Int. Archs Allergy appl. Immun. 48: 364-371 (1975)
Cell-Mediated Immunity to Insulin and its Polypeptide Chains in Insulin-Treated Diabetics1 W. P age F aulk, J. P. G irard and H. D. W elscher MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire, Department of Medicine, University of Geneva School of Medicine, Geneva, and Swiss Serum and Vaccine Institute, Bern
Abstract. 35 insulin-treated diabetics and 25 control persons were studied for in vivo and in vitro manifestations of hypersensitivity to insulin and its isolated polypeptide chains. Delayed hypersensitivity reactions were determined by skin testing, and blastogénie conversion tests were done with peripheral blood lympho cytes. None of the controls and 62.9% of the patients gave positive reactions in the blastogénie test, most of these reacted to the intact molecule, fewer patients responded to B chains, and very few responded to A chains. None of the controls and only three of the patients produced positive delayed hypersensitivity reactions to insulin, and all of these were to the intact insulin molecule.
Introduction There is not a great deal of information about cell-mediated immune (CMI) reactions to insulin. Delayed hypersensitivity reactions have been studied in guinea pigs by M c D evitt [1964], and similar studies by C lark and M unoz [1970] showed that immune animals responded to insulin as well as to B chain, but not to A chain. This has been con firmed by F ederlin [1971] using a B chain fragment. There are no comparable studies in man. Immediate hypersensitivity reactions to var ious insulins have been reported by L ieberman et al. [1971], and de-
Received: September 11, 1974.
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1 Part of this work was done with the financial support of the Swiss National Council for Scientific Research (grant No. 3.439.70).
F aulk/G irard/W elscher
365
layed hypersensitivity reactions to the intact insulin molecule have been reported by F rei et al. [1965], R enold et al. [1965] and D evlin [1968]. Antipancreatic cellular hypersensitivity has also been shown us ing the microsomal fraction of islet cells in the leukocyte migration test with leukocytes from diabetic patients [N erup et al., 1971], The present investigation was undertaken to test parameters of CMI in man to insu lin and its isolated B and A chains. For this purpose we chose delayed hypersensitivity tests in vivo and blastogénie tests of lymphocytes in vi tro. Many of our patients produced a positive blastogénie test, and some of them presented positive immediate and delayed skin test reactions. The intact insulin molecule produced more CMI reactions in our tests than did either B or A chains, and many more positive reactions were obtained with B than with A chains. This is in agreement with Clark and M unoz [1970] and F ederlin [1971] for delayed hypersensitivity re actions in guinea pigs. Materials and Methods
Preparation of Insulin A and B Chains A mixture of 5X crystallized bovine and porcine insulin (Novo, Copenhagen) was reduced with dithiothreitol (DTT; Calbiochem, Los Angeles Calif.) and alky lated with iodoacetamide (IAA; Fluka AG, Buchs) and A and B chains were sepa rated by gel filtration and ion-exchange chromatography. This was done by dis solving 580 mg insulin (0.1 mmol) in 10 ml distilled water by dropwise addition of 1 m HC1 until pH 3.0 was reached. 60 mg DTT (0.4 mmol) were then added under N2-atmosphere and the mixture kept at 37 °C for 1 h. The solution was cooled to 0° C and 150 mg IAA (0.8 mmol) were added, and the pH of this mixture was kept constant by adding appropriate amounts of glycine. To separate A and B chains, one-half of the reduced and alkylated material was chromatographed on a Sephadex G-75 column in IM propionic acid, and the elution profile of a typical separation is shown in figure 1. The A and B chains were separately concentrated and freed of excess propionic acid by pervaporation, and pH adjusted to 3.0. The second half of the reduced and alkylated material was fractionated on carboxymethylcellulose (CM-52, Whatman) equilibrated with 0.25°/o acetic acid. A chains were eluted with the 0.25% acetic acid solvent, and B chains were eluted from the column with 0.15 m Tris-HCl buffer, pH 7.4. Purity
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Patients 35 insulin-treated diabetics were randomly chosen from the diabetic clinic at the Cantonal Hospital, University of Geneva School of Medicine, Geneva. The age range for this group was 21-77 years, and the duration of their disease ranged from 1-30 years. 25 randomly selected non-insulin-treated, non-diabetic controls were also studied.
