Aust h L' J Wed (1979). 9, pp__370 377
_.
Immune Complexes in Diabetes Mellitus J. A. Charlesworth*, L. V. Campbell:, J. Quin:.
L. Lazarus" and G. J. Macdonald++
From the Department of Medicine, Prince Henry Hospital, University of New South Wales and the Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney
Summary:
Immune complexes in diabetes mellitus. J. A. Charlesworth, L. V. Campbell, J. Quin, L. Lazarus and G. J. Macdonald, Aust. N.Z.J . Med., 1979, 9, pp. 370-373.
Sera from 86 well controlled diabetics were examined for the presence of immune complexes. Thirty-six patients were receiving standard insulins, 19 monocomponent preparations, 24 oral hypoglycaemic agents and seven dietary restriction alone. Three methods were used to detect complexes: measurement of complement components, a Clq binding assay (BA) and the Raji cell radioimmunoassay (RIA). Complement components were normal in all patients. Eleven (31 Yo)of the group on standard insulins had a positive Raji cell RIA; none had an abnormal Clq-BA. One patient on monocomponent therapy had a mildly positive Raji cell RIA; ClqB A was negative in each patient of this group. Thirteen (54%) of the patients on oral hypoglycaemic agents were positive on one or both assays while one patient on diet alone was abnormal on both assays. These data show that immune complex production is common in both insulin-requiring and non-insulinrequiring diabetics and that this phenomenon is strikingly less frequent in patients on monocomponent insulins. Such observations could bear relevance to the pathogenesis of microvascular complications in diabetes.
A number of clinical and experimental observations suggest that insulin-requiring diabetes (IRD) has an autoimmune aetiology.132However, serological evidence of immune pancreatic .
__
'Renal Physician, Prince Henry Hospital.' +Research Fellow in Endocrinology, Garvan Institute of Medical Research. :Senior Scientific Officer, Prince Henry Hospital. '* Director, Garvan Institute of Medical Research. t+Senior Lecturer in Medicine, Prince Henry Hospital. Correspondence: Dr. J. A. Charlesworth, Renal Unit, Prince Henry Hospital, Little Bay, NSW 2036 Accepted for publication: 17 January, 1979
damage is usually transient or absent in such cases.3 On the other hand allergic reactions to therapeutic agents, and in particular insulin, are well recognised. Insulin antibodiec can be detected in virtually all insulin-treated diabetics' and recent work reports the finding of serum immune complexes in this group of patient^.^ Such complexes have been proposed to cause tissue injury in diabetes although evidence remains inconclusive. Most observations in this regard have bccn made in relationship to diabetic nephropathy: certain authors have reported proliferative glomerular lesions on light microscopy'. while others have shown binding of gamma-globulin7, complement8 and insulin9 using immunofluorescent techniques. Wehner" has reported the appearance of hump-like projections of glomcrular basement membrane following the administration of hetcrologous insulins to guinea pigs and rabbits. The incidence of the lesions rose as insulin antibodies increased. Animals treated with monocomponent insulins did not show changes in glomerular structure, nor did they develop detectable insulin antibodies. With the advent of sensitive methods for detecting immune complexes in the circulation it is possible to examine the frequency of these phenomena in different groups of diabetic subjects. In our study, complexes were sought in 86 well controlled diabetic patients attending a clinic. Fifty-five were receiving insulin, 24 were taking oral hypoglycaemic agents and seven had only dietary restriction. Nineteen of the insulintreated group were using only monocomponent insulins. Three methods were used to demonstrate immune complex reactivity: measurement of complement components; a binding assay involving the precipitation of 1251. Clq with polyethylene glycol (PEG)" and a radioimmunoassay (RIA) using the C3b receptor on a lymphoblastoid cell-line (Raji cells).I2
371 Patients and Methods Eighty-six patients with proven diabetes niellitus were sludied. Certain clinical details are included in Table I . All were clinically stahle at the time of investigation; none had overt infection. Fifty-five (age range: 13 81 years; mean: 52 years) were insulin-treated. Nineteen of these had used exclusively monocomponent preparations for at least six months (duration of all forms of insulin therapy: 1 32 years; mean: 1 I years). The other 36 were receiving combinations of standard short and long-acting bovine and porcine insdins (duration of insulin therapy: 1 27 years; mean: 10 years). Nineteen of these 55 patients had one o r more complications attributable to diabetes: seven peripheral neuropathy, ten retinopathy, eight nephropathy and two peripheral vascular 'e (I'VD). I'VD was not regarded cation in patients over 60 years. None of this group was receiving oral hypoglycaemic agents. Twenty-four patients (age range: 43 86 years: mean: 6 3 years) were receiving oral hypoFlycaeinic agents. P'ifteen of these were taking glihenclamidealone' or in combination with a biguanide. The other nine were using various regimens of biguanides and other sulphonylureas. All had been stabilised o n such treatment for at least three months. None had ever received insulin. Five of this group had one or more diabetic complications: three nephropathy. three retinopathy and one neuropathy. Seven patients (age range: 40 86 years; mean: 59 years) were on alone. Patients with evidence of co-existing es were not studied. An antinuclear factor (ANF) was performed on all positive sera to exclude systemic lupus erythematosus (SLE).
