TO DISEASE ACTIVITY

966

satisfactory serological careful

response in young

infants;

a more

of short and long term side-effects is of antibody responses and of and the duration necessary clinical protection will also have to be determined before this vaccine can be advocated for immunisation of young infants. Another issue that needs to be addressed is whether the vaccine, when given to young infants, alters the immune response to subsequent measles vaccination at a later age. Several researchers have reported that children in the United States when first vaccinated at less than 10 months of age with the Moraten strain of virus have had a depressed HI antibody response when revaccinated later.19,20 Our limited data pertaining to Gambian children given either E-Z vaccine or Schwarz vaccine at 18-20 wk of age do not support this concept, for all these children responded successfully to revaccination. The E-Z vaccine holds promise that, when given in the correct dose, it will successfully immunise young infants against measles. If this proves true the way is open for more efficient and effective immunisation campaigns in the developing world. Will the goal of global measles eradication2l then be translated from fantasy to fact? assessment

We thank Mr S. K. Rahman for his skilled technical help and Mr B. Sarr, M. Daffeh, P. Cham, and Ms M. Jooffor help in the field. We also thank Dr B. M. Greenwood and Dr R. Whitehead for their advice and encouragement.

Correspondence

should be addressed

to

H. C.

W., MRC Laboratories,

Fajara, The Gambia, West Africa. REFERENCES

Expanded Programme on Immunization Measles-spots that kill. EPI Update, Geneva: WHO, 1986. 2. Shahid NS, Clauquin P, Shaik K, Zimicki S. Long-term complications of measles in rural Bangladesh. J Trop Med Hyg 1983; 86: 77-80. 3. Morley D. Paediatric priorities in the developing world London Butterworths, 1973: 1.

220-22. 4. Foster A, Sommer A. Childhood blindness from corneal ulceration in Africa: causes, prevention, and treatment. Bull WHO 1986, 64: 619-23. 5. Aaby P, Bukh J, Lisse IM, Smits AJ. Measles mortality, state of nutrition, and family structure. A community study from Guinea-Bissau. J Infect Dis 1983; 147: 693-701. 6. Aaby P, Bukh J, Lisse IM, da Silva MC. Measles mortality: Further community studies on the role of overcrowding and intensive exposure. Rev Infect Dis (in

press) 7. World Health Organisation. Weekly Epidemiol Rec 1979; 44: 337-39. 8. Ministry of Health of Kenya and World Health Organization. Measles immunity in the first year after birth and the optimum age for vaccination in Kenyan children. Bull WHO 1977; 55: 21-30. 9. Whittle HC, Rowland MGM, Mann GF, Lamb WH, Lewis RA. Immunisation of 4-6 month old Gambian infants with Edmonston-Zagreb measles vaccine. Lancet

1984; ii: 834-37. 10. Walsh JA. Selective primary health care: Strategies for control of disease in the developing world. (IV) Measles Rev Infect Dis 1983; 5: 330-40. 11. Klein-Zabban ML, Foulon G, Gaudebout C, Badoual J, Adou A. Fréquence des rougeoles nosocomiales dans un centre de protection maternelle et infantile d’ Abidjan. Bull WHO 1987; 65: 197-201. 12. Sinha WP. Measles in children under 6 months of age: an epidemiological study J Trop Paediatr 1981; 27: 120-22. 13. Loening WEK, Coovadia HM. Age in urban, peri-urban, and rural environments implications for time of vaccination. Lancet 1983, ii: 324-26 14. Ikic D, Juzbasic M, Beck M, Hraber A, Cimbur-Scheriber T. Attenuation and characterization of Edmonston-Zagreb measles virus. Ann Immunol Hung 1972,16; 175-81 15. Ikic DM. Edmonston-Zagreb strain of measles vaccine: epidemiologic evaluation in Yugoslavia. Rev Infect Dis 1983, 5: 558-63. 16. Sabin AB, Arechiga AF, de Castro JF, et al. Successful immunization of children with and without maternal antibody by aerosolized measles vaccine. JAMA 1983; 249: 2651-62. 17. Khanum S, Uddin N, Garelick H, Mann G, Tomkins A. Comparison of Edmonston-Zagreb and Schwarz strains of measles vaccine given by aerosol or subcutaneous injection. Lancet 1987, i: 150-53. 18. Mann GF, Allison LMC, Compeland JA, Agostini CFM, Zuckerman AJ. A simplified plaque assay system for measles virus. J Biol Standard 1980; 8: 219-25. 19. Wilkins J, Wehrle PF. Additional evidence against measles vaccine administration to infants less than 12 months of age: altered immune response following active/ passive immunization. J Pediatr 1979; 94: 865-69. 20. Linnemann CC, Dine MS, Roselle GA, Asky PA. Measles immunity after re-vaccination: results m children vaccinated before 10 months of age. Pediatrics 1983; 67: 332-35 21 Hopkins DR, Hinman AR, Koplan JP, Lane JM. The case for global measles eradication. Lancet 1982; i: 1396-98.