366
F aulk/G irard/W elscher
Fig.l. Separation of insulin A and B chains by Sephadex chromatography. Sephadex G-75 gel filtration of 290 mg of a reduced and alkylated mixture of bovine and porcine insulin in 1 m propionic acid. Column 3X60 cm, fraction volume 7 ml at 4 fractions/h.
of the isolated chains was confirmed by amino acid analysis. As both chains from the Sephadex separation appeared as individual peaks when separately rechromato graphed on CM-52, and both chains from CM-52 appeared as individual peaks when separately rechromatographed on Sephadex G-75, the Sephadex preparations were used for the immunological studies. Aliquots of A and B chains and insulin were aseptically placed in multiple-dispenser sealed bottles in 7.4 pH phosphate buffered saline in such concentrations that 0.1 ml of solution contained 10/,ug of A chain, B chain, or insulin.
Delayed Hypersensitivity Each patient was injected intracutaneously with 10/«g insulin, B chain, and A chain, and skin reactions were measured at 15 min, 6 h and 36 h. A description and measurement to the nearest millimetre of each reaction were recorded. Urti-
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Lymphocyte Blastogénie Tests 50 ml of blood was drawn in heparinized syringes (50 units of phenol-free heparin/ml whole blood) from each patient, and lymphocytes were separated using a Ficoll-Isopaque gradient, washed three times in Eagle’s minimum-essential-medium, and four different cultures containing 1X106 lymphocytes were placed in amino acid enriched growth medium according to standard techniques. Following prelimi nary dose-response analysis, 50/fig of intact insulin, 50/,«g of insulin B chain, or 50/fig of insulin A chain were added to separate culture tubes, the fourth tube being a control. None of the antigens were found to be mitogenic for non-diabetic lymphocytes. Each experiment was done in triplicate, pulsed with 1.0 nCi of tritiated thymidine (New England Nuclear, Frankfurt) for 16 h at day 4, and at day 5 each culture was stopped and its DNA extracted and counted in a Beckman li quid-scintillation counter for tritiated thymidine incorporation. The mean value for all control experiments was 824 cpm+294. A value of 50% over the controls in the presence of antigen was thus considered as positive, i.e., antigen stimulated.
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
367
cariai and/or weal-and-flare reactions of less than 5 mm were recorded as nega tive, erythema of less than 5 mm at 6 h were recorded as negative, and erythema and induration of less than 5 mm at 36 h were recorded as negative. Reactions of 5 mm or greater were listed as positive.
Results Lymphocyte Blastogenic Tests Preliminary dose-response testing showed that insulin, B chain or A chain did not react as a mitogen with non-diabetic control lymphocytes, and that measurable and optimal responses were given with 50//rg insu lin, B chain or A chain when mixed with lymphocytes from insulintreated diabetics. The results of these experiments are recorded in ta ble I. Of the 35 patients tested, 22 (62.9%) gave a positive reaction in vitro with insulin or its isolated polypeptide fragments, 21 positive reac tions were recorded to the intact molecule, 16 reactions to B chain, and 8 reactions to A chain. Analysis of these data showed that 6 patients re sponded to the intact molecule alone, 1 patient responded only to B chain, and no patient responded to A chain alone. Furthermore, 8 pa tients responded to the intact molecule as well as to A and B chains, 7 patients responded only to intact molecule and B chain, and no patient responded to A and B chains only or to A chain and intact molecule only (table I). These data suggest that intact insulin molecules are the most immu nogenic for insulin-treated diabetics, and that sensitivity for B chains is developed in those individuals who already have sensitivity to the intact molecule, and, that A chain sensitivity develops only in those patients who are already sensitized to intact molecule and to B chains.
