- Email: [email protected]
HIDDEN RHEUMATOID FACTOR IN SERONEGATIVE NODULAR RHEUMATOID ARTHRITIS
878
possibility of measles immunisation before cyclophosphamide is used. Such immunisation evokes a satisfactory antibody response in children with the nephrotic syndrome1o; and, although the exact site and nature of immune suppression by cyclophosphamide is uncertain, measles immunisation may possibly provide some protection for these children. We are grateful to Dr. P. R. Evans and Dr. J. S. Cameron for permission to record this case; to Dr. Frank Perkins for the antibody titres; and to Dr. M. T. Cwynarski for the immunoglobulin
estimations. REFERENCES
Scheinman, J. I., Stamler, F. W. J. Pediat. 1969, 74, 117. Ettledorf, J. N., Roy, S., Summitt, R. L., Sweeney, N. J., Wall, H. P., Berton, W. M. ibid. 1967, 70, 758. 3. Lipsey, A. L., Kahn, M. J., Bolande, R. P. Pediatrics, Springfield, 1967, 39, 659. 4. Mitus, A., Enders, J. F., Craig, J. M., Holloway, A. New Engl. J. Med. 1959, 261, 882. 5. Hobbs, J. R. Proc. R. Soc. Med. 1968, 61, 883. 6. Waterson, A. P., Cruickshank, J. G., Laurence. G. D., Kanarek, A. D. Virology, 1961, 15, 379. 7. Nakai, T., Shand, F. L., Howatson, A. F. ibid. 1969, 38, 50. 8. Janeway, C. A., Moll, G. H., Armstrong, S. H., Wallace, W. M., Hallman, N., Barnes, L. A. Trans. Ass. Am. Physns, 1948, 61, 108. 9. Moncrief, M. W., White, R. H. R., Ogg, C. S., Cameron, J. S. Br. med. J. 1969, i, 666. 10. Yuceoglu, A. M., Berkovich, S., Chiu, J. J. Pediat. 1969, 74, 291. 1. 2.
Preliminary
Communications
HIDDEN RHEUMATOID FACTOR IN SERONEGATIVE NODULAR RHEUMATOID ARTHRITIS RODNEY BLUESTONE
LEONARD S. GOLDBERG
ANDREA CRACCHIOLO III
Department of Medicine, U.C.L.A. Center for the Sciences, Los Angeles, California
Health
of three patients with seronodular rheumatoid arthritis negative were subjected to gel filtration and the eluted fractions were tested for rheumatoid factor (R.F.) activity. In contrast to the whole sera, R.F. in high titre was detected in the eluted IgM of these three subjects. The revealed R.F. was inhibited in vitro by autologous serum and by aggregated IgG. These findings indicate that the sera of certain patients, presumed to be seronegative, contain hidden R.F. Hidden R.F. should be considered in any clinico-immunological study of rheumatoid disease.
Sum ary
The
sera
INTRODUCTION
RHEUMATOID factor (R.F.) can be detected in the sera of most patients with rheumatoid arthritis.1 Since the presence and titre of R.F. usually correlate with severity of disease, patients with gross joint destruction and rheumatoid nodules are almost invariably seropositive.2,3 A few rare patients with nodular rheumatoid arthritis, however, appear to be seronegative by all conventional antiglobulin tests. R.F.s are typically IgM-type antibodies 4 which are directed against antigenic sites on the IgG immuno-
globulin.5 It has previously been demonstrated that the in-vitro activity of R.F. may be inhibited by the presence of autologous IgG, resulting in falsely low antiglobulin titres. 6 It is also thought that the in-vivo formation of complexes of R.F. and IgG may sometimes
mask in-vitro R.F. activity-i.e., R.F. tightly bound to its antigen would not be detected by conventional assays. Dissociation of these complexes by acidic gel filtration has exposed antibodies to native IgG in certain rheumatoid sera; the whole sera of these patients reacted with aggregated but not with native IgG.’ Thus, R.F. activity in the sera of some patients with severe rheumatoid disease might be completely masked by the presence of autologous IgG or by the formation of R.F.-IgG complexes. We therefore studied three patients with severe, seronegative, nodular rheumatoid arthritis and seven others with various joint diseases. Their whole sera were fractionated by gel filtration at neutral and at acidic pH(s) in an attempt to reveal hidden R.F. MATERIALS AND METHODS
