The Role of the Complement System in Rheumatoid Inflammation

The Role of the Complement System in, Rheumatoid InHammation SHAUN RUDDY, M.D.* K. FRANK AUSTEN, M.D.**

The complement system consists of a group of nine serum proteins which act sequentially to produce cell destruction and to generate certain factors which produce inflammation. 16 . 20 Several lines of evidence suggest that the complement system may contribute to the pathogenesis of rheumatoid arthritis. This report will review some of the recent advances in the understanding of the complement system as it relates to the general phenomenon of immunologically induced tissue injury, and analyze the existing evidence that this system may be operative in the particular problem of rheumatoid arthritis. Finally, preliminary measurements of the activities of individual complement components in rheumatoid synovial fluid will be presented. The nature and extent of the depletion of individual components supports the view that local activation of the complement system results from intraarticular immunologic events which occur in certain joint diseases.

REACTION MECHANISMS OF THE COMPLEMENT SYSTEM During the past 10 years, considerable progress has been made in understanding the means by which the complement system mediates the effects of antigen-antibody interaction .. Although most of this information has come from studies on the lysis of sensitized sheep erythrocytes by guinea pig 20 or human complement,t6 a variety of other cell types, including mast cells,28 tumor cells,t2 and gram-negative bacteria,t9 have From the Department of Medicine, Harvard Medical School at the Robert B. Brigham Hospital, Boston, Massachusetts 'Investigator, Howard Hughes Medical Institute; Instructor in Medicine, Harvard Medical School; Assistant Physician, Robert B. Brigham Hospital '*Professor of Medicine, Harvard Medical School at the Robert B. Brigham Hospital; Physician-in-Chief, Robert B. Brigham Hospital Supported by grant No. 5-23 from the Massachusetts Chapter of the Arthritis and Rheumatism Foundation and by National Institutes of Health grants Nos. AI-07722 and AM-12051.

Surgical Clinics of North America- Vol. 49, No.4, August, 1969

741

742

SHAUN RUDDY,

K.

FRANK AUSTEN

been shown to be susceptible to the cytotoxic action of the complement system. Similarly, although the terminal step of the complement sequence results in cell membrane damage and cell death by osmotic lysis, a variety of other noncytolytic reactions occurring earlier in the sequence may result in increased vascular permeability, histamine release/ enhancement of phagocytosis,lO or chemotaxis for polymorphonuclear leukocytes. 3o The first step in the complement reaction sequence occurs when complexes formed by the interaction of an antigen with antibody bind the first component (Cl) and convert it from an inactive precursor form to an active esterase. A normal inhibitor of this enzyme is present in the alpha-2 globulin fraction of human serum!4 Cl acts on its two natural substrates, the fourth (C4) and second (C2) components, and binds them to the cell surface. Studies with radioactive C4 have shown that the reaction between C 1 and C4 is very inefficient, in that only one of approximately every 40 C4 molecules becomes bound to the cell surface; the balance remain free in the fluid phase as antigenically intact but hemolytically inactive C4i. 17 It is also clear that Cl splits the C2 molecule and binds only a portion of it to the cell membrane, the split products remaining free in the fluid phase. 15 The action of CIon C4 and C2 not only binds them to the cell surface, but also generates from their combination a new enzyme, C3 convertase. 18 This enzyme splits the third (C3) component and binds the major fragment of it to the cell surface. Like the binding of C4, the action of C3 convertase on C3 is also inefficient, and it is estimated that only one of approximately every hundred C3 molecules which participate in this reaction is bound to the cell surface, the remainder persisting in the fluid phase as hemolytically inactive C3i. At the C3 step, biologic activity becomes apparent for the first time. The fragment bound to the cell surface (C3b) enhances the ability of this cell to stick to primate erythrocytes (the immune adherence phenomenon)20 and increases the susceptibility of this cell to phagocytosis by polymorphonuclear leukocytes. lo Furthermore, a fragment not bound to the cell surface, C3a (anaphylatoxin), is a potent liberator of histamine. Although less is known about the reaction mechanisms of C5, C6, and C7 with the EAC1423 cell, it is clear that these components are also physically bound to the cell surface. During the course of this reaction, a new biologic activity appears in the fluid phase. This activity, which appears to comprise a macromolecular complex of C5, C6, and C7, directs the migration of polymorphonuclear leukocytes, and is known as a chemotactic factor. 3o It has been shown in vivo that the proliferative phase of nephrotoxic nephritis due to heterologous antibody requires the participation of an active complement system, capable of generating this C5, C6, and C7 chemotactic factor.5 The binding of C8 to the membrane of a cell which has reacted fruitfully with the first seven components results in damage to this membrane. 27 Such cells will gradually lyse on prolonged incubation. The rate of lysis is vastly increased, however, when active C9 combines with the EACl-8 cell,27 The result is the production of a morphologically

