Sobotka Postgraduate Fellowship of the Univer- sity of Western Australia. ... Dr R D Harkness (London): One thing that seems to be missing fiom what has been ...
118 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
Penicillamine Treatment of Rheumatoid Arthritis: Relationship of Proteinuria and Autoantibodies to Immune Status
25r 201
4
IgG
IgA 9/L
9/L 3
15
by Dr P J Zilko, Dr R L Dawkins and Dr M L Cohen (Department of Clinical Immunology, Perth Medical Centre, Shenton Park, Western Australia 6008)
I
Penicillamine is an accepted form of therapy in rheumatoid arthritis and is known to suppress disease activity (Lancet 1975). Despite its effectiveness, little is known of the mode of action of the drug, although some work suggests that it may influence the immune system (Bluestone & Goldberg 1973, Huskisson & Berry 1974). It has also become apparent that penicillamine treatment causes a surprising variety of side-effects, many of which appear to be immunologically mediated. Among these are immune complex nephritis (Jaffe 1968), Goodpasture's sydrome (Sternlieb et al. 1975), systemic lupus erythematosus (Camus et al. 1974), pemphigus, pemphigoid and myasthenia gravis (Bucknall et al. 1975). We have examined the relationship between some measurements of immune function and the development of autoantibodies and proteinuria. We have assessed these with the aim of shedding some light on the action of the drug both therapeutic and in the production of these sideeffects. -
Patients and Methods The 37 patients studied fulfilled the American Rheumatism Association criteria for 'classical'
10
2
5
I
Proteinuria
No proteinuria
No proteinuria
Fig 1 IgG and IgA levels in patients with and without proteinuria
'definite' rheumatoid arthritis (Committee of the American Rheumatism Association 1959). All patients were treated with penicillamine, starting at 250 mg daily and increasing by 250 mg every two to four weeks, until a dose varying from 750 mg to 1.5 g per day was reached. This dose was maintained for at least three months, unless side-effects intervened. Depending on the clinical response the dose was then varied to maintain the maximum clinical effect at the smallest dose possible. Immunological studies were carried out before treatment and then at approximately twomonthly intervals during the study period. The length of observation ranged from 5 to 21 months, with a mean of 8.8 months. Immunoglobulin and complement levels were estimated by immunodiffusion using IgG, IgA, IgM and C3 plates (Behringwerke). Rheumatoid factor was estimated by a latex method (Hyland) or
Table I Mean baseline immunological parameters to penicillamine-treated RA patients Mean dose
(glday) (months)
Articular index
Increased skin test index (>2)(n = 5)
1.5
8.4
19.8
Rheu- RA % IgG IgA IgM C3 (g/l) (g/l) (g/l) (g/l) maton latex Seroneg ANF 0 2 4.0 20% 11.6 2.57 1.78 1.17 6.0 1+ 1 2+ 1
No change in skin test (n=7)
1.5
8.4
18.3
14.5
Proteinuriagroup (n=7)
1.2
7.3
10.0
10.2
1.89
1.48
1.16
3.4
3.5
29%
Non-proteinuriagroup (n=30)
1.2
9.1
14.4
13.1
2.65
2.03
1.34
4.6
4.7
17°
Duration
Other autoantibodies
Neg 5
3+ 1 2.80
1.74
1.41
5.2
3.0
14%
0
3
2+ 2 3+ 1 5+ 1
4+ 1 3+ 1 1+ 2 0
5+ 4+ 3+ 2+ 1+ 0
APC 1 Neg 6
Neg7
3 1 ASM 2 1 APC 1 2 4 8 14
119
Penicillamine After treatment
Before treatment
No change ANF
+ve
t
patients
20
titre
ANF
6
(16%)
8
(22%)
(54%)
/,,Ititre
lost ANF 6
or
(166%)
37 patients change ~~~~~~~~~No
\
ANP -ve
17
( 30%)
11
C
7atients i46%)
Developed