lymphoma. Details of this y were identical to those described elsewhere.'2 Results were expressed as p g equivalent of aggregated human IgG ml of serum (normal range: 4k 16 pg mean + 2 SD---in 30 healthy age-matched, controls). Kaji cells were supplied by Dr. J. Pope, Queensland Institute of Medical Research. Immunoglobulin content of washed PEG precipitates was determined by radial inimunodiffusion using antisera to IgG, IgM and IgA. Antigen and antibody components of precipitates of samples from four patients on oral therapy w t h a positive ( X - B A were separated by a standard method using Crlycine-hydrochloric acid. pl1 2 . S1" and samples reconcentrated h j pressurised nitrogen evaporation at 37 C'. Insulin content was then nieasurcd by KIA using similarly processed controls and a porcine insulin standard supplied by
-1 Iq-Bk RAJl Clq-Bk
>loo 80 0
TABI,E 1 Summary of patient sub-groups Age range modal clays
Compltcations ("J
Positive assays
("4,)
Standard insdins (n = 36)
13 81 50- 60
39
31
MC - - I n d i n s (n = 19)
15 74 45 55
20
5
Oral theiapy (n - 24)
43 86 60-70
21
54
Diet alone (n = 7)
40-86 50 60
14
14
E
3
a
: d,
0
L40
!
IRD
+
t
6 o I
0
Coniplern(wt Stirdic~s All sera were stored at - - 20°C and analysed within one week of coliection. clq.C4. c3,Factor B and C5 were measured by radial immunodiffusionL3 using monospecific antisera. Values were expressed as a percentage of pooled normal human serum (NHS). Sera for immune complex analysis were stored at - 20 'C with at least two control sera being kept under identical conditions for each assay. Samples were thawed once only (at 37 'C) and tested within 48 hours of collection. Two assays were used. Firstly, quantitation by binding of complexes to purified '''I. Clq (normal range: 13 6.5";,- mean 2 sd in 35 healthy age-matched controls). Secondly. an RIA using Kaji cells, a lymphoblastoid cell line from Burkitt's
% -
IRD-MC
FIGURE 1. Results of Clq-BA and Raji RIA in patients on (i) standard insulin therapy (IRD) and, (ii) monocomponent preparations (IRD-MC)
ResuIts
Complement components in each group were normal or mildly elevated. Results of immune complex assays are shown in Figures 1 and 2 and Table 1. In the IRD group, 11 out of 36 patients (3 108) on standard insulins had a positive result on Raji cell RIA; the Clq-BA was negative for all patients in this group. One out of 19 patients on mono-component therapy had a positive Raji cell RIA: Clq-BA was negatiye in each patient of this group. In the group on oral hypoglycaemic therapy. five (i.e. 21%) were positive on both assays while a further eight (330.;) had one positive result (one Clq-BA and seven Raji cell RIA). The following medications were being used by these patients: seven glibenclamide; two phenformin; two tolbutamide: one gliben-
37 2
_-
CHARLESWORTH Kl' AL.
- -...-.