THE COMPLEMENT SYSTEM IN RHEUMATOID INFLAMMATION

743

characteristic 90 Angstrom lesion in the cell membrane,4 with concomitant breakdown in the function of this membrane and death of the cell by osmotic lysis. In summary, it is important to note that the sequential interaction of the nine components of the complement system results in the production of most of the essential ingredients of acute inflammation-namely, change in vascular permeability, attraction of polymorphonuclear leukocytes, enhancement of phagocytosis, and cell destruction. It is equally important to recall that the participation of an individual component in this reaction sequence involves depletion of the activity of this component from the fluid phase; small quantities of this component are deposited on the surface of the reacting cell, and the bulk of the component protein remains unbound in the fluid as the hemolytically inactive product.

EVIDENCE IMPLICATING THE COMPLEMENT SYSTEM IN RHEUMATOID ARTHRITIS Although measurements of serum complement levels in patients with rheumatoid arthritis have repeatedly yielded normal or slightly elevated values, two lines of evidence suggest that an intra-articular disturbance of complement metabolism may exist in some patients with this disease. Measurements of total hemolytic complement (CH50) and second component (C2) activities have demonstrated depressed values in rheumatoid synovial fluid when compared with fluids from normal individuals or patients with osteoarthritis. More recently, immunofluorescent studies with fluorescein-conjugated antibody specific for C3 and C4 have demonstrated deposits of complement components in synovial tissue obtained from rheumatoid joints. In 1964, Pekin and Zvaifler 2 measured the CH50 levels of joint fluids from 26 patients with rheumatoid arthritis, 16 with osteoarthritis, 14 with gouty arthritis, and 6 normal individuals. For patients without rheumatoid arthritis, the synovial CH50 was directly correlated with the number of polymorphonuclear leukocytes and the concentration of protein present in the joint fluid. By contrast, rheumatoid synovial fluids did not contain the CH50 activities which would have been expected on the basis of polymorphonuclear cells and protein concentration. The authors suggested that their data constituted evidence for a local antigenantibody reaction utilizing complement in rheumatoid arthritis. Synovial fluid CH50 determinations by Hedberg 13 in a total of 174 patients confirmed the observations of Pekin and Zvaifler. Hedberg adjusted the observed CH50 for synovial fluid protein concentration, and related it to the serum CH50. He found that rheumatoid patients with negative rheumatoid factor tests tended to have synovial fluid C activities which were not significantly different from those of a group with nonrheumatoid seronegative arthritides. For patients with a positive rheumatoid factor test, he found a highly significant degree of negative correlation between the synovial fluid C activity and the rheumatoid factor titers of serum and synovial fluid: patients with the highest rheumatoid factor titers had the lowest synovial fluid C activity.

744

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K.

