IgG subclass antibody production in human serum sickness

IgG subclass antibody serum sickness

prodwtion

in human

L. Bielory, MD,* D. M. Kemeny, PhD,** D. Richards, M. H. Lessof** Newark, N. J., and London, England

MD, FRCP,** and

The role of IgG-subclass antibodies in the spectrum of immunologic disorders has not yet been de$ned. In an attempt to understand its role in an immune complex-mediated disease, we studied patients who developed serum sickness (SSX) after treatment with an equine-derived immunoglobulin, antithymocyte globulin (ATG), for bone marrow failure. The predominant IgG subclass produced was IgGl, respresenting nearly 80% of all IgG anti-ATG activity present. The appearance of IgG anti-ATG antibodies and Clq-containing immune complexes wus closely correlated with symptoms of SSX. Although other antibody isotypes were present and may have contributed to the patients’ symptoms, it is evident that IgGl is the predominant IgG subclas,, produced in human SSX caused by a heterologous protein. (.I ALLERGY CLIN IMMUNOL 1990;85:573-7.)

fully

Human IgG is comprised of four subclasses.‘-3 These subclassesdiffer in their effector functions, principally complement fixation, mainly becauseof differences in the structure of the hinge region.4-7To determinethe function of IgG subclasses,studieshave been made of their role in IgE-mediated (type I) processes,including food allergy, bee sting anaphylaxis, and inhalant allergy.*, 9 However, there has been no prospective analysis of IgG-subclassformation in immune complex-mediated (type III) disorders. Therefore, we analyzed the sera from patients who subsequently developed SSX to a hererologousprotein to determine the predominant IgG-subclass antibody formed. SSX was described at the turn of the century in children treated with horse antidiphtheria antiserum.‘0 Eight to 12 days after subcutaneousinjections of horse

From the *Division of Allergy and Immunology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, N.J., and **Department of Medicine, United Medical and Dental Schools,Guy’s Hospital, London, England. Supportedin part by the Hnmdation of the University of Medicine and Dentistry of New Jersey Grant No. 10-87. Presentedat the Forty-fiftb Annual Meeting of the American Academy of Allergy and Immunology, San Antonio, Texas, Feb. 27, 1989. Received for publication April 7, 1989. Revised July 29, 1989. Accepted for publication Sept. 11, 1989. Reprint requests:Leonard Bielory, MD, Director, Division of Allergy and Immunology, UMDNJ, New Jersey Medical School, 185 South Grange Ave., MSB I-512, Newark, NJ 07103-2757. l/l/17554

antiserum, these children developedsymptomsof fever, cutaneouseruptions, lymphadenopathy, and arthralgia. For the next 30 years, the administration of heterologousantiserawas focusedon the treatmentof various infectious epidemic disorders, including diphtheria (whooping cough), pnemnococcus (pneumonia) , meningococcus (meningitis), clostridia (tetanus), Streptococcus(scarlet fever), and many other infectious disorders. This focusing led to the detailed analysis and classification of SSX as a type III (immune complex-mediated) hypersensitivity reaction.“.” However, with the advent of antibiotics, the use of heterologous antisera decreased,and further analysesof this well-defined immun&gic diseasewas restricted to animal models. Recently, with currently available techniques and methodotogy. human SSX was reanalyzed in patients with bone marrow failure who received infusions of equine ATG. “. ” As an extension of this SSX study, @G-subclassantibody responsewas analyzedin five patients with bone marrow failure treated with ATG for 10 or 28 days. MATERtAlANDN?Emoos patlent clum~&Mcs Five patients(four female and one malt patient) with

General

aplastic anemia (mean values of hemoglobin. 7.4 gm!dl; 573

574

J. ALLERGY

Bielory et al.

TABLE I. Patient demographics

and clinical

parameters

noted during

CLIN. IMMUNOL. MARCH 1990

their course of SSX Day of onset GI

F’t No.

Age

Sex

A

HR

cut

PIP

1

57

66 27 12 23

F M F F

10 10 10 10 28

+ + 0 + 0

7

2 3 4 5

9 8

11 15

12 -

9

13 -

-

10

13

-

F

5 10 6 5

Rheum

-

7

PC, Patient; ATG, days of ATG treatment; HR, hematopoietic response; + , positive; Cut, cutaneouslesions; P/P, palmar/plantar sign;

rheum, rheumatologic complaints; and GI, gastrointestinalcomplaints. Day of onset refers to the number of days after the initiation of ATG that the reported signs or symptoms of SSX appeared.