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Delayed Hypersensitivity None of the 25 control subjects reacted to insulin or its fragments. As recorded in table II, 14 of the 35 insulin-treated diabetic patients produced a positive reaction at 15 min, 6 reacted at 6 h, and 3 patients produced erythema and induration at 36 h. Of the 14 patients who had a reaction at 15 min, 13 reactions were obtained to intact molecule, 13 to B chain, and 7 to A chain (table II). Of the 6 patients who produced a reaction at 6 h, 3 reactions were obtained to intact molecule, 5 to B chain, and 2 to A chain (table II). Of the 3 patients who produced an
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368
Table 1. Lymphocyte blastogénie tests. Insulin stimulation In vitro with insulin antigens. Insulin antigens, cpm backA chains ground counts no antigen
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
480 882 517 387 872 1,064 413 326 1,349 658 513 382 784 740 642 464 396 886 794 528 513 374 670 572 504 384 383 732 910 556 379 820 451 781 927
Mean
629.15
890 976 612 366 912 1,631 472 315 2,122 594 492 351 912 856 1,117 710 352 913 919 513 592 592 740 692 602 313 572 713 1,048 513 1,118 944 430 692 1,840 783.6
B chains
intact insulin molecule
Ratio of insulin antigen/no antigen A chain B chain intact no no insulin antigen antigen no antigen
756 1,211 643 392 1,006 2,009 436 409 2,076 609 538 424 692 1,211 1,024 728 370 901 1,211 917 486 618 792 876 812 486 711 1,642 813 498 784 1,415 492 638 1,925
920 1,436 908 415 1,229 2,217 786 576 3,004 672 604 596 714 1,944 1,241 912 415 1,372 980 984 474 691 647 1,040 986 412 1,086 1,510 973 612 1,225 1,740 529 744 2,214
1.85 1.10 1.18 0.94 1.04 1.53 1.14 0.96 1.57 0.90 0.95 0.91 1.16 1.15 1.73 1.53 0.88 1.03 1.15 0.97 1.15 1.58 1.10 1.20 1.19 0.81 1.49 0.97 1.15 0.92 2.94 1.15 0.95 0.88 1.98
872.98
1.57 1.37 1.24 1.01 1.15 1.90 1.05 1.25 1.53 0.92 1.04 1.10 0.88 1.63 1.59 1.56 0.93 1.01 1.52 1.73 0.94 1.65 1.18 1.53 1.61 1.26 1.85 2.24 0.89 0.89 2.06 1.72 1.09 0.81 2.07
1.91 1.62 1.75 1.07 1.40 2.08 1.90 1.76 2.22 1.02 1.17 1.56 0.91 2.62 1.93 1.96 1.04 1.54 1.23 1.86 0.92 1.84 0.96 1.81 1.95 1.07 2.83 2.06 1.06 1.10 3.23 2.12 1.17 0.95 2.38
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Patient No.
369
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
Table II. Hypersensitivity reactions to insulin and its polypeptide chains by skin-testing 35 Insulin-treated diabetics Number of patients with positive reactions
Number of positive reactions to insulin at B chain at 15 min 6h 35^ 15 min 6h
36h
A chain at 15 min 6h
36h
17
13
0
7
0
3
3
13
5
2
Number of patients with positive reactions at
Number of patients with positive reactions to insulin B chain A chain AandB A chain and chains only only only insulin only only
15 min 6h 36 h
1 1 3
1 2 0
0 0 0
0 1 0
0 0 0
B chain and insulin only
insulin and A and B chains
5 1 0
7 1 0
erythema and induration reaction at 36 h, all reacted only to the intact molecule (table II). Distribution analysis of these data are recorded in table III. These data suggest that intact molecule and B chain express the same immunogenicity at 15 min, that B chain seems to be slightly more immunogeneic at 6 h, and, as with the in vitro lymphocyte blastogénie tests, A chains produced reactions only in those patients who were positive for either intact molecule and/or B chain (table III). All 36 h reactions were elicit ed only with the intact insulin molecule. Results from the blastogénie tests were compared to the skin test re sults. Positive skin tests with insulin, B chain, or A chain at 15 min, 6 h, or 36 h were usually associated with positive blastogénie tests for the same antigen. However, positive blastogénie tests for insulin, B chain, or A chain were not always associated with positive skin tests. For exam ple, of the 13 patients in table I that were negative for all antigens by blastogenesis, all of them were also negative to all antigens at all times
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Table III. Distribution of hypersensitivity reactions to insulin and its polypeptide chains by skin-testing 35 insulin -treated diabetics
370
F aulk/G ir ardAVelscher
by skin testing. Alternatively, of the 21 patients who were negative for all antigens at all times by skin testing, only 13 of them were negative in the blastogénie test. This suggests that blastogénie tests are more sensi tive than delayed hypersensitivity reactions when used to measure CMI to insulin and its polypeptide chains.