FRANK AUSTEN

Measurements of total hemolytic complement (CH50) reflect the ability of the serum or joint fluid being assayed to lyse a standardized suspension of sensitized sheep erythrocytes. The CH50, therefore, is the resultant of the sequential interaction of all nine of the components of complement. In order to locate more precisely the abnormalities of this system reflected by the reduced CH50, Fostiropoulos, Austen, and Bloch9 measured the activity of the second component with a hemolytic assay, and identified the fourth and third components by immunoelectrophoresis with specific antiserum. The CH50 and C2 activities in 22 of the 23 rheumatoid joint fluids were markedly lower than those of control joint fluids from patients with traumatic, degenerative, or gouty arthritis. On immunoelectrophoresis, C4 was readily demonstrable in the control fluids, but little or no C4 protein was observed in five of seven effusions from patients with rheumatoid arthritis. C3 protein arcs obtained on immunoelectrophoretic analysis of rheumatoid fluids were indistinguishable from those of the controls. More recently, Barnett, Bienenstock, and Bloch! have confirmed the observations of low C2 activities in synovial fluid from rheumatoid effusions. It is now clear that the C2 assay used by Fostiropolous et al. and by Barnett et al. reflects the activities of both C4 and C2 in the test fluid, so that a more precise interpretation of this work would be that either C4 or C2 (or both) were diminished. Immunofluorescent studies of synovial tissue or synovial fluid leukocytes have revealed deposits of C3 and C4 in specimens from patients with rheumatoid arthritis. Rodman et al. 23 ,24 stained samples of synovial tissue from eight patients with rheumatoid arthritis with specific fluorescein-conjugated antibodies for IgG, IgA, IgM, albumin, C4, and C3. In addition, they used fluorescein-conjugated aggregates of human IgG to localize tissue-bound rheumatoid factors. All eight synovial membranes stained extensively for IgG, in a diffuse pattern. Studies with serial sections showed that the complement proteins, C3 and C4, tended to localize in the same areas as the IgG, and did not follow the distribution of either IgA or IgM. Deposits of rheumatoid factor were most often found in the cytoplasm of mononuclear or plasma cells, and were not accompanied by deposits of complement protein. In none of eight normal control samples of synovia was significant staining for IgG, IgA, IgM, C4, or C3 observed. The deposition of complement protein in conjunction with IgG in rheumatoid synovia was confirmed by Fish et al. B These workers studied samples from eight children and seven adults with rheumatoid arthritis, using antibody against IgG, IgA, IgM, C3, fibrin, and albumin. Aggregated gamma globulin conjugates were also used for the detection of rheumatoid factor. They classified patterns of staining into three groups: (a) three patients had homogeneous deposits of IgG and C3 in large synovial cells, thought to be tissue mast cells; (b) four patients had discrete noncellular focal deposits of IgG and C3 within the connective tissue stroma; and (c) seven patients had large, amorphous noncellular deposits in areas which corresponded to hyalinized and noncellular zones seen on routine light microscopy. Of particular interest were the

THE COMPLEMENT SYSTEM IN RHEUMATOID INFLAMMATION

745

findings in the eight specimens from patients with juvenile rheumatoid arthritis. Seven of these had deposits of C3 protein, even though four of these seven had apparently normal levels of total hemolytic complement in the joint fluids. Vaughan et al. 29 examined joint fluid leukocytes for the presence of immunoglobulins and C3. Cytoplasmic inclusions containing IgG, IgM, or C3 were found in 15 of 18 samples of leukocytes from the joints of patients with adult rheumatoid arthritis, but similar inclusions were also noted in about one half the control samples from patients who did not have rheumatoid arthritis. A strong association was, however, observed between reduced joint fluid CH50, a positive serum latex fixation test, and the presence of cytoplasmic inclusions of IgG in the synovial leukocytes. The presence of IgM deposits in the leukocytes was also strongly associated with a positive serum latex fixation test. There was no apparent relationship between demonstrable C3 in the joint fluid leukocyte inclusions and the CH50 in the joint fluid, but only half of the cell preparations from the latex positive rheumatoid patients were examined for the presence of C3.