TABLE II. Immunoglobulin, immune ATG-induced SSX (mean + SEMI

MA

W

(loo-250

(700-1690 mgldl)

Baseline 1014 + 59 Peak 1882 + 265 Day 11

mgldl)

complex,

and complement

IgM (50-200 mgldl)

levels in patients

CIC-Clq (nl -40%)

with

CIC-Rji (nl cl.00 TiNi)

CH, (nl60%-100%)

144+21

113 * 30

13 2 1%

0.08 + 0.02

85+ 12

346 + 78

526 + 166

53?4%

1.55 * 0.45

13 + 23*

11

8*

11

11

11

CIC, Circulating immune complexes; CH,,, total hemolytic complement. *Nadir value and day.

white blood cell count, 2640 cells per milliliter; and platelets 33,OOO/ml)were evaluated prospectively in this protocol with agesranging from 12 to 66 years. Patientswith anemia secondaryto radiation, cytotoxic chemotherapy,neoplastic disease,storagedisease,or nutritional deficiencies, and patients who had severerenal, cardiac, or metabolic complications were not included. All patients were evaluatedseveral times each day for the developmentof subjective and objective findings of SSX.” Daily samplesof serum were obtained from each patient before the infusion of ATG (15 mg/kg/day) and during their subsequentdevelopment of SSX. Samples were stored at -70” C until the samples were tested. lmmunoglobulin

levels

Serum IgG, IgA, and IgM were measuredin serial samples with nephelometry. Circulating

immune

complexes

Serial samples of serum were tested for IgG and IgM containing immune complexes in the Clq-binding assay.18 The upper limit of normal (95% confidence limit) in the control population was 10% binding. IgG immune complexes were also measuredby the Raji cell immunoassay, as previously described.19Results are expressedas TilUi ([counts per test sample]I [counts per minute of daily upper 95% confidence limit of normal control subjects]). The upper limit of normal is 1.O Ti/Ui.”

Reagents Immunoplate I microtiter plates (Nunc) were purchased from Gibco Ltd. (Uxbridge, England). Goat serum was purchasedfrom Sera Laboratories Ltd. (Sussex, England). Tween 20 and p-nitrophenyl phosphatewere purchasedfrom Sigma Ltd. (Poole, Dorset, England). All other reagents were purchasedfrom BDG Chemicals, Ltd. (Poole, Dorset, England). Antigens

and antisera

Horse IgG affinity-purified, horseradish peroxidaseconjugated rabbit anti-MIG and alkaline phosphataseconjugated rabbit anti-MIG were purchased from Sigma Ltd. Monoclonal antibody to humanIgG and IgG subclasses 1 to 4 were a kind gift from Dr. J Lowe (University of Birmingham, Birmingham, England), and supplies were subsequently purchased from Oxoid Ltd. (London, England). The clones used were 8a4 (IgG), NL16 (IgGl), GOM 2 (IgG2), Zg4 (IgG3), and RJ4 (IgG4). ELISA IgG and IgG-subclass antibodies were measured by ELISA as previously described elsewhere.2o,Horse IgG (1 kg/ml) in pH 9.6 carbonatelbicarbotiate buffer was bound to microtiter plates (Nunc Immun@late I) overnight. All incubation volumes were 50 ~1, and temperaturewas 4” C. Between each incubation or step there were three washeswith phosphate-bufferedsaline, pH 7.4, 0.1 mol/L

VOLlJMk NUMBER

85 3

containing 0.05% Tween 20. Assay diluent was phosphatebuffered saline per 0.5% Tween 20 per 0.5% normal goat serum. The test sample was addedafter 2 hours, the plates were washed,and monoclonal anti-IgG or IgG l-4 was added ( 111000 dilution). After I hour, alkaline phosphataseconjugated rabbit anti-MIG was added (l/500). After an additional l-hour incubation, substrate,P-nitrophenyl phosphate, 1mg/ ml in pH 9.8 diethanolaminebuffer, was added. Incubation at 37” C was carried out with P-nitrophenyl phosphate for 1% hours, and the color reaction was stoppedby addition of 50 pl of sodium hydroxide (3M Center, St. Paul, Minn.). Optical density was read in a Titertek Multiscan (Flow Laboratories, Ricksmansworth, Hertfordshire, England) at 405 nm. Results were expressedas arbitrary units of units per milliliter by reference to an internal standard serum, as describedelsewhere.” The interassaycoefficient of variation was 1.5%.