GALACTOSYLATION OF IgG ASSOCIATED OLIGOSACCHARIDES: REDUCTION IN PATIENTS WITH ADULT AND JUVENILE ONSET RHEUMATOID ARTHRITIS AND RELATION TO DISEASE ACTIVITY

RAJ B. PAREKH1 DAVID A. ISENBERG2 BARBARA M. ANSELL3

IVAN M. ROITT2 RAYMOND A. DWEK1 THOMAS W. RADEMACHER1

Glycobiology Unit, Department of Biochemistry, University of Oxford;1 Departments of Immunology and Rheumatology Research, University College and Middlesex School of Medicine, London;2 Department of Rheumatology, Northwick Park Hospital, Harrow3

prevalence of agalactosyl N-linked oligosaccharides on serum IgG was determined for patients with juvenile onset and with adult rheumatoid arthritis. A. significant difference in the prevalence of these structures from age matched controls was found in both types of arthritis. In patients with adult onset rheumatoid arthritis, the results showed a strong correlation between the prevalence of IgG-associated agalactosyl oligosaccharides and disease activity. A correlation between disease activity and agalactosyl structures was also seen in a retrospective analysis of serial IgG samples from patients with juvenile onset disease. The finding that childhood onset arthritis and adult rheumatoid arthritis share a defect of glycosylation of serum IgG suggests that there may be a greater similarity between these Summary

The

varieties of rheumatoid arthritis than has been hitherto considered. The observation that the incidence of agalactosyl oligosaccharides on IgG fluctuates with disease activity provides indirect evidence for a seminal role for this change of glycosylation in the inflammatory process which, in rheumatoid arthritis, is focused on the synovial tissues and results in bone erosions and joint destruction. two

Introduction VARIOUS immunological indices have been found to be abnormal in rheumatoid arthritis, but the aetiology of this disease is not established:1 both infection and heredity are believed to play a part. Juvenile arthritis is characterised by an onset of arthritis under the age of 16 yr, and its differential diagnosis requires the active exclusion of separate diseases such as infection, congenital anomalies of the musculoskeletal system, and haematological disorders. Juvenile arthritis is currently subclassified into three types on the basis of the pattern of illness in the first three months.2 "Systemic onset" involves a high swinging fever associated with an atypical rash, and in many cases with lymphadenopathy, hepatosplenomegaly, and occasionally pericarditis: at this stage there may be no joint symptoms (or merely arthralgia), but polyarthritis subsequently develops. A "polyarthritic onset" is recognised when five or more joints are involved from the outset. A "pauciarticular onset" is described when the arthritis is confined to four or fewer joints: this third subgroup, the largest, is further subdivided into those with a young age of onset (under 9 yr, usually girls, and often associated with chronic iridocyclitis), and those older (usually boys, who carry HLA B27, and whose symptoms relate more to spondyloarthritis). A previous study of patients with adult onset rheumatoid arthritis demonstrated characteristic changes in the Nglycosylation of serum IgG,3.4 with an elevated relative incidence of agalactosyl N-linked oligosaccharides, both outer arms terminating in N-acetylglucosamine. We now

967

of agalactosyl monosaccharide G(0), from the IgG of patients with juvenile onset and

Fig I-Percentage prevalence sequences,

adult

onset

rheumatoid arthritis related to age.