-
.-
--
Iq-Eh 3AJI Iq-BI RAJl *a-
100, CT
V
a
a
,3100
80
O
In-
.-d 0
{
.60
60
40
o\" 20
0
...
a
a
-40
a
:
-I - .20
:In Oral Therapy
-0
Diet
FIGURE 2. Results of Clq-BA and Raji RIA in patients on (i) oral hypoglycaemic agents and, (ii) diet alone.
clamide and phenforniin and one tolbutamidc and metformin. One patient on diet alone had an abnormal result for both assays. Five of the 11 patients on standard insulins with a positive Raji cell RIA had diabetic complications: two retinopathylnephropathy; one neuropathyi retinopathy; one nephropathy, and one PVD. Three of the non-insulin-requiring group with assay-abnormalities had complications: two retinopathy: one nephropathy. In all, 16 of 60 patients with negative assays had complications. No patient in the study had a positive ANF. Analysis of PEG precipitates for immunoglobulin class showed only IgG. Patients with a negative Clq-BA did not form detectable precipitates. No insulin activity was detected in dissociated PEG precipitates. Discussion
A significant number of both insulin-requiring and non-insulin-requiring diabetics had abnormal amounts of complement binding substances in serum. It is known that each of the radio-binding assays is complement-dependent and may react with a variety of substances; however, recent work15 suggests that both show a high degree of preferential reactivity with immune complexes. The findings in the group
.~...
..
VOI.. .
9. KO.. .4-
receiving standard insulin therapy are in agreement with lrvine and co-workers' who found the Kaji cell RIA to be positive in 53"/(,of such patients. The higher incidence of positive results in their series is explained by the inclusion of eight patientb (2%;) whose value was at the upper limit of normal. They did not examine patients on monocomponent insulins and no other methods of immune complex detection were used. In contrast. the) found the Raji cell RIA to be negative in 52 patients on oral therapy or diet alone. The age range of the patients \\as not stated. In our study, two different patterns of immune complex reactivity were observed: firstl,, a positive Raji cell RIA and a negative Clq-BA in the IRD group and secondly, positivity of either or both assays in the non-1RD group (including one patient on diet alone). In established immune complex diseases-. such as active SLE or acute glomcruloncphritis both assays have been reported to be positive in a high percentage of ~ a s e s . " ~ 'The ~ ~ reason '~ for the different assaypatterns In our patients is not clear. One explanation would be the molecular size of complexes in the two groups. It is possible that the antigenic component in insulin-treated diabetes is insulin'" (or insulin polymers) implying a rclat;vely low molecular weight for these complexes. Such material might not activate the Clq-BA which detects high molecular weight reactants." On the other hand, the Raji cell RIA has been shown to react with complexes of widely varying molecular size.I2 Positivity of both assays, therefore suggests that the serum reactants in the group on oral hypoglycaemic therapy are of high molecular weight. It is also possible that the complexes in the IRD group activate the alternative pathway of complement; such a proposal is unproven, but would explain the selective positivity of the Raji cell RIA. In contrast, results on patients using monocomponent insulins were essentially negative although our methods do not exclude the prescncc of non-complement-fixing complexes. This observation would also be consistent with the hypothesis that some component other than insulin monomer is the antigen in complexes seen in patients on standard therapy, as monocomponent insulins have been
shown to produce less antibody than conventional preparations.16 Immunoglobulin-analysis of PEG precipitates in the non-1RD group showed only IgG. The antibody specificity in the Raji cell RIA implies that the antibody in the I R D group is similarly IgG. The nature of the antigen(s) particularly in the non-IRD patients remains to he determined. The observation that such complexes can occur in a patient on diet alone suggests that this antigen is unrelated to therapy. Although a number of patients with positive assays in both groups showed peripheral diabetic complications their occurrence was comparable with others where both assays were negative. Their tissue-damaging effect therefore remains unproven, although complement-fixation and large molecular size are two factors known to influence antigen-antibody mediated injury." If immune complexes in IRD prove to be associated with diabetic complications then their absence in monocomponent-treated patients suggests such therapy to be a major advance in the management of this disease. Further longitudinal studies of such patients and analysis of immune complex structure and behaviour may help define the role of this immune mechanism. Acknowledgements We wish to thank Miss C. Boon and Miss S. Pickup for skilled technical assistance and Dr. U'.Kidson for providing sera.
References I R V l s t . W. W. JJ.. 11975) 11975) Clinicdl Clinicdl Aspects Aspects of of Immunology, Immunology, Hlackweil Blackwell Scientific Scientific 1.1. IRVlst.
2. 2.