Blastogénie reactions were obtained from 22 of the 35 patients (ta ble I) and most of these were to the intact molecule, followed by B chain and then by A chain. There were no reactions to A chain alone or to A chain in combination with B chain or insulin. A chain reactions were only obtained when both insulin and B chain were positive. These observations suggests a progression in the development of CMI to insu lin beginning with immunity to the intact molecule, then to B chains and then to A chains. Development of these sensitivities may, however, be independent events. Also, future studies of this problem might consider the development of such sensitivities as a function of time since the initi ation of insulin therapy. Although many patients produced positive skin tests most of these were immediate reactions to either the intact insulin molecule or to B chain. Most of the positive immediate hypersensitivity reactions repre sented combinations of B chain with intact molecule and the three pa tients who responded in a classical delayed hypersensitivity reaction re sponded only to insulin at 36 h. All patients who produced positive skin test reactions also gave a positive blastogénie test at 6 or 36 h but not all patients with positive blastogénie tests had positive skin tests. The 5X crystallized insulin was not further purified, so it is possible that some of the reactions to intact molecule could have been reactions to contaminants. Recent studies by A rquilla et al. [1969], B rugman and A rquilla [1973], and others have indicated that the immunological specificity of the humoral response to insulin is directed to conformational sites on the intact molecule. Our results with the blastogénie test have shown that some patients’ lymphocytes also respond to B chains, and, in rather fewer cases, to A chains. This is consistent with the basic observations of G ell and B enacerraf [1961] and H enney [1969] that animals with delayed hypersensitivity to defined antigens respond to conformational
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Discussion
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
371
determinants which also elicit immediate responses, as well as to deter minants in the molecule which do not react with circulating antibody. The implications of these observations for understanding aspects of the pathophysiology and complications of diabetes remain to be investigat ed. References Arquilla , E. R.; Bromer , W. W., and M ercola, D.: Immunological conformation
and biological activity of insulin. Diabetics 18: 193 (1969). Arquilla , E. R.; O oms , H., and F inn , J.: Genetic differences of combining sites
Dr. W. P age F aulk , MRC Rheumatism Unit, Taplow, Maidenhead, Berkshire SL6 OMN (England)
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of insulin antibodies and importance of c-terminal portion of the A-chain to biological and immunological activity of insulin. Diabetologia 2: 1 (1966). Brugman, T. M. and A rquilla , E. R.: Circular dichroic and immunologic studies of structure relationships of insulin and derivatives. Biochem. 12: 727 (1973). C lark, C. and M unoz , J.: Delayed hypersensitivity to insulin and its component polypeptide chains. J. Immunol. 105: 574 (1970). D evlin , J. G.: Hormone resistance and hypersensitivity; in G ell and Coombs Clinical aspects of immunity, p. 672 (Blackwell, Oxford 1968). F aulk, W. P.; Karam, J. H., and F udenberg , H. H.: Human anti-insulin antibodies. J. Immunol. 106: 1112 (1971). F ederlin , K.: Immunopathology of insulin (Springer, Berlin 1971). F rei, P. C.; Cruchaud , S. et V anotti, A.: Allergie k l’insuline de type cellulaire. Rev. frang. Et. clin. biol. 10: 1083 (1965). G ell , P. G . and Benacerraf, B.: Delayed hypersensitivity to simple protein anti gens. Adv. Immunol. 1: 319 (1961). Henney, C. S.: The specificity of the early immune response. I. A comparison of the specificity of delayed hypersensitivity and humoral responses following im munization with human gamma G globulin. J. Immunol. 103: 519 (1969). L ieberman, P.; P atterson , R.; M etz , R., and Lucena, G.: Allergic reactions to insulin. J. amer. med. Ass. 215: 1106 (1971). McDevitt, H. O.: Delayed hypersensitivity to insulin in guinea pigs. Fed. Proc. 23: 259 (1964). N erup , J.; A nderson , O. D.; Bendixen , G.; E geberg, J., and P ulsen , J. E.: Antipancreatic cellular hypersensitivity in diabetes mellitus. Diabetes 20: 424 (1971). R enold , A. E.; Steinke , J.; Soeldner , J. S.; G onet , A., and Le C omfte , P.: Insulite experimentale chez la genisse; in M iescher and G rabar 4th Int. Symp. Im munopathology, p. 349 (Schwabe, Basel 1965).