All juvenile onset patients had active disease and were separated as to the mode of onset: systemic x ; polyarticular 0; pauciarticular . Data from individual adult onset patients is separated into those with active disease 0; and those without W. The solid line depicts the regression function for normal subjects (n = 111). The hatched lines depict the regression functions for 2 SD from the mean (for ages with n > 3). Values of G(O) for patients with either juvenile onset or adult onset rheumatoid and active disease were significantly different from age-matched controls (p < 0-01) by analysis of covariance. G(O) values for patients with inactive disease were not significantly different from the G(O) values obtained for normal subjects.

report the prevalence of such structures on serum IgG in patients with juvenile arthritis, and the association between the prevalence of such structures and disease activity in both juvenile and adult onset cases. Patients and Methods

Fig 3-A) Retrospective analysis

All of the adult onset cases (n = 53; 19 male, 34 female) met the American Rheumatism Association’s criteria for classic or definite disease.5 The clinical activity in these adult onset cases was independently assessed according to a previously published index.6

onset

of

G(0)

in

patients with juvenile

arthritis.

in remission at the time of the second serum sample. Mode of onset systemic in 6, x ; polyarticular in 3, 0; and pauciarticular in 1,
were

was

B) Retrospective analysis of G(0) in systemic onset juvenile arthritis.

a

patient presenting

with

Progression to symmetrical polyarthritis, before inactive disease ( .and (A).

eventual remission

Active disease

was associated with active synovitis, and was usually accompanied by a raised erythrocyte sedimentation rate (ESR). For

this study, we defined remission as a lack of clinical activity associated with a normal ESR and requiring no drug therapy for 2 yr.

Fig 2-Percentage prevalence of agalactosyl monosaccharide sequences from IgG of patients with adult onset rheumatoid arthritis related

Clinical 3

=

score:

severely active,

1 n

to =

=

clinical

score.

inactive/mild, 4.

n

=

3; 2

=

moderately active,

n

=

7;

Serum IgG from 27 patients with juvenile onset arthritis was studied (9 male, 18 female). 14 patients had presented with systemic onset; 9 patients with pauciarticular arthritis, which in 5 cases was associated with chronic iridocyclitis; and 4 patients with

polyarticular onset. 22 patients progressed to symmetrical polyarthritis; 5 remained with pauciarticular disease, 3 of whom had an asymmetric presentation. The age of onset was between 6 mo and 11 yr. Synovitis, with or without a raised ESR, was required for

968 definition of active disease. Inactivity was defined as no evidence of active synovitis and a normal ESR, although joint limitation may still have been present. Remission was defined as for subjects with adult onset arthritis. Medications included penicillamine, gold, steroids, and chlorambucil. IgG was purified from fresh or stored (— 20°C, 1-15 yr) serum samples as reported previously.’ The N-linked oligosaccharides were released from the IgG samples using anhydrous hydrazine, purified and 3H labelled by reduction as reported previously.’ The percentage incidence of N-linked monosaccharide sequences which contained no terminal galactose residues—G(0)—was determined with an exoglycosidase mixture.8 Linear regression analysis was performed on the G(0) vs age data for normal subjects and for each of the disease groups. In each case, juveniles (age 1 to 19 yr) and adults (age 20 yr and over) were analysed separately. Differences between the regression lines for the diseased groups and the corresponding normal patients were assessed by analysis of covariance. Probabilities are reported for the null hypothesis that the normal and disease data lie on a coincidental line. All statistics were calculated with the Minitab programme running on an IBM