and the Rheumaton test (Denver). Autoantibodies were determined using standard indirect immunofluorescent methods. Cell-mediated immunity was assessed in 11 patients by measuring skin induration at 48 h in response to four antigens: candida, mumps, varidase (streptokinase/streptodornase), and tuberculin. A skin test index was calculated by adding the values for the four tests, scored as follows: 0-4 mm=0, 5-10 mm=1, 11 -20 mm =2and > 21 mm =3. The articular index used was a variation of that described by Ritchie et al. (1968). Statistical evaluation employed Student t and paired t tests. Results
Side-effects developed in 11 of the 37 patients during the observation period, 7 developing proteinuria of > 1 g/day, 2 developing thrombocytopenia and 2 developing mouth ulcers. All these patients, apart from one with mouth ulcers, had the drug withdrawn, although some patients were subsequently restarted on penicillamine, usually at lower doses. The baseline immune status of the patients developing proteinuria differed from that of the other patients in that immunoglobulin concentrations tended to be lower, particularly in the case of IgG and IgA (Fig 1). Two patients had subnormal levels of IgG and the values for the group ranged from 5.4 to 15.1 g/l. The mean IgG and range in the other patients was 14.1 g/l (7.0-23.0 g/l) with only one of the 30 patients having a subnormal level. The mean fgA concentration in the proteinuria group was 1.81 g/l compared with 2.65 g/l in the remaining patients.
ANF
6
(16%)
Fig 2 Change in ANF in r-heumatoid patients treated with penicillamine
None of these patients had subnormal IgA levels (i.e. < 0.7 g/l) (Table 1). The only differences with regard to. sequential changes in the groups was that there was no fall in IgA and no improvement in the articular index in the proteinuria group (Table 2). The incidence of changes in antinuclear antibody (ANF) during treatment is shown in Fig 2. Fifty-four per cent of the patients had ANF before treatment and approximately equal proportions showed an increase, decrease or no change in titre. In those patients without ANF before treatment, only a third subsequently developed detectable antibody. Of the 14 patients developing or increasing their titre of ANF, only 7 showed an increase of at least two dilutions (log 5), and as it may be argued that an increase of only one dilution could be within experimental error, only those showing an increase of two dilutions or more are considered in Table 3. The only striking differences between the three groups is that those with increased ANF had a much higher incidence of negative rheumatoid factor. When one considers the changes occurring (Table 4) the most significant findings were that the increased ANF group showed the lowest falls in IgA. None of the patients who acquired or increased ANF showed any evidence of clinical SLE, and there was no correlation between ANF and proteinuria. The incidence and type of autoantibodies other than ANF which developed are shown in Fig 3. Apart from one patient who developed a weak antimitochondrial antibody (AMC) all the other antibodies were to muscle components. A total of
Table 2 Change in mean clinical and laboratory parameters in penicillamine-treated RA patients Duration Change in treatment articular (g/day) (months) index -11.2 8.4 Increased skin 1.5 test index (>2) Mean dose
IgG
IgA
(g/l) (gll) -3.22 -0.77
-0.52
C3 (g81) -0.03
Rheumaton RA latex (no. of tubes (no. of tubes change in change in titre) titre) -2.7 -2.5
IgM
(gll)
(n = 5) 1.5 Nochangein skin test (n = 6) 1.2 Proteinuria (n =7)
7.1
-5.0
-1.66
-0.42
-0.70
-0.06
-1.0
0
7.3
0
-2.51
-0.04
-0.60
-0.06
-1.0
-0.8
Development
of other autoantibodies ANF 3+O(1) 2+ 3 (1)
IncreaseinANF(3) Decrease inANF(l)
2+ASM(l) 1 +ASM (1) 3+AStr(l)
120 Proc. roy. Soc. Med. Volume 701977 Supplement 3 Before Treatment
After Treatient
2 + APC I patient -> 2 + ASM I patient -> I + ASMH Ioatient ->
37 patients
No change Became negative (ANF also 2 + ASM
-ve
ASM -ve
I
)
25 patients *ve
4
ASH
patients
+ *
patients
34
I
ASS 3 tr.