MEASUREMENT OF INDIVIDUAL COMPLEMENT COMPONENT ACTIVITIES IN SYNOVIAL FLUID Advances in the isolation and purification of the nine components of complement, and in the techniques for the preparation of cellular intermediates of the hemolytic system, have made av3.ilable methods for the measurement of individual component activities in complex biologic fluids. Stoichiometric hemolytic assays have been described for Cl,2 its two natural substrates C4 25 and C2,3 and for C3,26 the substrate of the complex formed by the action of CIon C4 and C2. A technique for the measurement of C 1 inhibitor has also been describedY The principles of these assays are similar: they measure the activity of an individual component in a system in which all of the components except the one being measured are supplied in large excess, either bound to a cellular intermediate or free in the fluid phase of the reaction. The assay for C4, for example, requires the interaction of the fluid being tested with sheep erythrocytes s~nsitized by optimal amounts of antibody and the purified first component of complement (EACI cells). The interaction of the fluid being tested with these cells converts a portion of them to the state EACI4, and the extent of this conversion is a function of the amount of C4 contained in the test fluid. Then the system is flooded with an excess of C2, followed by C3, 5, 6, 7,8, and 9, and lysis ensues. The proportion of cells lysed is determined from spectrophotometric measurements of the released hemoglobin, and the proportion is directly related to the amount of C4 contained in the test fluid. This assay is exquisitely sensitive, normal human serum being diluted 40,OOO-fold before use. Dose responses are linear over a wide range of concentrations of C4, and the specificity of the reaction for C4 has been demonstrated by its ability to measure C4 activity in highly purified preparations of this component. Analogous assays of equally

-..}

~

al

Table 1.

Complement Component and Inhibitor Activities in Paired Serum and Synovial Fluids

PATIENT

Cl'"

DIAGNOSIS

NUMBER

C4':'

C2*

C3*

C5t

C6t

C7t

C9t

Osteoarthritis

Serum Fluid

103,000 46,900

90,000 36,000

805 545

6000t 1500

8000 4000

1600 500

16,000 3,000

6400 400

2

Adult rheumatoid arthritis

Serum Fluid

256,000 86,000

128,000 44,000

840 640

3000t 1500

4000 1500

2400 400

16,000 1,200

1600 300

3

Adult rheumatoid arthritis

Serum Fluid

354,000 10,250

47,000 5,700

780 106

4500 560

8000 2650

N.D. N.D.

N.D. N.D.

9600 '" 100

rJl

II: II>

4

5

Juvenile rheumatoid arthritis

Juvenile rheumatoid arthritis

Serum Fluid

244,000 56,500

158,000 56,000

736 488

3900 1380

8000 1340

3200 300

32,000 2,400

2400 1200

Serum Fluid

176,000

42,000

< 10,000

< 1,000

398 63

2840 214

5350 1340

2400 300

48,000 4,800

1600 600

d 2i

:;:i

d ti ti .>
~ "!j

:>d

·Stoichiometric titrations.

II>

tMicrotiter plate determinations.

> d

N.D., Not done.