Cell-Mediated Immunity to Insulin and its Polypeptide Chains in Insulin-Treated Diabetics1 W. P age F aulk, J. P. G irard and H. D. W elscher MRC Rheumatism Unit, Canadian Red Cross Memorial Hospital, Taplow, Maidenhead, Berkshire, Department of Medicine, University of Geneva School of Medicine, Geneva, and Swiss Serum and Vaccine Institute, Bern
Abstract. 35 insulin-treated diabetics and 25 control persons were studied for in vivo and in vitro manifestations of hypersensitivity to insulin and its isolated polypeptide chains. Delayed hypersensitivity reactions were determined by skin testing, and blastogénie conversion tests were done with peripheral blood lympho cytes. None of the controls and 62.9% of the patients gave positive reactions in the blastogénie test, most of these reacted to the intact molecule, fewer patients responded to B chains, and very few responded to A chains. None of the controls and only three of the patients produced positive delayed hypersensitivity reactions to insulin, and all of these were to the intact insulin molecule.
Introduction There is not a great deal of information about cell-mediated immune (CMI) reactions to insulin. Delayed hypersensitivity reactions have been studied in guinea pigs by M c D evitt [1964], and similar studies by C lark and M unoz [1970] showed that immune animals responded to insulin as well as to B chain, but not to A chain. This has been con firmed by F ederlin [1971] using a B chain fragment. There are no comparable studies in man. Immediate hypersensitivity reactions to var ious insulins have been reported by L ieberman et al. [1971], and de-
Received: September 11, 1974.
Downloaded by: MacQuarie University 137.111.162.20 - 1/16/2020 5:35:54 AM
1 Part of this work was done with the financial support of the Swiss National Council for Scientific Research (grant No. 3.439.70).
F aulk/G irard/W elscher
365
layed hypersensitivity reactions to the intact insulin molecule have been reported by F rei et al. [1965], R enold et al. [1965] and D evlin [1968]. Antipancreatic cellular hypersensitivity has also been shown us ing the microsomal fraction of islet cells in the leukocyte migration test with leukocytes from diabetic patients [N erup et al., 1971], The present investigation was undertaken to test parameters of CMI in man to insu lin and its isolated B and A chains. For this purpose we chose delayed hypersensitivity tests in vivo and blastogénie tests of lymphocytes in vi tro. Many of our patients produced a positive blastogénie test, and some of them presented positive immediate and delayed skin test reactions. The intact insulin molecule produced more CMI reactions in our tests than did either B or A chains, and many more positive reactions were obtained with B than with A chains. This is in agreement with Clark and M unoz [1970] and F ederlin [1971] for delayed hypersensitivity re actions in guinea pigs. Materials and Methods
Preparation of Insulin A and B Chains A mixture of 5X crystallized bovine and porcine insulin (Novo, Copenhagen) was reduced with dithiothreitol (DTT; Calbiochem, Los Angeles Calif.) and alky lated with iodoacetamide (IAA; Fluka AG, Buchs) and A and B chains were sepa rated by gel filtration and ion-exchange chromatography. This was done by dis solving 580 mg insulin (0.1 mmol) in 10 ml distilled water by dropwise addition of 1 m HC1 until pH 3.0 was reached. 60 mg DTT (0.4 mmol) were then added under N2-atmosphere and the mixture kept at 37 °C for 1 h. The solution was cooled to 0° C and 150 mg IAA (0.8 mmol) were added, and the pH of this mixture was kept constant by adding appropriate amounts of glycine. To separate A and B chains, one-half of the reduced and alkylated material was chromatographed on a Sephadex G-75 column in IM propionic acid, and the elution profile of a typical separation is shown in figure 1. The A and B chains were separately concentrated and freed of excess propionic acid by pervaporation, and pH adjusted to 3.0. The second half of the reduced and alkylated material was fractionated on carboxymethylcellulose (CM-52, Whatman) equilibrated with 0.25°/o acetic acid. A chains were eluted with the 0.25% acetic acid solvent, and B chains were eluted from the column with 0.15 m Tris-HCl buffer, pH 7.4. Purity
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Patients 35 insulin-treated diabetics were randomly chosen from the diabetic clinic at the Cantonal Hospital, University of Geneva School of Medicine, Geneva. The age range for this group was 21-77 years, and the duration of their disease ranged from 1-30 years. 25 randomly selected non-insulin-treated, non-diabetic controls were also studied.