A.
Striational
3
patienta.Z.
1 + A Str. A.
AMC
2
Heart I patient I patient
Fig 3 Autoantibodies apart from ANFdeveloping in rheumatoidpatients treated with penicillamine
8 out of 34 patients developed these antibodies 1974). The patients with proteinuria had the drug and of these, 3 patients were among those who stopped, but some were restarted on penicillamine developed proteinuria. These 8 antimuscle without the recurrence of unacceptable proteinpositive patients showed a similar baseline uria. The tendency of these patients to have immune status to the other groups (Table 3) but low immunoglobulin levels suggests that this did show a striking change over the observation humoral depression may be a factor in the period by increasing IgA levels compared to falls development of this complication. The falls in in IgA in the other groups (Table 4). There was immunoglobulins in these patients despite the also a relatively small fall in IgM. None of the preexisting low levels may indicate that the patients who developed antistriational antibody penicillamine may have caused some humoral developed overt clinical evidence of myasthenia depression. gravis or X-ray evidence of thymoma, but 3 were One of these 7 patients had a renal biopsy considered to have subclinical myasthenia and performed which showed minimal histological will be followed further. changes but heavy deposits of IgG and C3 on Of the 11 patients skin-tested sequentially none immunofluorescence. The nature of such immune showed any significant depression of skin complexes in this condition is not known, reactivity, but 5 showed an increased skin test although it has been shown that successful index (>2). There was no difference in the two penicillamine treatment is accompanied by a fall skin test groups with respect to baseline immune in the detectable levels of circulating immune status (Table 1), but those manifesting increased complexes (Mohammed et al. 1976). It is possible skin responses showed larger falls in IgG and that penicillamine-induced humoral depression rheumatoid factor, and showed more marked in these patients caused a change in the antigenclinical improvement as reflected in the articular antibody ratio of the circulating immune complexes, thus causing deposition in the kidney index (Table 2). leading to proteinuria. The opposing view that these complexes consist of a drug as a hapten and Discussion The incidence of serious side-effects found here is its antibody has also been suggested, but to date in accord with that found in other studies there have been no definitive studies of the nature (Multicentre Trial Group 1973, Day & Golding of the immune complexes found in the kidney.
Table 3 Mean baseline immunological parameters in penicillamine-treated RA patients Mean dose
Duration
(g/day) (months) 1 Decrease in ANF
1.4
8
Articular IgG IgA IgM C3 index (gll) (g/l) (g/l) (gll) Rheumaton 16.5 13.3 2.13 1.87 1.27 4.8
RA latex 4.3
Other auto% Seroneg ANF antibodies 0
(n=6) II No change (n= 17)
1.2
III Increase in ANF (n=7) Developed smooth muscle, striational or heart antibodies
1.3
(n=8)
1.3
7.5
10 8
9.9
17.9 9.6
11.4
15.2 14.3
2.37
2.54
2.44
2.0
1.6 1.95
1.26
1.58 1.35
4.7
3.7 4.2
5.2
3.5 4.6
29%
43%
33%
1+ 2+ 3+ 5+
2 1 2 1 0 11 I+ 2 2+ 1 3+ 1 4+ 2 0 4 I+ 2 2+ 1 0 5 2+ 1 4+ 3
ASM 1
Neg 5 ASM 1
Neg 16 APC I
Neg 6 Neg 9
121
Penicillamine
Table 4 Change in mean clinical and laboratory parameters in penicillamine-treated RA patients Mean dose
Duration Change in treatment articular
(glday) (months) 1 Decrease in ANF (n =6) II No change
(n=17) III Increase in ANF (n = 7) Developed smooth muscle, striational or heart antibodies (n =8)
IgG
IgA
(gll)
(gll)
-2.62
-0.13
Rheumaton (no. of tubes change in titre) -2.4
RA latex (no. oftubes change in titre) -0.6
C3
(gll)
-0.27
igM (g/l) -0.67
1.4
8.0
index -5
1.2
7.5
-3.6
-0.50
-0.34
-0.63
+0.05
-2.4
-1.6
1.25
10.0
-5.3
-1.04
-0.65
-0.13
-0.17
+0.25
-0.3
1.20
10.2
-4.7
-1.10
+0.62
-0.26
+0.16
-1.0
-1.0