2i ~

[Jl

>-3 t
2i

THE COMPLEMENT SYSTEM IN RHEUMATOID INFLAMMATION

747

high sensitivity and specificity are available for Cl, C2, C3, and Cl inhibitor. In the case of components reacting subsequently to C3, specific assays are available,21 but their stoichiometry has not been well studied, and limitations in the availability of purified components have restricted their application to the form of microtiter assays, which are semiquantitative because they involve the visual estimation of end points from sets of doubling dilutions. Applications of these techniques to the analysis of joint fluids from patients with rheumatoid arthritis are currently in progress. Table 1 shows the preliminary results of such analyses for paired sera and synovial fluids from five patients, one with osteoarthritis, two with adult rheumatoid arthritis, and two with juvenile rheumatoid arthritis. Patient 3 had a positive serum latex fixation test for rheumatoid factor, and patient 2 a negative test. Both juvenile rheumatoid arthritis patients had negative latex tests. In general, patients 2 and 4 resemble the patient with osteoarthritis with respect to the concentrations of the individual complement components in their joint fluids. The synovial fluid component levels of patients 3 and 5, however, differ markedly from those of patients 2 and 4, and from the patient with osteoarthritis. Joint fluid Cl levels are sharply reduced, and concomitant reductions in the levels of C4 and C2 are also apparent. This suggests that there may be intraarticular fixation of Cl, which removes this component from the fluid phase. Activation of the C 1 enzyme in association with this fixation may result in the fixation and destruction of C4 and C2, the natural substrates of this enzyme. Generation of C3 convertase by this process may account for the reduced levels of C3 observed in the joint fluids of patients 3 and 5. Although no important differences were found among the five patients in levels of C5, 6, and 7, it is noteworthy that a reduction in C9 was observed for patient 3, the one with a positive latex test. Complete interpretation of the changes in component activities of synovial fluid will require the study of a larger number of carefully selected patients, and the application of stoichiometric quantitative techniques to the measurement of components reacting after C3. The information presently available does, however, indicate that the reductions in CH50 levels previously reported are more likely due to the fixation and activation of Cl, and the sequential action of the remaining components than to nonspecific destruction of a single isolated component in the sequence. The preliminary data are compatible with an immunologic event occurring within the joint space of some patients with rheumatoid arthritis.

SUMMARY The complement system comprises a group of nine serum proteins which mediate the effects of antigen-antibody interaction. Among these effects are changes in vascular permeability, enhancement of phagocytosis, attraction of polymorphonuclear leukocytes, and cell death due to immune lysis. Abundant evidence indicates that there is intra-articular

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SHAUN RUDDY,

K. FRANK AUSTEN

utilization of complement in some patients with rheumatoid arthritis. Measurements of total hemolytic complement activity show marked reductions, particularly in patients with positive serum tests for rheumatoid factor. Immunofluorescent studies have demonstrated deposits of C4 and C3 in synovial tissue, in association with deposits of IgG. Preliminary measurements of individual complement component activities suggest that the reductions observed, both in adult rheumatoid arthritis and juvenile rheumatoid arthritis, resemble those which would be expected from the intra-articular fixation and activation of the complement system by an immunologic process.