PC/AT.9 Results

G(0) is age-related, so the values of G(0) for patients with arthritis are presented as a function of age. The results shown in fig 1 confirm the original observation that patients with adult onset rheumatoid arthritis with active disease are characterised by an increase in IgG-associated agalactosyl monosaccharide sequences (ie, terminating with residues other than galactose, predominantly with Nacetylglucosamine). No differences between the G(0) values of normal subjects and adult rheumatoid patients with inactive disease were found. Fig 1 also shows the G(0) values for patients with juvenile onset arthritis: clearly, the G(0) values in patients with active juvenile onset and adult rheumatoid arthritis are similar in magnitude. Fig 2 shows the relation between G(0) and clinical score for patients with adult onset rheumatoid arthritis: some of the patients with active disease (score 2 and 3) had low ESR values, despite the presence of active synovitis. The G(0) values seem to be independent of the mode of onset in the juvenile patients. Fig 3A shows the G(0) values found at the time of disease onset and at a subsequent time of inactivity (or remission) for juvenile onset cases: the G(0) values obtained at the time of disease inactivity are within the normal range for this age group. To further evaluate the relation of G(0) to disease progression, serial IgG samples were analysed from a single individual (fig 3B). The G(0) values vary progressively, and again there appears to be a correlation between this variable and disease activity, with a dramatic range of variation in G(0) during the course of disease progression and remission in this patient. Discussion The N-glycosylation defect of serum IgG (namely decreased outer-arm galactosylation), first described amongst adult onset patients with rheumatoid arthritis, is shown by this study to be present almost uniformly in juvenile onset cases, and is detectable irrespective of the mode of onset in this younger age group. The defect was related to disease activity both in serial studies amongst the juvenile onset patients and amongst adult onset patients whose clinical activity was independently assessed. These observations raise questions both about the importance of the N-glycosylation defect in patients with rheumatoid arthritis, and about the relation between the adult and juvenile modes of onset.

Studies with a large number of healthy subjects have shown that the number of oligosaccharide chains on serum IgG whose outer arms lack galactose and terminate predominantly in N-acetylglucosamine is age-related.8 In particular, there is significantly more agalactosyl IgG between the ages 1-15 and 40-70 yr than in adolescence and young adult life. This observation makes it essential to relate every result that is obtained to age. When this is done, no abnormality is discernible amongst individuals with osteoarthritis (as was originally reported)4, but both the adult and juvenile onset patients with active rheumatoid arthritis remain well outside the normal limits. An analysis of over 300 sera from patients with approximately 25 different autoimmune rheumatic diseases, seronegative arthropathies, and infectious diseases showed that the defect in galactosylation is virtually confined to rheumatoid arthritis, tuberculosis, and Crohn’s disease (Rademacher TW et al, unpublished). The low galactose values are not a consequence of chronic excessive IgG synthesis, since normal glycosylation was seen in primary systemic lupus erythematosus (SLE) and leprosy--diseases with persistently raised IgG levels far in excess of those seen in rheumatoid arthritis. Furthermore, the defect does not arise as a simple acute phase reaction, since many of the SLE patients with a normal oligosaccharide arrangement of their IgG had an ESR greater than 70 mm/hr. The defect is not simply linked to HLA DR status, since the high prevalence of HLA DR4 in adult onset patients with rheumatoid arthritis is not found in most juvenile onset cases,10 or in tuberculosis (de Vries RBP, personal communication). The lack of terminal galactose residues in the Fc portion of IgG molecules could contribute to the autoantigenicity of this region.4 Possible mechanisms for this include exposure of particular aminoacids in the Fc region which are usually "concealed" by the overlying oligosaccharide structure; and shortened oligosaccharides terminating in Nacetylglucosamine acting as an epitope. Autosensitisation to the Fc portion of IgG remains an important concept in adult onset rheumatoid disease, and approximately 70% of these patients are IgM rheumatoid factor positive; most of the others have IgG and, to a lesser extent, IgA rheumatoid factors. Although conventional agglutination tests for rheumatoid factors are usually negative in juvenile onset cases, IgG rheumatoid factors are commonly detected by solid phase assays.11,12 However, the juvenile onset cases have several clinical features which distinguish them from patients with adult onset disease. For example, major systemic disease in the absence of overt synovitis is very rare in adult onset rheumatoid arthritis, unlike the juvenile onset cases. The pattern of joint involvement also differs between the childhood and adult onset cases. In adults, rheumatoid arthritis is principally a symmetrical disease of the small joints of the feet and hands, especially the proximal interphalangeal and metacarpophalangeal joints; children more often have knee and distal or proximal interphalangeal arthropathy, but rarely metacarpophalangeal involvement. Despite the clinical differences between juvenile and adult onset rheumatoid arthritis, we have now shown that they share the same defect in respect of the glycosylation of their IgG and that there may be a greater similarity between these two types of arthritis than was previously thought. The observation that the agalactosyl IgG levels fluctuate with disease activity is also new: we are not yet certain whether this fluctuation is relevant to the causation of the inflammatory process in rheumatoid arthritis. The genetic and environmental factors leading to altered galactosylation