The occurrence of ANF with penicillamine therapy has been noted before, and a proportion of these cases may develop clinical systemic lupus erythematosus (SLE) (Camus et al. 1974). In our series there was no consistent pattern of changes, and, in fact, nearly as many patients lost their ANF as developed it, or increased the titre. It is notable that in the group who had a significant rise in ANF titre there was no clinical evidence of SLE. Anti-DNA binding by the Farr technique was estimated in some of these cases and was always found to be within the normal range. The group of patients who developed antibody to muscle components, particularly smooth and striated muscle, proved more interesting. They showed a much higher incidence of proteinuria (43 %) than the group without antibody (13 %), and although there was nothing to distinguish them in comparing the baseline immune status, they did show a rise in IgA levels compared with falls in IgA in other groups. The explanation for this correlation is not obvious, although it may be postulated that these changes could reflect disturbances in immune homeostasis. It has recently been suggested that penicillamine may be an immunostimulant and act in a similar manner to levamisole (Huskisson et al. 1976). We have shown that penicillamine, although not immunosuppressant in terms of skin test reactivity, has a variable effect with only some patients showing increased responses (Zilko & Dawkins, unpublished). Those patients showing an increase in skin test reactivity had larger falls in IgG and rheumatoid factor, and more marked clinical improvement as shown by the articular index. There was no correlation with proteinuria, but this group with augmented skin tests had a lower incidence of ANF. None of these 11 patients skin-tested developed muscle antibodies. We would suggest that the augmentation of cell-mediated immunity seen with penicillamine treatment may be due to a suppressive effect on
Development ofother autoantibodies 2+ASM(l) 1 +AH (1) 2+ASM>0(1) 1 +ASM(2) 3 +AStr (1) 1 +AMC(1) 1 +AStr(1) ASM (4) AStr (3) AH (1)
humoral immunity rather than a direct effect on T-cell function (Zilko & Dawkins, unpublished). The data presented here of the patients with augmented skin tests having more intense suppression of some humoral parameters would be in accord with the above hypotheses. It has been argued elsewhere, without any evidence, that the falls in immunoglobulins and rheumatoid factors are secondary manifestations of a general suppression of disease activity. The mode of action of penicillamine in inducing proteinuria and antibodies has not been clarified in this study, but it would appear that relatively low immunoglobulin levels in some patients may be one of the factors predisposing to proteinuria. It is also apparent that the appearance of muscle antibodies has much more clinical significance in terms of side-effects than that of antinuclear antibodies, which occur commonly but do not seem to be associated very frequently with any particular side-effect.
Summary Thirty-seven patients were studied before and during treatment with respect to immune status, clinical response and development of adverse effects and autoantibodies. The baseline immune status was not predictive in terms of the above features, apart from the fact that the group of 7 patients developing proteinuria had a tendency to low or subnormal IgG levels. The most marked clinical improvement was recorded in the group who had augmented skin test responses sometime during the treatment period. These patients also had the largest falls in the IgG, IgA and rheumatoid factor. Antinuclear antibody persisted or increased in titre in 38% of patients, but was not associated with poor prognosis or liability to side-effects. Autoantibodies to striational or smooth muscle occurred in 20% of patients, and there was a
122 Proc. roy. Soc. Med. Volume 70 1977 Supplement 3
much higher incidence of proteinuria in this group. We have previously suggested that penicillamine may act by depressing humoral function, leading to augmentation of cell-mediated immunity. Although the present findings suggest that penicillamine does cause humoral depression in some cases, it is not clear how the drug induces the side-effects described.