REFERENCES 1. Barnett, E. U., Bienenstock, J., and Bloch, K. J.: Antinuclear factors in synovia J.A.M.A., 198:163,1966. 2. Borsos, T., and Rapp, H. J.: Chromatographic separation of the first component of complement and its assay on a molecular basis. J. Immunol., 91 :851-858, 1963. 3. Borsos, T., and Rapp, H. J.: Immune hemolysis. A simplified method for the preparation of EAC'4 with guinea pig or human complement. J. Immunol., 99:263-268, 1967. 4. Borsos, T., Dourmashkin, R R, and Humphrey, J. H.: Lesions in erythrocyte membranes caused by immune cytolysis. Nature, 202:251, 1964. 5. Cochrane, C. C.: Immunologic tissue injury mediated by neutrophilic leukocytes. Progress in Allergy, 11:1, 1967. 6. Cooper, N. R, and Muller-Eberhard, H. J.: A comparison of methods for the molecular quantitation of the fourth component of human complement. Immunochemistry, 5:155,1968. 7. Dias da Silva, W., and Lepow, I. H.: Complement as a mediator of inflammation. II. Biological properties of anaphylatoxin prepared with purified components of human complement. J. Exper. Med., 125:921-946, 1967. 8. Fish, A. J., Michael, A. F., Gewurz, H., and Good, R A.: Immunopathologic changes in rheumatoid arthritis. Arth. Rheum., 9:267, 1966. 9. Fostiropoulos, G., Austen, K. F., and Bloch, K. J.: Total hemolytic complement (CH50) and second component of complement (C2): Activity in serum and synovial fluid. Arth. Rheum., 8:219, 1965. 10. Gigli, I., and Nelson, R A.: The role of complement in immune phagocytosis. Exper. Cell. Res., 51 :45, 1968. 11. Gigli, I., Ruddy, S., and Austen, K. F.: The stoichiometric measurement of the serum inhibitor of the first component of complement by the inhibition of immune hemolysis. J. ImmunoI., 100:1154, 1968. 12. Green, H., Barrow, P., and Goldberg, B.: Effect of antibody and complement on permeability control in ascites tumor cells and erythrocytes. J. Exper. Med., 100:699, 1959. 13. Hedberg, H.: Studies on synovial fluid in arthritis. Acta Med. Scandinav., supplement, 1967, p. 479. 14. Levy, L. R, and Lepow, I. H.: Assay and properties of a serum inhibitor of C1 esterase. Proc. Soc. Exper. BioI. Med., 101 :608, 1959. 15. Mayer, M. M.: Mechanism of hemolysis by complement. In Wolstenholme, G. E. W., and Knight, J., eds.: Ciba Foundation Symposium on Complement. London, J. & A. Churchill, 1965, p. 4. 16. Muller-Eberhard, H. J.: The chemistry and reaction mechanisms of complement. Adv. Immunol., 8:1, 1968. 17. Muller-Eberhard, H. J., and Biro, C. F.: Isolation and description of the fourth component of human complement. J. Exper. Med., 118:447, 1963. 18. Muller-Eberhard, H. J., Polley, M. J., and Calcott, M. A.: Formation and functional significance of a molecular complex derived from the second and the fourth component of human complement. J. Exper. Med., 125:359,1967. 19. Muschel, L. H., and Treffers, H. P.: Quantitative studies on the bactericidal actions of serum and complement. J. Immunol., 76:1, 1956. 20. Nelson, R A.: The role of complement in immune phenomena. In Zweifach, B. H., Grant, L. H., and McCluskey, R T., eds.: The Inflammatory Process. New York, Academic Press, 1965, p. 819. 21. Nelson, R A., Jensen, J., Gigli, I., and Tamura, R: Methods for the separation, purification, and measurements of the nine components of hemolytic complement in guinea pig serum. Immunochemistry, 3:111,1966.

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22. Pekin, T. J., and Zvaifler, N. S.: Hemolytic complement in synovial fluid. J. Clin. Invest., 43:1372,1964. 23. Rodman, W. W., Williams, R. C., Jr., Bilka, P. J., and Muller-Eberhard, H. J.: Immunofluorescent localization of i3,C and i3,E globulin complement components in synovial tissues from rheumatoid arthritis patients. Arth. Rheum., 7: 749,1964. 24. Rodman, W. W., Williams, R. C., Bilka, P. J., and Muller-Eberhard, H. J.: Immunofluorescent localization of the third and the fourth component of complement in synovial tissue from patients with rheumatoid arthritis. J. Lab. Clin. Med., 69:141,1967. 25. Ruddy, S., and Austen, K. F.: Stoichiometric measurement of the activity of the fourth component of complement (C'4) in whole human serum. J. Immunol., 99:1162,1967. 26. Ruddy, S., and Austen, K. F.: C3 inactivator of man. I. Hemolytic measurement by the inactivation of cell-bound C3. J. Immunol., 102:533, 1969. 27. Stolfi, R.: Immune lytic transformation: A state of irreversible damage generated as a result of the reaction of the eighth component of the guinea pig complement system. J. Immunol., 100:46, 1968. 28. Valentine, M. D., Bloch, K. J., and Austen, K. F.: Mechanisms of immunologic injury of rat peritoneal mast cells. III. Cytotoxic histamine release. J. Immunol., 99:98, 1967. 29. Vaughan, J. H., Barnett, E. V., Sobel, M. V., and Jacox, R. F.: Intracytoplasmic inclusions of immunoglobulins in rheumatoid arthritis and other diseases. Arth. Rheum., 9:127, 1968. 30. Ward, P. A., Cochrane, C. G., and Muller-Eberhard, H. J.: The role of serum complement in chemotaxis of leukocytes in vitro. J. Exper. Med., 122:327-346, 1965. 125 Parker Hill A venue Boston, Massachusetts 02120