Publications. Oxford Oxford Publications. x5Kl:P.J.. Pi..AIL. Pi..AIL. PP ,,44VWDXt R s L sS.. 0 0 0.. 0..CCHHRRIISSIIYY.. hl.. hl.. LI .YYVu a o E , II ,, P(H~I.sl\, PlH~i.Si\, N5Ri:P.J.. E., RYIXR, RYOER, I.. PP ,, STAt'B STA1.B NlriS€.S. NlriS€.S. L., L., TIIOMs1,s. TI~OMs1,s.\1 \1 and and SVIICAARD, SVIICAARD, A. J.J. E., I.. A. 11974): 11L-A 11L-A antigens antigens and and diabetes diabetes inellitus, inellitus, Luricer Luricer 2, 2, XX6644 866. 866. 11974): J., GRAY,R S , ~AMl'Ill.I.1.. C. J., DUXAS, HA%, H and M(IRRIS,P. J (1977) Pancreatic islet-cell antihcdxs in diahefes mellifus corr d with the dUrdt!On and type of diabetes, co-existent .+utoiminurie disea md HLA type. 1)iuherP.s 26, 138 147 A,. YYA AIi rI oOwW, ,RR SS .. HAI HAIHAS. HAS. A A .. RCIII~S(.HILD. RCIIHS(.HILD.M M A A and and SS A,. IY. K. (19%): Insulin 1"' me~dbdism in h n m m suhlects. trailon of Iiiwlin hiiiiiing globulin in the circulatum ~ i f i n \ i i l ntrcdtcd i
pathogenesis of dmhetx glomerulmxlerosir. II Deinonstraimn of ins Innding cdpdcity capacity of o ithe the Yd~iOu5 vdrmu5 histopathological histopathological components components of of the the dis disease hinding by fluorescence fluorescence microscopy. microscopy. Diuheres Diuheres 11, 11, 30X 30X 317. 317. by 88 HBcnaHl~i IIER. P. P. \ \I I. .(1965). (1965). IImmunuhistopatholOBic 01 localised localised c R a H i l i IXR. m m u n o h i s t o p a i h o l ~ ~ study istudy c of plasma proteins and fiaation of guinra-pig complement in renal lesions of diabetic elomerulasclerosis. D i u h ~ r e s14. 7 5 5 ,770. 9. FAKKANI. P. C. and S i m ) i x . ~W , I H (1965). Ohrcrvationa (in the uptake (it insulin conjugated with fluorescein isoihioqanale b) diahetx kidncy tissuc. Drohcrv.$ 14, 14, 274 274 278. 278. Drohctv~ 10 WI 1191:R, €1. (1976). Immunological aspects ofdiabetes niellitus. Actuendorr. 83 1Suppl. 205). 241 253
I?.
Circulating immune complexes in the serum 111 systemic lupus erythcmatosus and in carrier* of hepatitis B antigen. J . clrn inrcsr. 54, 297 309. ' I i l i ~ ~ ~ i I . ~ A. ) ~S~ ~WII.sos, ~ ~ i . ~C ~ . 13 and Dixr)\, F.J f I 97hi: The Rajj cell detecting immune complexes In human sera. J d m .
.
AKA. A. 0 and I1ini.MANs. J . I . (1965) Immunon o i mtifcns by single radial Irnrnunodiifusion. I l , I l , l u l l o ~ ~ h r l , l r .2, ~ r 235 ~~
254 14. f'At.i.ti, W. P.. KAR.W, J. TI and P~JI)I\IER(;, JI. H (1971) Human antiinsulin antibodies. J In,murrol 106. I I I ? 11 I6 I C WOOI)ROIC~.. A J., HOnmK, W A , THMI~~I~OPOIJLOS A . S . , tiorzi:. 0 .
tir.nssocx, R. J., Dixos. F. J . and WILSON,C. B. (1977): I k t e c t m n uf circulating immune complexes in patients with glomerulonephntis, Kidtiiar hrf. 12, 268-278. 16. Y-r:t, D. K . andTmric, I. K . f 1 9 7 3 New formsoiinsulin andthcirusein the treatment of diabetes. Diuheres 26, 341 345. (1971). Mechanisms involved in thcdepositiun of.irnrnune 17. C'lK'HKAht, C. 0. complexes in tissues, J . e l p .%fed 1.34, -5s 89s
_.