The Patient Group Ten patients were studied. Three had classical rheumatoid arthritis with gross, erosive joint destruction; two of these had histologically proven subcutaneous rheumatoid nodules, and the third had gross joint destruction, fibrosing alveolitis, and a previous history of nodules. One patient had non-nodular rheumatoid arthritis, two had systemic lupus with prominent articular manifestations, one severe psoriatic arthropathy, one Reiter’s disease with widespread peripheral joint involvement, and two had ankylosing spondylitis with peripheral joint involvement, iritis, and aortitis. Gel Filtration Each serum (7-10 ml.) was subjected to ’Sephadex G-200 ’ gel filtration. Initial elution was performed with 0-15 M sodium-phosphate buffer, pH 7-35. The peaks eluted in the void volume, measured spectrophotometrically at 280 m, were concentrated by vacuum dialysis at 4°C. After removing small aliquots, the residual volumes were resubjected to gel filtration on sephadex G-200 columns equilibrated with 0-2 M glycine/hydrochloric-acid buffer, pH 3-0. The void volume peaks eluted at pH 3-0 were dialysed against 0-15 M sodium phosphate, pH 7-35, prior to concentration. The immunoglobulins present in the sera and eluted peaks were detected by agar-gel double diffusion using monospecific antisera for IgG, IgA, and IgM. Immunoglobulin concentrations were determined by radial diffusion in agar-antibody plates. Tests for Rheumatoid Factor Four tests for rheumatoid factor were performed on each serum and void volume peak simultaneously. All samples were heat-inactivated at 56°C for 30 minutes before testing. R.F. activity was determined at 22°C using latex particles coated with human aggregated IgG, human type-0 positive erythrocytes sensitised with anti-CD serum Ripley, sheep red cells sensitised with rabbit y-globulin, and F II-coated tanned sheep cells. Known positive and negative sera were included in each assay. Latex agglutination and anti-CD Ripley titres greater than 1/40, sensitised sheep-cell titres greater than 1/2, and F II sheep cell titres greater than 1/160 were considered abnormal.
Inhibition Tests The specificity of the R.F.s eluted in the IgM peaks was tested by inhibition of agglutination. Four agglutinating units of the eluted R.F. were incubated with an equal volume of: (1) phosphate-buffered saline solution, (2) autologous serum, (3) autologous native IgG (10 mg. per ml.) prepared by D.E.A.E.-chromatography, and (4) heat-aggregated human IgG (10 mg. per ml.). After incubation for 5 minutes at room-temperature, the mixtures were re-tested against latex particles coated with aggregated IgG.
879 RESULTS
All ten sera were negative for R.F. by all assay methods. The void volume peaks eluted at pH 7.35 from the
sera
of three patients with nodular rheumatoid
arthritis showed high titres of R.F. by the latex agglutination, anti CD-Ripley, and F II tests (see accompany-
ing table). The sensitised sheep-cell tests on these peaks were also positive, but in lower titre. The eluted peaks from the seven other patients were RHEUMATOID-FACTOR TITRES OF WHOLE SERA AND ELUTED PEAKS
contained either altered IgG . or some other factor capable of antiglobulin inhibition. The masking of R.F. was not due to the presence of circulating complexes of R.F. and IgG since R.F. activity was not enhanced by acid-gel filtration of the initial eluates. The presence of such complexes has been previously shown to account for hidden R.F. activity specific for native IgG in some seropositive rheumatoid subjects.’7 Much emphasis has been placed on the relationship between the severity of rheumatoid arthritis and the presence of R.F.8,9 The association of high-titre R.F. with severity of disease and with extra-articular manifestations has been stressed.2,3 Indeed, the distinction between seropositive and seronegative rheumatoid arthritis is thought by some to be of great
prognostic significance. Furthermore, it has been postulated that seropositive and seronegative rheuma-
mg. per ml. t Reciprocal of the highest
serum
dilution giving
a
positive
test.