366
F aulk/G irard/W elscher
Fig.l. Separation of insulin A and B chains by Sephadex chromatography. Sephadex G-75 gel filtration of 290 mg of a reduced and alkylated mixture of bovine and porcine insulin in 1 m propionic acid. Column 3X60 cm, fraction volume 7 ml at 4 fractions/h.
of the isolated chains was confirmed by amino acid analysis. As both chains from the Sephadex separation appeared as individual peaks when separately rechromato graphed on CM-52, and both chains from CM-52 appeared as individual peaks when separately rechromatographed on Sephadex G-75, the Sephadex preparations were used for the immunological studies. Aliquots of A and B chains and insulin were aseptically placed in multiple-dispenser sealed bottles in 7.4 pH phosphate buffered saline in such concentrations that 0.1 ml of solution contained 10/,ug of A chain, B chain, or insulin.
Delayed Hypersensitivity Each patient was injected intracutaneously with 10/«g insulin, B chain, and A chain, and skin reactions were measured at 15 min, 6 h and 36 h. A description and measurement to the nearest millimetre of each reaction were recorded. Urti-
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Lymphocyte Blastogénie Tests 50 ml of blood was drawn in heparinized syringes (50 units of phenol-free heparin/ml whole blood) from each patient, and lymphocytes were separated using a Ficoll-Isopaque gradient, washed three times in Eagle’s minimum-essential-medium, and four different cultures containing 1X106 lymphocytes were placed in amino acid enriched growth medium according to standard techniques. Following prelimi nary dose-response analysis, 50/fig of intact insulin, 50/,«g of insulin B chain, or 50/fig of insulin A chain were added to separate culture tubes, the fourth tube being a control. None of the antigens were found to be mitogenic for non-diabetic lymphocytes. Each experiment was done in triplicate, pulsed with 1.0 nCi of tritiated thymidine (New England Nuclear, Frankfurt) for 16 h at day 4, and at day 5 each culture was stopped and its DNA extracted and counted in a Beckman li quid-scintillation counter for tritiated thymidine incorporation. The mean value for all control experiments was 824 cpm+294. A value of 50% over the controls in the presence of antigen was thus considered as positive, i.e., antigen stimulated.
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
367
cariai and/or weal-and-flare reactions of less than 5 mm were recorded as nega tive, erythema of less than 5 mm at 6 h were recorded as negative, and erythema and induration of less than 5 mm at 36 h were recorded as negative. Reactions of 5 mm or greater were listed as positive.
Results Lymphocyte Blastogenic Tests Preliminary dose-response testing showed that insulin, B chain or A chain did not react as a mitogen with non-diabetic control lymphocytes, and that measurable and optimal responses were given with 50//rg insu lin, B chain or A chain when mixed with lymphocytes from insulintreated diabetics. The results of these experiments are recorded in ta ble I. Of the 35 patients tested, 22 (62.9%) gave a positive reaction in vitro with insulin or its isolated polypeptide fragments, 21 positive reac tions were recorded to the intact molecule, 16 reactions to B chain, and 8 reactions to A chain. Analysis of these data showed that 6 patients re sponded to the intact molecule alone, 1 patient responded only to B chain, and no patient responded to A chain alone. Furthermore, 8 pa tients responded to the intact molecule as well as to A and B chains, 7 patients responded only to intact molecule and B chain, and no patient responded to A and B chains only or to A chain and intact molecule only (table I). These data suggest that intact insulin molecules are the most immu nogenic for insulin-treated diabetics, and that sensitivity for B chains is developed in those individuals who already have sensitivity to the intact molecule, and, that A chain sensitivity develops only in those patients who are already sensitized to intact molecule and to B chains.