Immune Complexes in Diabetes Mellitus J. A. Charlesworth*, L. V. Campbell:, J. Quin:.
L. Lazarus" and G. J. Macdonald++
From the Department of Medicine, Prince Henry Hospital, University of New South Wales and the Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney
Summary:
Immune complexes in diabetes mellitus. J. A. Charlesworth, L. V. Campbell, J. Quin, L. Lazarus and G. J. Macdonald, Aust. N.Z.J . Med., 1979, 9, pp. 370-373.
Sera from 86 well controlled diabetics were examined for the presence of immune complexes. Thirty-six patients were receiving standard insulins, 19 monocomponent preparations, 24 oral hypoglycaemic agents and seven dietary restriction alone. Three methods were used to detect complexes: measurement of complement components, a Clq binding assay (BA) and the Raji cell radioimmunoassay (RIA). Complement components were normal in all patients. Eleven (31 Yo)of the group on standard insulins had a positive Raji cell RIA; none had an abnormal Clq-BA. One patient on monocomponent therapy had a mildly positive Raji cell RIA; ClqB A was negative in each patient of this group. Thirteen (54%) of the patients on oral hypoglycaemic agents were positive on one or both assays while one patient on diet alone was abnormal on both assays. These data show that immune complex production is common in both insulin-requiring and non-insulinrequiring diabetics and that this phenomenon is strikingly less frequent in patients on monocomponent insulins. Such observations could bear relevance to the pathogenesis of microvascular complications in diabetes.
A number of clinical and experimental observations suggest that insulin-requiring diabetes (IRD) has an autoimmune aetiology.132However, serological evidence of immune pancreatic .
__
'Renal Physician, Prince Henry Hospital.' +Research Fellow in Endocrinology, Garvan Institute of Medical Research. :Senior Scientific Officer, Prince Henry Hospital. '* Director, Garvan Institute of Medical Research. t+Senior Lecturer in Medicine, Prince Henry Hospital. Correspondence: Dr. J. A. Charlesworth, Renal Unit, Prince Henry Hospital, Little Bay, NSW 2036 Accepted for publication: 17 January, 1979
damage is usually transient or absent in such cases.3 On the other hand allergic reactions to therapeutic agents, and in particular insulin, are well recognised. Insulin antibodiec can be detected in virtually all insulin-treated diabetics' and recent work reports the finding of serum immune complexes in this group of patient^.^ Such complexes have been proposed to cause tissue injury in diabetes although evidence remains inconclusive. Most observations in this regard have bccn made in relationship to diabetic nephropathy: certain authors have reported proliferative glomerular lesions on light microscopy'. while others have shown binding of gamma-globulin7, complement8 and insulin9 using immunofluorescent techniques. Wehner" has reported the appearance of hump-like projections of glomcrular basement membrane following the administration of hetcrologous insulins to guinea pigs and rabbits. The incidence of the lesions rose as insulin antibodies increased. Animals treated with monocomponent insulins did not show changes in glomerular structure, nor did they develop detectable insulin antibodies. With the advent of sensitive methods for detecting immune complexes in the circulation it is possible to examine the frequency of these phenomena in different groups of diabetic subjects. In our study, complexes were sought in 86 well controlled diabetic patients attending a clinic. Fifty-five were receiving insulin, 24 were taking oral hypoglycaemic agents and seven had only dietary restriction. Nineteen of the insulintreated group were using only monocomponent insulins. Three methods were used to demonstrate immune complex reactivity: measurement of complement components; a binding assay involving the precipitation of 1251. Clq with polyethylene glycol (PEG)" and a radioimmunoassay (RIA) using the C3b receptor on a lymphoblastoid cell-line (Raji cells).I2
371 Patients and Methods Eighty-six patients with proven diabetes niellitus were sludied. Certain clinical details are included in Table I . All were clinically stahle at the time of investigation; none had overt infection. Fifty-five (age range: 13 81 years; mean: 52 years) were insulin-treated. Nineteen of these had used exclusively monocomponent preparations for at least six months (duration of all forms of insulin therapy: 1 32 years; mean: 1 I years). The other 36 were receiving combinations of standard short and long-acting bovine and porcine insdins (duration of insulin therapy: 1 27 years; mean: 10 years). Nineteen of these 55 patients had one o r more complications attributable to diabetes: seven peripheral neuropathy, ten retinopathy, eight nephropathy and two peripheral vascular 'e (I'VD). I'VD was not regarded cation in patients over 60 years. None of this group was receiving oral hypoglycaemic agents. Twenty-four patients (age range: 43 86 years: mean: 6 3 years) were receiving oral hypoFlycaeinic agents. P'ifteen of these were taking glihenclamidealone' or in combination with a biguanide. The other nine were using various regimens of biguanides and other sulphonylureas. All had been stabilised o n such treatment for at least three months. None had ever received insulin. Five of this group had one or more diabetic complications: three nephropathy. three retinopathy and one neuropathy. Seven patients (age range: 40 86 years; mean: 59 years) were on alone. Patients with evidence of co-existing es were not studied. An antinuclear factor (ANF) was performed on all positive sera to exclude systemic lupus erythematosus (SLE).