IgG subclass

AVERAGE SWCLASS IgG ANTI-HORSE

antibodies

w SSX

575

DlSTFWLSUTIO)SOF

GAMMA GL0fM.H

% TOTAL IgG ANTIBODY

80

70

80

50 IRESULTS

Four of the five patients received a lo-day course of ATG, and one patient received a 28-day course of ATG. All patients developed signs and symptoms of SSX 5 to 10 days after the initiation of the ATG that included the palmar and plantar sign, urticarial and morbilliform eruptions, and rheumatologic and gastrointestinal complaints (Table I). Concomitantly, levels of circulating immune complexes and immunoglobulin levels (IgG, IgA, and IgM) increasedand complement levels decreased(Table II). ” Specific IgG antibodies to horse gamma globulin were produced in all five patients with the predominance of IgGl (Fig. 1) as comparedto IgG2, IgG3, and IgG4. Of the total arbitrary units of IgG sublcasses specific for horse IgG measuredin the five patients, 77% belonged to IgGl. IgG2 subclass comprised ~5% of the total specific IgG-subclass antibody against the horse immunoglobulin. IgG3 comprised 10% and 21% of the total IgG-subclassantibody specific for horse IgG in two of the patients but only comprised about 6% of the total in the other three patients. IgG4 comprised about4% of the total in four of the five patients. In the fifth patient who received a 28-day course of ATG in the treatment protocol for aplastic anemia, IgG4 comprised 36% of the total antibody specific for horse IgG. The appearance of these antibodies during the course of treatment is illustrated in Fig. 2 for two representativepatients. In patient No. 2 (Fig. 2, A) IgG 1, IgG2, IgG3, and IgG4 antibodies appearin the serum between days 4 and 8. The rise in antibody

40 I-

3a I-

2c I-

1cl-

CI-

4 3 1 -2 SUBCLASS OF IgG ANTI-HORSE GAMMA GLOBLJLIN

FIG. 1. lgG1-4 antibodies to horse gamma globulin in patients treated with horse ATG. These data represent the geometric mean of 12 patients and include all 113 serum samples tested whether positive or not. Samples in which no antibody could be detected were assigned a value equal to the lower cutoff of the assay.

sponse associated with immune complexesand symptoms. In this patient and in two other patients, IgG4 antibody levels were increasedat the end of treatment, a feature of repeatedexposure to antigen.” DISCUSSKIN Antibody response to different types of antigens are

appearanceof IgGl antibodies and the presence of Clq-binding immune complexes coincided with the

effected by a predominant IgG subclass,and subclass deficiencies tend to be associatedwith certain diseases.??.” Various researchershave demonstratedthat

onset of symptoms of SSX and their fall with remission. Patient No. 4 (Fig. 2, B) demonstrated a modest response but again exhibited an IgGl-dominated re-

antibodies to proteinaceous antigens. such as toxins and viral antigens, belong primarily to IgGl as compared to the IgG2 subclass formed to bacterial poly-

concentration for IgGl covered a 4 log,, range. The

576

Bielory

et al.

J. ALLERGY

CLIN. IMMUNOL. MARCH 1990

FIG. 2. The relationship between IgG subclass antihorse gamma globulin, immunization, Clqbinding immune complexes, and SSX. A, In patient No. 2, antihorse gamma globulin antibody could be detected in all four subclasses. B, Patient No. 4 demonstrated a weaker response that was restricted to subclasses 1 and 4; IgGl (O--O), lgG2 (A . . . A), lgG3 (A - - -A), lgG4 (0 - - - o), immunization (U), and Clq-binding immune complexes and SSX Cm).