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Delayed Hypersensitivity None of the 25 control subjects reacted to insulin or its fragments. As recorded in table II, 14 of the 35 insulin-treated diabetic patients produced a positive reaction at 15 min, 6 reacted at 6 h, and 3 patients produced erythema and induration at 36 h. Of the 14 patients who had a reaction at 15 min, 13 reactions were obtained to intact molecule, 13 to B chain, and 7 to A chain (table II). Of the 6 patients who produced a reaction at 6 h, 3 reactions were obtained to intact molecule, 5 to B chain, and 2 to A chain (table II). Of the 3 patients who produced an
F aulk/G irard/W elscher
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Table 1. Lymphocyte blastogénie tests. Insulin stimulation In vitro with insulin antigens. Insulin antigens, cpm backA chains ground counts no antigen
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
480 882 517 387 872 1,064 413 326 1,349 658 513 382 784 740 642 464 396 886 794 528 513 374 670 572 504 384 383 732 910 556 379 820 451 781 927
Mean
629.15
890 976 612 366 912 1,631 472 315 2,122 594 492 351 912 856 1,117 710 352 913 919 513 592 592 740 692 602 313 572 713 1,048 513 1,118 944 430 692 1,840 783.6
B chains
intact insulin molecule
Ratio of insulin antigen/no antigen A chain B chain intact no no insulin antigen antigen no antigen
756 1,211 643 392 1,006 2,009 436 409 2,076 609 538 424 692 1,211 1,024 728 370 901 1,211 917 486 618 792 876 812 486 711 1,642 813 498 784 1,415 492 638 1,925
920 1,436 908 415 1,229 2,217 786 576 3,004 672 604 596 714 1,944 1,241 912 415 1,372 980 984 474 691 647 1,040 986 412 1,086 1,510 973 612 1,225 1,740 529 744 2,214
1.85 1.10 1.18 0.94 1.04 1.53 1.14 0.96 1.57 0.90 0.95 0.91 1.16 1.15 1.73 1.53 0.88 1.03 1.15 0.97 1.15 1.58 1.10 1.20 1.19 0.81 1.49 0.97 1.15 0.92 2.94 1.15 0.95 0.88 1.98
872.98
1.57 1.37 1.24 1.01 1.15 1.90 1.05 1.25 1.53 0.92 1.04 1.10 0.88 1.63 1.59 1.56 0.93 1.01 1.52 1.73 0.94 1.65 1.18 1.53 1.61 1.26 1.85 2.24 0.89 0.89 2.06 1.72 1.09 0.81 2.07
1.91 1.62 1.75 1.07 1.40 2.08 1.90 1.76 2.22 1.02 1.17 1.56 0.91 2.62 1.93 1.96 1.04 1.54 1.23 1.86 0.92 1.84 0.96 1.81 1.95 1.07 2.83 2.06 1.06 1.10 3.23 2.12 1.17 0.95 2.38
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Patient No.
369
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
Table II. Hypersensitivity reactions to insulin and its polypeptide chains by skin-testing 35 Insulin-treated diabetics Number of patients with positive reactions
Number of positive reactions to insulin at B chain at 15 min 6h 35^ 15 min 6h
36h
A chain at 15 min 6h
36h
17
13
0
7
0
3
3
13
5
2
Number of patients with positive reactions at
Number of patients with positive reactions to insulin B chain A chain AandB A chain and chains only only only insulin only only
15 min 6h 36 h
1 1 3
1 2 0
0 0 0
0 1 0
0 0 0
B chain and insulin only
insulin and A and B chains
5 1 0
7 1 0
erythema and induration reaction at 36 h, all reacted only to the intact molecule (table II). Distribution analysis of these data are recorded in table III. These data suggest that intact molecule and B chain express the same immunogenicity at 15 min, that B chain seems to be slightly more immunogeneic at 6 h, and, as with the in vitro lymphocyte blastogénie tests, A chains produced reactions only in those patients who were positive for either intact molecule and/or B chain (table III). All 36 h reactions were elicit ed only with the intact insulin molecule. Results from the blastogénie tests were compared to the skin test re sults. Positive skin tests with insulin, B chain, or A chain at 15 min, 6 h, or 36 h were usually associated with positive blastogénie tests for the same antigen. However, positive blastogénie tests for insulin, B chain, or A chain were not always associated with positive skin tests. For exam ple, of the 13 patients in table I that were negative for all antigens by blastogenesis, all of them were also negative to all antigens at all times
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Table III. Distribution of hypersensitivity reactions to insulin and its polypeptide chains by skin-testing 35 insulin -treated diabetics
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by skin testing. Alternatively, of the 21 patients who were negative for all antigens at all times by skin testing, only 13 of them were negative in the blastogénie test. This suggests that blastogénie tests are more sensi tive than delayed hypersensitivity reactions when used to measure CMI to insulin and its polypeptide chains.