lymphoma. Details of this y were identical to those described elsewhere.'2 Results were expressed as p g equivalent of aggregated human IgG ml of serum (normal range: 4k 16 pg mean + 2 SD---in 30 healthy age-matched, controls). Kaji cells were supplied by Dr. J. Pope, Queensland Institute of Medical Research. Immunoglobulin content of washed PEG precipitates was determined by radial inimunodiffusion using antisera to IgG, IgM and IgA. Antigen and antibody components of precipitates of samples from four patients on oral therapy w t h a positive ( X - B A were separated by a standard method using Crlycine-hydrochloric acid. pl1 2 . S1" and samples reconcentrated h j pressurised nitrogen evaporation at 37 C'. Insulin content was then nieasurcd by KIA using similarly processed controls and a porcine insulin standard supplied by
-1 Iq-Bk RAJl Clq-Bk
>loo 80 0
TABI,E 1 Summary of patient sub-groups Age range modal clays
Compltcations ("J
Positive assays
("4,)
Standard insdins (n = 36)
13 81 50- 60
39
31
MC - - I n d i n s (n = 19)
15 74 45 55
20
5
Oral theiapy (n - 24)
43 86 60-70
21
54
Diet alone (n = 7)
40-86 50 60
14
14
E
3
a
: d,
0
L40
!
IRD
+
t
6 o I
0
Coniplern(wt Stirdic~s All sera were stored at - - 20°C and analysed within one week of coliection. clq.C4. c3,Factor B and C5 were measured by radial immunodiffusionL3 using monospecific antisera. Values were expressed as a percentage of pooled normal human serum (NHS). Sera for immune complex analysis were stored at - 20 'C with at least two control sera being kept under identical conditions for each assay. Samples were thawed once only (at 37 'C) and tested within 48 hours of collection. Two assays were used. Firstly, quantitation by binding of complexes to purified '''I. Clq (normal range: 13 6.5";,- mean 2 sd in 35 healthy age-matched controls). Secondly. an RIA using Kaji cells, a lymphoblastoid cell line from Burkitt's
% -
IRD-MC
FIGURE 1. Results of Clq-BA and Raji RIA in patients on (i) standard insulin therapy (IRD) and, (ii) monocomponent preparations (IRD-MC)
ResuIts
Complement components in each group were normal or mildly elevated. Results of immune complex assays are shown in Figures 1 and 2 and Table 1. In the IRD group, 11 out of 36 patients (3 108) on standard insulins had a positive result on Raji cell RIA; the Clq-BA was negative for all patients in this group. One out of 19 patients on mono-component therapy had a positive Raji cell RIA: Clq-BA was negatiye in each patient of this group. In the group on oral hypoglycaemic therapy. five (i.e. 21%) were positive on both assays while a further eight (330.;) had one positive result (one Clq-BA and seven Raji cell RIA). The following medications were being used by these patients: seven glibenclamide; two phenformin; two tolbutamide: one gliben-
37 2
_-
CHARLESWORTH Kl' AL.
- -...-.