Dr Zilko: We did not observe that sort of reaction, although it is interesting that you have also noted low immunoglobulins in those patients with proteinuria.
Acknowledgments: This work was supported by the Western Australia Arthritis and Rheumatism Foundation. P J Zilko is a recipient of a P F Sobotka Postgraduate Fellowship of the University of Western Australia. We are grateful to the WAARF Study Group for allowing us to study their patients.
Dr R D Harkness (London): One thing that seems to be missing fiom what has been discussed this morning is any mention of gut bacteria and dietary intake. Sometimes these patients get nausea. This seems to have been missed out in accounts of changes taking place under this treatment. Has anyone any information about it?
REFERENCES Bluestone R & Goldberg L S (1973) Annals of the Rheumatic Diseases 32, 50 Bucknall R L, Dixon A St J, Glick E N, Woodland J & Zutshi D W (1975) British Medical Journal i, 600 Camus J-P, Cruzet J, Beniclose C & Lievre J A (1974) Annales de mcdecine interne 125, 9 (Committee of the American Rheumatism Association (1959) Arth,itis and Rheumatism 2, 16 Day A T & Golding J R (1974) Postgraduate Medical Journal 50, Suppl. 2, 71 Huskisson E C & Berry H (1974) Postgraduate Medical Journal 50, Suppl. 2, 59 Huskisson E C, Dieppe P R, Scott J, Trapnell J, Balme H W & Willoughby D R (1976) Lancet i, 393 Jaffe I A (1968) Postgraduate MedicalJournal 44, October Suppl., p 15 Lancet (1 975) i, 1123 Mohammed I, Barraclough D, Holborow E J & Ansell B M (1976) Annals of the Rheumatic Diseases 35,458 Multicentre Trial Group (1973) Lancet i, 274 Ritchie D M, Boyle J A, McInnes J M, Jasani M J, Dalakos J G, Grierson P & Buchanan W W (1968) Quartet ly Journal of Medicine 37, 393 Sternlieb I, Bennett B & Scheinberg I H (1975) Annals of Internal Medicine 82, 673
Dr Zilko: I have no information.
DISCUSSION
Dr J Watkins (Sheffield): We are particularly interested in this work because we are doing something similar. I was interested in the group which showed proteinuria. What were the normal ranges of immunoglobulins in this group? Dr Zilko: With IgG 8-16 g/l, with IgA 0.82.3 g/l, and with IgM 0.9-3.2 g/l. Dr Watkins: Those are similar to our levels. We have found that there is a group of patients who develop proteinuria who have these rather lower levels of immunoglobulin. They are not ab-
normally low, but low normal. They develop transient rises in IgG, perhaps shooting up to 20 g/l, or even 25 g/l, and then develop these adverse reactions.
Dr R N Maini (London): The incidence of antinuclear antibodies in Dr Zilko's study seems to be exceptionally high - 57 % of the rheumatoid patients had antinuclear antibodies. Dr Zilko: That is with a screening dilution of one
in five. Dr Maini: I am not aware of any other series in which such a high prevalence rate has been described: 20% is generally about the highest reported rate. Certainly our patients would not exceed that figure, and might be less (at 1/10 dilution).
Dr Zilko: Of course the patients we studied were severe rheumatoids with longstanding disease. If one studied rheumatoids with mild disease the incidence would be lower. Dr Maini: We have seen double-strand DNA antibodies in our RA patients treated with penicillamine. There is no doubt that these antibodies occasionally develop during treatment and disappear following withdrawal of the drug. In one case we found immunoglobulin and C3 deposits at the dermo-epidermal junction in noninvolved skin, which were rather typical of lupus. It was unusual, again, that Dr Zilko did not find raised anti-DNA antibodies in the large number of patients he has treated. Dr Watkins: May 1 support Dr Zilko, in response to Dr Maini s question. We have seen several hundred rheumatoid patients at the Protein Reference Laboratory, and the incidence of significant ANF titres is almost 50%. It is extremely high in rheumatoid arthritis.