negative for R.F. by all methods. R.F. activity was not enhanced following elution of the IgM at pH 3-0. The IgM concentrations of the void volume peaks eluted at pH 7-35 were equal to, or twice as high as, their respective serum levels. Small amounts of IgG were also detected in the void volume peaks by agar-gel double diffusion, but the amounts were too low to be measured by radial diffusion. The R.F. activity in the eluted IgM was completely inhibited by autologous serum and by heat-aggregated IgG. It was not inhibited by the addition of saline solution or autologous native IgG. DISCUSSION
This study has clearly demonstrated the presence of hidden R.F. in the sera of three patients with nodular rheumatoid arthritis, each of whom had previously been considered seronegative. In contrast to the whole sera, the IgM fractions isolated by gel filtration contained R.F. activity in high titre. Identical treatment of the sera from seven other patients with erosive arthritis caused in various ways failed to reveal R.F. One of these patients had classical rheumatoid arthritis without nodules; the other six had diseases usually characterised as seronegative-viz., ankylosing spondylitis, Reiter’s disease, systemic lupus, and psoriatic
arthropathy. It is most likely that R.F. activity in the whole serum inhibited by the presence of its specific antigen (IgG) or perhaps by other, unknown serum factors. Separation of the IgM containing the R.F. activity from the remainder of the serum components apparently revealed the hidden R.F. Such high titre activity could not be explained by an increased IgM concentration alone. Indeed, in one patient the IgM level of the seropositive eluate was identical to that of the seronegative whole serum. The antibody activity was directed primarily against antigenic sites present on denatured IgG, since the activity was inhibited in vitro by denatured IgG, but not by native autologous IgG. The R.F. activity, however, was inhibited by autologous whole serum, suggesting that the serum was
toid arthritis may represent two distinct diseases.1o The results of our study indicate that certain individuals, presumed to be seronegative, have hidden serum-R.F. and that the presence of hidden R.F. should be considered in any future clinico-immunological study of rheumatoid disease. We thank Mrs. Sharon Meresman and Miss Jo Ellen Cunningham for their valued technical assistance. This work was supported by U.S.P.H.S. grant no. GM15759 and by a clinical investigatorship from the Veterans Administration (no. 15-69). R. B. is the recipient of an Arthritis and Rheumatism Council of Great Britain travelling fellowship and a Wellcome Research travel grant. Requests for reprints should be addressed to L. S. G., Department of Medicine, U.C.L.A. Center for the Health Sciences, Los Angeles, California 90024. R. B.’s present address is Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W.12. REFERENCES
Ziff, M. J. chron. Dis. 1957, 5, 644. Hill, A. G. S. in Rheumatic Diseases (edited by J. J. R. Duthie and W. R. M. Alexander); p. 144. Edinburgh, 1968. 3. Bywaters, E. G. L. ibid. p. 96. 4. Franklin, E. C., Kunkel, H. G., Müller-Eberhard, H. J., Holman, H. R. Ann. rheum. Dis. 1957, 16, 315. 5. Kunkel, H. G., Tan, E. M. Adv. Immun. 1964, 4, 351. 6. Oreskes, I., Plotz, C. M. J. Immun. 1965, 94, 567. 7. Allen, J. C., Kunkel, H. G. Arthritis Rheum. 1966, 9, 758. 8. Ragan, C., Farrington, E. J. Am. med. Ass. 1962, 181, 663. 9. Duthie, J. J. R., Brown, P. E., Knox, J. D. E., Thompson, M. Ann. rheum. Dis. 1957, 16, 411. 10. Mongan, E. S., Cass, R. M., Jacox, R. F., Vaughan, J. H. Am. J. Med. 1969, 47, 23. 1. 2.
CARDIOVASCULAR DYNAMICS IN WOMEN RECEIVING ORAL CONTRACEPTIVE THERAPY Y. L. LIM
W. A. W. WALTERS
Department of Obstetrics and Gynæcology, Monash University, Queen Victoria Memorial Hospital, Lonsdale Street, Melbourne, Australia serial study of hæmodynamic changes in the first 6 of 30 healthy young women being investigated while receiving combined œstrogen-progestogen oral contraceptive therapy are presented. Significant increases in cardiac output (expressed as cardiac index), systolic and mean blood-pressures, and plasma-volume occurred during the 2-3 months of therapy. Heart-rate and
Summary
The results of
a
diastolic-blood-pressure changes The increase in cardiac output
were not was
significant.