969 of the N-linked oligosaccharides of serum IgG in these diseases are not known, but recent results indicate a modulation of p-galactosyltransferase activity in B

specific 0-galactosyltransferase which lymphocytes: transfers UDP-Gal to an asialo-agalacto IgG has been reported to be present in B lymphocytes.13,14 The affinity of this enzyme for UDP-Gal in the B lymphocytes of patients with rheumatoid arthritis is lower than in B lymphocytes from a control group, and the specific activity of the galactosyltransferase towards asialo-agalacto IgG was found

2. Ansell BM. Juvenile chronic arthritis. Curr Orthop 1986; 1: 81-89. 3. Parekh RB, Dwek RA, Rademacher TW. Rheumatoid arthritis as 4.

a

to

be reduced

to

50-60% of control levels in adult

rheumatoid patients. Further analysis of the mechanism underlying the galactosylation defect may yield important insights into the pathogenesis of both disorders. We thank Daryl Fernandes and David Ashford for help with the statistical analysis and Mrs P. M. Rudd for technical assistance. R. B. P., R. A. D., and T. W. R are members of the Oxford Glycobiology Unit, which is supported by the Monsanto Company.

Correspondence should be addressed to T. W. R., Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXl 3QU. REFERENCES 1. Morrow

WJW, Isenberg DA. Autoimmune rheumatic Scientific, 1987: 161-67.

disease. Oxford: Blackwell

TUMOUR NECROSIS FACTOR AS AN AUTOCRINE TUMOUR GROWTH FACTOR FOR CHRONIC B-CELL MALIGNANCIES F. T. CORDINGLEY A. BIANCHI A. V. HOFFBRAND J. E. REITTIE H. E. HESLOP A. VYAKARNAM A. MEAGER M. TURNER M. K. BRENNER

Department of Haematology, Royal Free Hospital, London; Department of Immunology, Sunley Research Institute, Charing Cross Hospital, London, and National Institute of Biological Standards and Control, South Mimms, Herts

Recombinant tumour necrosis factor (TNF) promotes survival and induces proliferation in the tumour cells from two malignancies of B lymphocytes—hairy-cell leukaemia and B-chronic lymphocytic leukaemia. Culture with TNF also induces TNF mRNA and protein, so the cytokine may act as an autocrine tumour growth factor. These growth promoting effects are antagonised by alpha but not by gamma interferon.

Summary

Introduction TUMOUR necrosis factor (TNF) may induce lysis of malignant cells and cell-lines and regression of some animal tumours;l.2 this effect has led to investigation of the therapeutic value of TNF in clinical oncology. More recently, however, TNF has been shown to act as a growth

factor for normal fibroblasts,3 T cells,4 and B cells. We now show that TNF can also act as a tumour growth factor and promote the survival and proliferation of tumour cells in two types of B-cell malignancy-hairy-cell leukaemia (HCL) and B-chronic lymphocytic leukaemia (B-CLL). Patients and Methods - PanMH.—Ten patients, three with HCL and seven with B-CLL, were studied on one to four occasions. Diagnosis of HCL was made on the basis of circulating cell morphology, phenotype

5. 6.

7.

8.

9.

a

glycosylation

disorder. Br J Rheumatol (in press). Parekh RB, Dwek RA, Sutton BJ, et al Association of rheumatoid arthritis and primary osteoarthritis with changes m the glycosylation pattern of total serum IgG. Nature 1985; 316: 452-57. Ropes MW, Bennett GA, Cobb S, Jacobs R, Jessar RA. 1958 revision of diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 1958; 9: 175-76. Isenberg DA, Martin P, Hajirousou V, Todd-Pokropek A, Goldstone AH, Snaith ML. Haematological re-assessment of rheumatoid arthritis. Br J Rheumatol 1986; 25: 125-27. Ashford D, Dwek RA, Welply JK, et al. The &bgr;1 →2-D-xylose and &agr;1 →3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Eur J Biochem 1987; 166: 311-20. Parekh RB, Isenberg DA, Roitt IM, Dwek RA, Rademacher TW. Age-specific galactosylation of N-linked oligosaccharides of human serum IgG. J Exp Med (in