Blastogénie reactions were obtained from 22 of the 35 patients (ta ble I) and most of these were to the intact molecule, followed by B chain and then by A chain. There were no reactions to A chain alone or to A chain in combination with B chain or insulin. A chain reactions were only obtained when both insulin and B chain were positive. These observations suggests a progression in the development of CMI to insu lin beginning with immunity to the intact molecule, then to B chains and then to A chains. Development of these sensitivities may, however, be independent events. Also, future studies of this problem might consider the development of such sensitivities as a function of time since the initi ation of insulin therapy. Although many patients produced positive skin tests most of these were immediate reactions to either the intact insulin molecule or to B chain. Most of the positive immediate hypersensitivity reactions repre sented combinations of B chain with intact molecule and the three pa tients who responded in a classical delayed hypersensitivity reaction re sponded only to insulin at 36 h. All patients who produced positive skin test reactions also gave a positive blastogénie test at 6 or 36 h but not all patients with positive blastogénie tests had positive skin tests. The 5X crystallized insulin was not further purified, so it is possible that some of the reactions to intact molecule could have been reactions to contaminants. Recent studies by A rquilla et al. [1969], B rugman and A rquilla [1973], and others have indicated that the immunological specificity of the humoral response to insulin is directed to conformational sites on the intact molecule. Our results with the blastogénie test have shown that some patients’ lymphocytes also respond to B chains, and, in rather fewer cases, to A chains. This is consistent with the basic observations of G ell and B enacerraf [1961] and H enney [1969] that animals with delayed hypersensitivity to defined antigens respond to conformational
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Discussion
Cell-Mediated Immunity to Insulin and its Polypeptide Chains
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determinants which also elicit immediate responses, as well as to deter minants in the molecule which do not react with circulating antibody. The implications of these observations for understanding aspects of the pathophysiology and complications of diabetes remain to be investigat ed. References Arquilla , E. R.; Bromer , W. W., and M ercola, D.: Immunological conformation
and biological activity of insulin. Diabetics 18: 193 (1969). Arquilla , E. R.; O oms , H., and F inn , J.: Genetic differences of combining sites
Dr. W. P age F aulk , MRC Rheumatism Unit, Taplow, Maidenhead, Berkshire SL6 OMN (England)
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of insulin antibodies and importance of c-terminal portion of the A-chain to biological and immunological activity of insulin. Diabetologia 2: 1 (1966). Brugman, T. M. and A rquilla , E. R.: Circular dichroic and immunologic studies of structure relationships of insulin and derivatives. Biochem. 12: 727 (1973). C lark, C. and M unoz , J.: Delayed hypersensitivity to insulin and its component polypeptide chains. J. Immunol. 105: 574 (1970). D evlin , J. G.: Hormone resistance and hypersensitivity; in G ell and Coombs Clinical aspects of immunity, p. 672 (Blackwell, Oxford 1968). F aulk, W. P.; Karam, J. H., and F udenberg , H. H.: Human anti-insulin antibodies. J. Immunol. 106: 1112 (1971). F ederlin , K.: Immunopathology of insulin (Springer, Berlin 1971). F rei, P. C.; Cruchaud , S. et V anotti, A.: Allergie k l’insuline de type cellulaire. Rev. frang. Et. clin. biol. 10: 1083 (1965). G ell , P. G . and Benacerraf, B.: Delayed hypersensitivity to simple protein anti gens. Adv. Immunol. 1: 319 (1961). Henney, C. S.: The specificity of the early immune response. I. A comparison of the specificity of delayed hypersensitivity and humoral responses following im munization with human gamma G globulin. J. Immunol. 103: 519 (1969). L ieberman, P.; P atterson , R.; M etz , R., and Lucena, G.: Allergic reactions to insulin. J. amer. med. Ass. 215: 1106 (1971). McDevitt, H. O.: Delayed hypersensitivity to insulin in guinea pigs. Fed. Proc. 23: 259 (1964). N erup , J.; A nderson , O. D.; Bendixen , G.; E geberg, J., and P ulsen , J. E.: Antipancreatic cellular hypersensitivity in diabetes mellitus. Diabetes 20: 424 (1971). R enold , A. E.; Steinke , J.; Soeldner , J. S.; G onet , A., and Le C omfte , P.: Insulite experimentale chez la genisse; in M iescher and G rabar 4th Int. Symp. Im munopathology, p. 349 (Schwabe, Basel 1965).