due mainly
to a
possibility of measles immunisation before cyclophosphamide is used. Such immunisation evokes a satisfactory antibody response in children with the nephrotic syndrome1o; and, although the exact site and nature of immune suppression by cyclophosphamide is uncertain, measles immunisation may possibly provide some protection for these children. We are grateful to Dr. P. R. Evans and Dr. J. S. Cameron for permission to record this case; to Dr. Frank Perkins for the antibody titres; and to Dr. M. T. Cwynarski for the immunoglobulin
estimations. REFERENCES
Scheinman, J. I., Stamler, F. W. J. Pediat. 1969, 74, 117. Ettledorf, J. N., Roy, S., Summitt, R. L., Sweeney, N. J., Wall, H. P., Berton, W. M. ibid. 1967, 70, 758. 3. Lipsey, A. L., Kahn, M. J., Bolande, R. P. Pediatrics, Springfield, 1967, 39, 659. 4. Mitus, A., Enders, J. F., Craig, J. M., Holloway, A. New Engl. J. Med. 1959, 261, 882. 5. Hobbs, J. R. Proc. R. Soc. Med. 1968, 61, 883. 6. Waterson, A. P., Cruickshank, J. G., Laurence. G. D., Kanarek, A. D. Virology, 1961, 15, 379. 7. Nakai, T., Shand, F. L., Howatson, A. F. ibid. 1969, 38, 50. 8. Janeway, C. A., Moll, G. H., Armstrong, S. H., Wallace, W. M., Hallman, N., Barnes, L. A. Trans. Ass. Am. Physns, 1948, 61, 108. 9. Moncrief, M. W., White, R. H. R., Ogg, C. S., Cameron, J. S. Br. med. J. 1969, i, 666. 10. Yuceoglu, A. M., Berkovich, S., Chiu, J. J. Pediat. 1969, 74, 291. 1. 2.
Preliminary
Communications
HIDDEN RHEUMATOID FACTOR IN SERONEGATIVE NODULAR RHEUMATOID ARTHRITIS RODNEY BLUESTONE
LEONARD S. GOLDBERG
ANDREA CRACCHIOLO III
Department of Medicine, U.C.L.A. Center for the Sciences, Los Angeles, California
Health
of three patients with seronodular rheumatoid arthritis negative were subjected to gel filtration and the eluted fractions were tested for rheumatoid factor (R.F.) activity. In contrast to the whole sera, R.F. in high titre was detected in the eluted IgM of these three subjects. The revealed R.F. was inhibited in vitro by autologous serum and by aggregated IgG. These findings indicate that the sera of certain patients, presumed to be seronegative, contain hidden R.F. Hidden R.F. should be considered in any clinico-immunological study of rheumatoid disease.
Sum ary
The
sera
INTRODUCTION
RHEUMATOID factor (R.F.) can be detected in the sera of most patients with rheumatoid arthritis.1 Since the presence and titre of R.F. usually correlate with severity of disease, patients with gross joint destruction and rheumatoid nodules are almost invariably seropositive.2,3 A few rare patients with nodular rheumatoid arthritis, however, appear to be seronegative by all conventional antiglobulin tests. R.F.s are typically IgM-type antibodies 4 which are directed against antigenic sites on the IgG immuno-
globulin.5 It has previously been demonstrated that the in-vitro activity of R.F. may be inhibited by the presence of autologous IgG, resulting in falsely low antiglobulin titres. 6 It is also thought that the in-vivo formation of complexes of R.F. and IgG may sometimes
mask in-vitro R.F. activity-i.e., R.F. tightly bound to its antigen would not be detected by conventional assays. Dissociation of these complexes by acidic gel filtration has exposed antibodies to native IgG in certain rheumatoid sera; the whole sera of these patients reacted with aggregated but not with native IgG.’ Thus, R.F. activity in the sera of some patients with severe rheumatoid disease might be completely masked by the presence of autologous IgG or by the formation of R.F.-IgG complexes. We therefore studied three patients with severe, seronegative, nodular rheumatoid arthritis and seven others with various joint diseases. Their whole sera were fractionated by gel filtration at neutral and at acidic pH(s) in an attempt to reveal hidden R.F. MATERIALS AND METHODS