press). Godfrey K. Simple linear regression

in medical research. N Engl J Med 1985; 313: 1629-36. 10. Miller ML, Glass DN. The major histocompatibility complex antigens in rheumatoid arthritis and juvenile arthritis. Bull Rheum Dis 1981; 31: 21-24. 11. Torrigiani G, Ansell BM, Chown EEA, Roitt IM. Raised IgG antiglobulin factors in Still’s disease. Ann Rheum Dis 1969; 28: 424-27. 12. Hay FC, Nineham LJ, Fletcher MR, Roitt IM. An improved method for the detection of IgG and IgM rheumatoid factors in rheumatoid diseases. In: Jayson MIV, ed. Still’s disease-juvenile chronic polyarthritis. London: Academic Press, 1976:

135-44. 13. Furukawa K, Matsuta

K, Takeuchi F, et al. Alteration of a galactosyltransferase in B cells of rheumatoid arthritis patients. From the IXth international symposium on glycoconjugates. Tourcoing (France): A. Lerouge, 1987: E56. 14. Axford JS, Mackenzie L, Lydyard PML, Hay FC, Isenberg DA, Roitt IM. Reduced B-cell galactosyltransferase activity in rheumatoid arthritis Lancet 1987; ii: 1486-88.

(with B-cell markers and a monoclonal antibody, FMC7, that detects a hairy-cell-associated marker), cytochemistry (positivity for tartrate-resistant acid phosphatase), and trephine or splenic histology, B-CLL was diagnosed by means of cell morphology, number, and phenotype (CD5 and CD20 positive). All patients were selected for high circulating HCL or B-CLL cell numbers. Two of the three HCL patients had undergone splenectomy 5 and 6 years previously. No patient was receiving therapy at the time of

study. Recombinant cytokines.-Recombinant (r) cytokines, expressed in Escherichia coli, were the gift of Knoll-AG (BASF) (rTNF), Biogen (interferon -IFN and Kirby Warwick (rIFN-ot). Specific activity of rTNF was 6-3 x 106 u/mg protein, of rIFN-y 3-3 x 106 u/mg protein, and of rIFN-a 2-5 x 106 u/mg protein. Cell preparation and culture.-Peripheral blood mononuclear cells were prepared by ’Ficoll’ sedimentation and then depleted of monocytes by adherence and of T cells by double-E rosetting. Cells used in this study contained < 0-5% T cells or monocytes; > 95 % of the mononuclear cell population from HCL patients and > 98 % from CLL patients were neoplastic cells. Viability counts were carried out daily in 96-well microculture plates (Nunc). 180 )1 of cell suspension at 106/ml in RPMI 1640 medium and 10% fetal calf serum selected for low mitogenicity were added to each well followed by 10 III of either cytokines or RPMI 1640. At intervals during incubation at 37°C in 5 % CO2, viability counts were carried out with 0 25% ’Nigrosine’. All estimations were carried out in triplicate. Proliferation was estimated by pulsing plates prepared as above with 1 pCi 3I-I-thymidine for 6 h at 37°C and then harvesting DNA and measuring thymidine incorporation in a beta-scintillation counter.

TNF receptor assay.-r TNF was iodinated with 1251 (Amersham Radiochemicals) by the iodogen method,6 producing a specific activity of > 1000 fCi/ug of protein. TNF receptor assays were carried out by the competitive displacement method7 and cellbound rTNF-125I was separated from unbound TNF by centrifugation through phthalate oil.a All estimations were carried out in triplicate and analysed with the LIGAND computer

program.9 Enzyme-linked immunosorbent assay (ELISA).-Microtitre plates (Micro Elisa, Dynatech, Billingshurst) coated with rabbit anti-human IgM (DAKO, Weybridge) or goat anti-human IgG chain-specific antiserum (Sigma) were incubated with supernatants from HCL or B-CLL cultures for 2 h; alkaline-phosphataseconjugated goat anti-human antibodies specific for IgM or IgG, or for anti-human kappa or lambda light chains (Sigma), were then