saccharidesor carbohydrates.*’ IgGl has been demonstrated to be the dominant subclassfor viral antigens, although other subclasses,predominantly IgG3, have also been formed to specific viral and bacterial proteins, for example, IgG3 restriction against polio, rubella, herpes simplex viruses, and streptococcal M-associated proteinN Although patients with bone marrow failure are further immunocompromised by their treatment with potent immunomodualtory agents, that is, ATG and steroids, these patients still developed a classic antibody and immune-complex responsereflecting the power of their immune reserve. In our study in patients who developedSSX, a classic immune complex-mediated (type III) condition to a heterologous protein, produced a predominance of IgGl antibodies specific for the inciting antigen. In view of a polyclonal increase in all immunoglobulin isotypes of thesepatients with ATG-induced SSX, the absenceof IgG, as comparedto IgM, IgE, and IgA and complementin the skin biopsy specimens of lesional skin, is intriguing, since it may reflect their pathophysiologic involvement in immune complexmediated reactions.I5 Immune complexes containing the ATG antigen and IgM antibody appearedto be the predominant immune complex, since the Raji cell assay, which specifically detects IgG immune complexes, was weakly positive as comparedto the Clq

binding assay,which detectsIgG- and IgM-containing immune complexes.I7 In our study, there was a close correlation between the presenceof circulating IgGl antibody and immune complexes and the clinical developmentof SSX. The finding of a predominant IgGl isotypic antibody with the early responseto an intravenously administered soluble proteinaceous antigen is in agreement with other studies, and the evaluation of a serum sample from a patient treated 20 years ago with equine antitetanus toxin (unpublished observationsfrom a sample provided by Dr. D. R. Stanworth, Birmingham University) in which the predominant IgG antihorse antibody was also of the IgGl isotype.‘, 26 Antibody responses are not distributed evenly among the subclasses.In adults, most antibodies to viral and bacterial antigensand heterologousproteins, for example, ATG, are primarily composed of the IgGl with a secondaryisotype in the IgG3 subclasses. In contrast, antibodies produced againstbacterial carbohydrate antigens are almost exclusively IgG2 and IgG4.23v2* The normal concentrationof IgG4, although it is approximately ,4% or less of the total IgG, may account for >50% of the total IgG antibody activity after prolonged immunization of atopic individuals.26 In three of our five patients, specific IgG4 remained elevatedseveralmonths after exposure.IgG4 hasbeen

VOLUME NUMBER

85 3

found to correlate after prolonged antigenic stimulation with specific IgE for individual allergens, but its role in allergic diseaseis unresolved.27 It is interesting to speculateon the purpose of isotypic antibodiesproducedat different times during the course of antigen exposure. IgGl , becauseof its neutrophil binding and complement fixing, may initiate a generalized inflammatory response,IgE may be required in the vasculardeposition of immune complex, and the “late” switching to IgG4 may lead to decreased inflammatory reaction, since it may block histamine releasefrom sensitizedbasophils in vitro and doesnot bind complement.29-32 Further analysis evaluating the role of other isotypes in immune-complex diseaseis warranted. We thank the secretarial assistance of Kathleen F. Donnelly in the preparation of this manuscript. REFERENCES

I Dray S.Three gamma globulins in normal serum revealed by monkey precipitins. Science 1960;132:1313-4. 2. Grey HM, Kunkel HG. H chain subgroupsof myelomaproteins and normal 7s globulin. J Exp Med 1964;120:253. 3. Terry WD, FaheyJL. Subclassesof humangamma-2-globulin basedon differencesin the heavy polypeptide chains. Science 1964;146:400-1. 4. Schur PH. Human gamma-G subclass.In: Schwartz RS, ed. Progress in immunology. New York: Grune & Stratton, 1972:71-104. 5. Isshizaka T, Stark AR, Ishizaka K. Demonstration of Fc receptors on human basophil granulocytes. J Immunol 1979; 123578-83. 6. Stanworth DR, Turner MW. Immunochemicalanalysis of human and rabbit immunoglobulins and their subunits. In: Weir DM, ed. Handbook of experimental immunology. Oxford: Blackwell Scientific, chap 12, 1987. 7. Burton Dr, Gregory L, Jefferies R. Aspects of the molecular structure of the IgG subclass.Monogr Allergy 1987;19:7-35. 8. Kemeny DM, Urbanek R, Amlot PL, Ciclitira PJ, Richards D, Lessof MH. Subclass of IgG in allergic disease. I. IgG subclassantibodies in immediate and nonimmediate food allergy. Clin Allergy 1986;16:571-81. 9. Urbanek R, Kemeny DM, Richards D. Subclassof IgG antibee venom antibody produced during bee venom immunotherapy and its relationship to long-term protection from bee stings and following termination of venom immunotherapy. Clin Allergy 1986;16:317-22. 10. Von Pirquet C, Schick B. Die semmkrankheit (serumsickness) 1905 [Translatedby B. Schick]. Baltimore: Williams & Wilkins, 1951. 11. WeaverGH. Serumdisease.Arch Intern Med 1909;3:485-513. 12. Kojis RG. Serum sicknessand anaphylaxis: analysis of cases OS62 11patientstreatedwith horseserumfor various infections. Am J Dis Child 1942;64:93-143. 13. Hunt LW. Recent observations in serum disease. JAMA 1932;99:909-12. 14. Gel1PGH, CoombsRRA, eds. Clinical aspectsof immunology. 1st ed. Oxford: Blackwell Scientific, 1953.