-
.-
--
Iq-Eh 3AJI Iq-BI RAJl *a-
100, CT
V
a
a
,3100
80
O
In-
.-d 0
{
.60
60
40
o\" 20
0
...
a
a
-40
a
:
-I - .20
:In Oral Therapy
-0
Diet
FIGURE 2. Results of Clq-BA and Raji RIA in patients on (i) oral hypoglycaemic agents and, (ii) diet alone.
clamide and phenforniin and one tolbutamidc and metformin. One patient on diet alone had an abnormal result for both assays. Five of the 11 patients on standard insulins with a positive Raji cell RIA had diabetic complications: two retinopathylnephropathy; one neuropathyi retinopathy; one nephropathy, and one PVD. Three of the non-insulin-requiring group with assay-abnormalities had complications: two retinopathy: one nephropathy. In all, 16 of 60 patients with negative assays had complications. No patient in the study had a positive ANF. Analysis of PEG precipitates for immunoglobulin class showed only IgG. Patients with a negative Clq-BA did not form detectable precipitates. No insulin activity was detected in dissociated PEG precipitates. Discussion
A significant number of both insulin-requiring and non-insulin-requiring diabetics had abnormal amounts of complement binding substances in serum. It is known that each of the radio-binding assays is complement-dependent and may react with a variety of substances; however, recent work15 suggests that both show a high degree of preferential reactivity with immune complexes. The findings in the group
.~...
..
VOI.. .
9. KO.. .4-
receiving standard insulin therapy are in agreement with lrvine and co-workers' who found the Kaji cell RIA to be positive in 53"/(,of such patients. The higher incidence of positive results in their series is explained by the inclusion of eight patientb (2%;) whose value was at the upper limit of normal. They did not examine patients on monocomponent insulins and no other methods of immune complex detection were used. In contrast. the) found the Raji cell RIA to be negative in 52 patients on oral therapy or diet alone. The age range of the patients \\as not stated. In our study, two different patterns of immune complex reactivity were observed: firstl,, a positive Raji cell RIA and a negative Clq-BA in the IRD group and secondly, positivity of either or both assays in the non-1RD group (including one patient on diet alone). In established immune complex diseases-. such as active SLE or acute glomcruloncphritis both assays have been reported to be positive in a high percentage of ~ a s e s . " ~ 'The ~ ~ reason '~ for the different assaypatterns In our patients is not clear. One explanation would be the molecular size of complexes in the two groups. It is possible that the antigenic component in insulin-treated diabetes is insulin'" (or insulin polymers) implying a rclat;vely low molecular weight for these complexes. Such material might not activate the Clq-BA which detects high molecular weight reactants." On the other hand, the Raji cell RIA has been shown to react with complexes of widely varying molecular size.I2 Positivity of both assays, therefore suggests that the serum reactants in the group on oral hypoglycaemic therapy are of high molecular weight. It is also possible that the complexes in the IRD group activate the alternative pathway of complement; such a proposal is unproven, but would explain the selective positivity of the Raji cell RIA. In contrast, results on patients using monocomponent insulins were essentially negative although our methods do not exclude the prescncc of non-complement-fixing complexes. This observation would also be consistent with the hypothesis that some component other than insulin monomer is the antigen in complexes seen in patients on standard therapy, as monocomponent insulins have been
shown to produce less antibody than conventional preparations.16 Immunoglobulin-analysis of PEG precipitates in the non-1RD group showed only IgG. The antibody specificity in the Raji cell RIA implies that the antibody in the I R D group is similarly IgG. The nature of the antigen(s) particularly in the non-IRD patients remains to he determined. The observation that such complexes can occur in a patient on diet alone suggests that this antigen is unrelated to therapy. Although a number of patients with positive assays in both groups showed peripheral diabetic complications their occurrence was comparable with others where both assays were negative. Their tissue-damaging effect therefore remains unproven, although complement-fixation and large molecular size are two factors known to influence antigen-antibody mediated injury." If immune complexes in IRD prove to be associated with diabetic complications then their absence in monocomponent-treated patients suggests such therapy to be a major advance in the management of this disease. Further longitudinal studies of such patients and analysis of immune complex structure and behaviour may help define the role of this immune mechanism. Acknowledgements We wish to thank Miss C. Boon and Miss S. Pickup for skilled technical assistance and Dr. U'.Kidson for providing sera.
References I R V l s t . W. W. JJ.. 11975) 11975) Clinicdl Clinicdl Aspects Aspects of of Immunology, Immunology, Hlackweil Blackwell Scientific Scientific 1.1. IRVlst.
2. 2.