The Patient Group Ten patients were studied. Three had classical rheumatoid arthritis with gross, erosive joint destruction; two of these had histologically proven subcutaneous rheumatoid nodules, and the third had gross joint destruction, fibrosing alveolitis, and a previous history of nodules. One patient had non-nodular rheumatoid arthritis, two had systemic lupus with prominent articular manifestations, one severe psoriatic arthropathy, one Reiter’s disease with widespread peripheral joint involvement, and two had ankylosing spondylitis with peripheral joint involvement, iritis, and aortitis. Gel Filtration Each serum (7-10 ml.) was subjected to ’Sephadex G-200 ’ gel filtration. Initial elution was performed with 0-15 M sodium-phosphate buffer, pH 7-35. The peaks eluted in the void volume, measured spectrophotometrically at 280 m, were concentrated by vacuum dialysis at 4°C. After removing small aliquots, the residual volumes were resubjected to gel filtration on sephadex G-200 columns equilibrated with 0-2 M glycine/hydrochloric-acid buffer, pH 3-0. The void volume peaks eluted at pH 3-0 were dialysed against 0-15 M sodium phosphate, pH 7-35, prior to concentration. The immunoglobulins present in the sera and eluted peaks were detected by agar-gel double diffusion using monospecific antisera for IgG, IgA, and IgM. Immunoglobulin concentrations were determined by radial diffusion in agar-antibody plates. Tests for Rheumatoid Factor Four tests for rheumatoid factor were performed on each serum and void volume peak simultaneously. All samples were heat-inactivated at 56°C for 30 minutes before testing. R.F. activity was determined at 22°C using latex particles coated with human aggregated IgG, human type-0 positive erythrocytes sensitised with anti-CD serum Ripley, sheep red cells sensitised with rabbit y-globulin, and F II-coated tanned sheep cells. Known positive and negative sera were included in each assay. Latex agglutination and anti-CD Ripley titres greater than 1/40, sensitised sheep-cell titres greater than 1/2, and F II sheep cell titres greater than 1/160 were considered abnormal.
Inhibition Tests The specificity of the R.F.s eluted in the IgM peaks was tested by inhibition of agglutination. Four agglutinating units of the eluted R.F. were incubated with an equal volume of: (1) phosphate-buffered saline solution, (2) autologous serum, (3) autologous native IgG (10 mg. per ml.) prepared by D.E.A.E.-chromatography, and (4) heat-aggregated human IgG (10 mg. per ml.). After incubation for 5 minutes at room-temperature, the mixtures were re-tested against latex particles coated with aggregated IgG.
879 RESULTS
All ten sera were negative for R.F. by all assay methods. The void volume peaks eluted at pH 7.35 from the
sera
of three patients with nodular rheumatoid
arthritis showed high titres of R.F. by the latex agglutination, anti CD-Ripley, and F II tests (see accompany-
ing table). The sensitised sheep-cell tests on these peaks were also positive, but in lower titre. The eluted peaks from the seven other patients were RHEUMATOID-FACTOR TITRES OF WHOLE SERA AND ELUTED PEAKS
contained either altered IgG . or some other factor capable of antiglobulin inhibition. The masking of R.F. was not due to the presence of circulating complexes of R.F. and IgG since R.F. activity was not enhanced by acid-gel filtration of the initial eluates. The presence of such complexes has been previously shown to account for hidden R.F. activity specific for native IgG in some seropositive rheumatoid subjects.’7 Much emphasis has been placed on the relationship between the severity of rheumatoid arthritis and the presence of R.F.8,9 The association of high-titre R.F. with severity of disease and with extra-articular manifestations has been stressed.2,3 Indeed, the distinction between seropositive and seronegative rheumatoid arthritis is thought by some to be of great
prognostic significance. Furthermore, it has been postulated that seropositive and seronegative rheuma-
mg. per ml. t Reciprocal of the highest
serum
dilution giving
a
positive
test.