IgG subclass antibadies in SSX

577

15 Morrison-Smith J, Gel1 PGH. Serum sicknessand acute anaphylaxis in man. In: Gel1PGH, CoombsRRA, LachmannPJ, eds. Clinical aspectsof immunology. 3rd ed. Oxford: Blackwell Scientific, 1975903-12. 16 Bielory L, Gascon P, Lawley TJ, Nienhuis A. Frank MM, Young NS. Serum sicknessand haematopoieticrecovery with antithymocyte globulin in bone marrow failure patients. Br J Haematol 1986;63:729-36. 17. Bielory L, Gascon P, Lawley TJ, Young NS, Frank MM. Human serumsickness:a prospective clinical, immunological, and immunopathological analysis of 35 patients treated with antithymocyte globulin. Medicine 1988;67:40.5’7. 18 Zubler RH, Lange G, Lambert PH. Miescher PA. Detection of immune complexes in unheatedseraby a modified “:“I-Clq binding test: effect of heating on the bonding of Clq by immune complexes and application of the test to systemic lupus erythematosus.J Immunol 1976;116:232-5. 19. Theofilopoulos AN. Wilson CB, Dixon FJ. The Raji cell radioimmune assay for detecting immune complexes in human sera. J Clin Invest 1976;57:169-82. 20. Kemeny DM, Urbanek R, Richards D, Grennall C. Development of a semiquantitative enzyme-linked immunosorbent assay(ELISA) for detectionof humanI&G subclassantibodies. J Immunol Methods 1987;96:47-56. 21. Kemeny DM, Lessof MH, Pate1S, Youlten LJF, Williams A, Lamboume E. IgG and IgE antibodies after immunotherapy with bee and wasp venom [in press]. Int Arch 4llergy Appl Immunol. 22. Heiner DC. Significanceof immunoglobulin G subclasses.Am J Med 1984;76:1-6. 23. Skvaril F. IgG subclassesand viral infections. Monogr Allergy 1986;19:134-43. 24. OchsHD, WedgewoodRJ. IgG subclassdeficiencies.Ann Rev Med 1987;38:329-34. 2.5. Bielory L, Yancey KB, Young NS, Frank MM, Lawley TJ. Cutaneous manifestations of serum sickness in patients receiving antithymocyte globulin. J Am Acad Dermatol 1985;13:41I-17. 26. Aalberse RC, Van der Gaag R, Van Leevwen J. Semlogic aspectsof IgG antibodies. I. Prolonged immunization results in an IgG4-restricted response.J Immunol 1983:130:722-6. 27. Bruynzeel PLB, BerrensL. IgE and IgG4 antibodiesin specific humanallergies. Int Arch Allergy Appl Immunol 1979:58:34452. 28. Hammarstmm L, Smith CIE. IgG subclassesand bacterial infections. Monogr Allergy 12986;19:122-33. 29. Benveniste J, Egido J, Gutierrez-Millet V. Evidence for the involvement of IgE-basophil system in acute strum sickness. Clin Exp Immunol 1978;26:449-56. 30. Siequiera M, Bier OG. Haemorrhagic reactions at sites of passive cutaneousanaphylaxis in guinea pig after intravenous inoculationsof unrelatedimmune complex Proc Sot Exp Biol Med 1%1;107:779-84. 31. Kniker WT, Cochmne CG. The localization of immune complexes in experimental serum sickness:The roie of vasoactive amines and hydrodynamic forces. J Exp Med 1967:127:11936. 32. Van der Zee JS, Swieten P, Aalberse RC. Inhibition of complement activation by IgG4 antibodies. Clin Exp Immunol 1986;64:415-22.