Publications. Oxford Oxford Publications. x5Kl:P.J.. Pi..AIL. Pi..AIL. PP ,,44VWDXt R s L sS.. 0 0 0.. 0..CCHHRRIISSIIYY.. hl.. hl.. LI .YYVu a o E , II ,, P(H~I.sl\, PlH~i.Si\, N5Ri:P.J.. E., RYIXR, RYOER, I.. PP ,, STAt'B STA1.B NlriS€.S. NlriS€.S. L., L., TIIOMs1,s. TI~OMs1,s.\1 \1 and and SVIICAARD, SVIICAARD, A. J.J. E., I.. A. 11974): 11L-A 11L-A antigens antigens and and diabetes diabetes inellitus, inellitus, Luricer Luricer 2, 2, XX6644 866. 866. 11974): J., GRAY,R S , ~AMl'Ill.I.1.. C. J., DUXAS, HA%, H and M(IRRIS,P. J (1977) Pancreatic islet-cell antihcdxs in diahefes mellifus corr d with the dUrdt!On and type of diabetes, co-existent .+utoiminurie disea md HLA type. 1)iuherP.s 26, 138 147 A,. YYA AIi rI oOwW, ,RR SS .. HAI HAIHAS. HAS. A A .. RCIII~S(.HILD. RCIIHS(.HILD.M M A A and and SS A,. IY. K. (19%): Insulin 1"' me~dbdism in h n m m suhlects. trailon of Iiiwlin hiiiiiing globulin in the circulatum ~ i f i n \ i i l ntrcdtcd i
pathogenesis of dmhetx glomerulmxlerosir. II Deinonstraimn of ins Innding cdpdcity capacity of o ithe the Yd~iOu5 vdrmu5 histopathological histopathological components components of of the the dis disease hinding by fluorescence fluorescence microscopy. microscopy. Diuheres Diuheres 11, 11, 30X 30X 317. 317. by 88 HBcnaHl~i IIER. P. P. \ \I I. .(1965). (1965). IImmunuhistopatholOBic 01 localised localised c R a H i l i IXR. m m u n o h i s t o p a i h o l ~ ~ study istudy c of plasma proteins and fiaation of guinra-pig complement in renal lesions of diabetic elomerulasclerosis. D i u h ~ r e s14. 7 5 5 ,770. 9. FAKKANI. P. C. and S i m ) i x . ~W , I H (1965). Ohrcrvationa (in the uptake (it insulin conjugated with fluorescein isoihioqanale b) diahetx kidncy tissuc. Drohcrv.$ 14, 14, 274 274 278. 278. Drohctv~ 10 WI 1191:R, €1. (1976). Immunological aspects ofdiabetes niellitus. Actuendorr. 83 1Suppl. 205). 241 253
I?.
Circulating immune complexes in the serum 111 systemic lupus erythcmatosus and in carrier* of hepatitis B antigen. J . clrn inrcsr. 54, 297 309. ' I i l i ~ ~ ~ i I . ~ A. ) ~S~ ~WII.sos, ~ ~ i . ~C ~ . 13 and Dixr)\, F.J f I 97hi: The Rajj cell detecting immune complexes In human sera. J d m .
.
AKA. A. 0 and I1ini.MANs. J . I . (1965) Immunon o i mtifcns by single radial Irnrnunodiifusion. I l , I l , l u l l o ~ ~ h r l , l r .2, ~ r 235 ~~
254 14. f'At.i.ti, W. P.. KAR.W, J. TI and P~JI)I\IER(;, JI. H (1971) Human antiinsulin antibodies. J In,murrol 106. I I I ? 11 I6 I C WOOI)ROIC~.. A J., HOnmK, W A , THMI~~I~OPOIJLOS A . S . , tiorzi:. 0 .
tir.nssocx, R. J., Dixos. F. J . and WILSON,C. B. (1977): I k t e c t m n uf circulating immune complexes in patients with glomerulonephntis, Kidtiiar hrf. 12, 268-278. 16. Y-r:t, D. K . andTmric, I. K . f 1 9 7 3 New formsoiinsulin andthcirusein the treatment of diabetes. Diuheres 26, 341 345. (1971). Mechanisms involved in thcdepositiun of.irnrnune 17. C'lK'HKAht, C. 0. complexes in tissues, J . e l p .%fed 1.34, -5s 89s