negative for R.F. by all methods. R.F. activity was not enhanced following elution of the IgM at pH 3-0. The IgM concentrations of the void volume peaks eluted at pH 7-35 were equal to, or twice as high as, their respective serum levels. Small amounts of IgG were also detected in the void volume peaks by agar-gel double diffusion, but the amounts were too low to be measured by radial diffusion. The R.F. activity in the eluted IgM was completely inhibited by autologous serum and by heat-aggregated IgG. It was not inhibited by the addition of saline solution or autologous native IgG. DISCUSSION
This study has clearly demonstrated the presence of hidden R.F. in the sera of three patients with nodular rheumatoid arthritis, each of whom had previously been considered seronegative. In contrast to the whole sera, the IgM fractions isolated by gel filtration contained R.F. activity in high titre. Identical treatment of the sera from seven other patients with erosive arthritis caused in various ways failed to reveal R.F. One of these patients had classical rheumatoid arthritis without nodules; the other six had diseases usually characterised as seronegative-viz., ankylosing spondylitis, Reiter’s disease, systemic lupus, and psoriatic
arthropathy. It is most likely that R.F. activity in the whole serum inhibited by the presence of its specific antigen (IgG) or perhaps by other, unknown serum factors. Separation of the IgM containing the R.F. activity from the remainder of the serum components apparently revealed the hidden R.F. Such high titre activity could not be explained by an increased IgM concentration alone. Indeed, in one patient the IgM level of the seropositive eluate was identical to that of the seronegative whole serum. The antibody activity was directed primarily against antigenic sites present on denatured IgG, since the activity was inhibited in vitro by denatured IgG, but not by native autologous IgG. The R.F. activity, however, was inhibited by autologous whole serum, suggesting that the serum was
toid arthritis may represent two distinct diseases.1o The results of our study indicate that certain individuals, presumed to be seronegative, have hidden serum-R.F. and that the presence of hidden R.F. should be considered in any future clinico-immunological study of rheumatoid disease. We thank Mrs. Sharon Meresman and Miss Jo Ellen Cunningham for their valued technical assistance. This work was supported by U.S.P.H.S. grant no. GM15759 and by a clinical investigatorship from the Veterans Administration (no. 15-69). R. B. is the recipient of an Arthritis and Rheumatism Council of Great Britain travelling fellowship and a Wellcome Research travel grant. Requests for reprints should be addressed to L. S. G., Department of Medicine, U.C.L.A. Center for the Health Sciences, Los Angeles, California 90024. R. B.’s present address is Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W.12. REFERENCES
Ziff, M. J. chron. Dis. 1957, 5, 644. Hill, A. G. S. in Rheumatic Diseases (edited by J. J. R. Duthie and W. R. M. Alexander); p. 144. Edinburgh, 1968. 3. Bywaters, E. G. L. ibid. p. 96. 4. Franklin, E. C., Kunkel, H. G., Müller-Eberhard, H. J., Holman, H. R. Ann. rheum. Dis. 1957, 16, 315. 5. Kunkel, H. G., Tan, E. M. Adv. Immun. 1964, 4, 351. 6. Oreskes, I., Plotz, C. M. J. Immun. 1965, 94, 567. 7. Allen, J. C., Kunkel, H. G. Arthritis Rheum. 1966, 9, 758. 8. Ragan, C., Farrington, E. J. Am. med. Ass. 1962, 181, 663. 9. Duthie, J. J. R., Brown, P. E., Knox, J. D. E., Thompson, M. Ann. rheum. Dis. 1957, 16, 411. 10. Mongan, E. S., Cass, R. M., Jacox, R. F., Vaughan, J. H. Am. J. Med. 1969, 47, 23. 1. 2.
CARDIOVASCULAR DYNAMICS IN WOMEN RECEIVING ORAL CONTRACEPTIVE THERAPY Y. L. LIM
W. A. W. WALTERS
Department of Obstetrics and Gynæcology, Monash University, Queen Victoria Memorial Hospital, Lonsdale Street, Melbourne, Australia serial study of hæmodynamic changes in the first 6 of 30 healthy young women being investigated while receiving combined œstrogen-progestogen oral contraceptive therapy are presented. Significant increases in cardiac output (expressed as cardiac index), systolic and mean blood-pressures, and plasma-volume occurred during the 2-3 months of therapy. Heart-rate and
Summary
The results of
a
diastolic-blood-pressure changes The increase in cardiac output
were not was
significant.
due mainly
to a