serum ige and igg antibodies to tetanus toxoid and candidin in immunodeficient children with the...

Journal of Clinical Immunology, Vol. 3, No. 2, 1983

Serum IgE and IgG Antibodies to Tetanus Toxoid and Candidin in Immunodeficient Children with the Hyper-IgE Syndrome

CHRISTIAN SCHMITT a,2 and JEAN JACQUES BALLET 1

Accepted: January 6, 1983

Serum IgG and IgE antibodies directed against tetanus toxoid and candidin were measured using a solid-phase radioimmunoassay in seven patients with the immunodeficiency syndrome with hyper-IgE. In parallel, six normal children, three normal adults, and eight patients with or without elevated serum IgE (including atopic diseases, Candida infections, and active schistosomiasis) were studied. Serum IgG antibodies to tetanus toxoid and candidin were present in the hyper-IgE patients in con- cordance with their immunization history. High concentrations of IgE antibodies against both antigens were found in the immune hyper-IgE patients but not in the controls. This suggests that elevated IgE antibody responses in the hyper-IgE syndrome results from a primary defect of IgE class regulation rather than an abnormal or deficient antibody response. KEY WORDS: Immunodeficiency; hyper-IgE syndrome; serum antibodies; tetanus toxoid; candidin.

INTRODUCTION

The immunodeficiency syndrome with hyper-IgE (HEIS) is characterized by a unique pattern of recurrent infections with Staphylococcus aureus and exceedingly high serum IgE concentrations (1- 4). Dermatitis is often part of the clinical history, with a demonstrated role of Candida albicans in some of the previously reported cases (5). Usually, the onset of the clinical manifestations is early in

life. The hyper-IgE production was generally con- sidered primary, possibly due to abnormal T-cell control since very high IgE levels were not observed in non-HEIS patients chronically infected with the same pathogens (5, 6). A role for elevated IgE production in the pathogenesis of the associated immunodeficiency has been proposed. The ab- normally elevated IgE antibody response to S. aureus in these patients (6), acting through IgE- mediated release of potent pharmacologic media- tors such as histamin, may explain the impaired neutrophil chemotaxis of some HEIS patients (2, 3, 7, 8). The demonstration of elevated anti-Candida IgE antibodies in a group of patients led to the consideration of C. albicans as another microorgan- ism capable of maintaining an immunodeficiency state (5).

In addition to the possible role of hyper-IgE in the recurrence of infections, it has also been suggested that IgG antibody responses may be deficient in HEIS patients (4, 9). The present study examines in parallel IgG and IgE serum antibodies in children with HEIS, in order to evaluate their response to (a) candidin (CDD), a ubiquitous antigen prepared from C. albicans; and (b) tetanus toxoid (TT), an immunization protein antigen. We have observed that antibodies of both immunoglobulin classes directed to both antigens were present in sensitized patients.

tLaboratoire d'Immunochimie et d'Imrnunopathotogie (IN- SERM U108); Institut de Recherches sur les Maladies du Sang, Universit6 Paris VII; Laboratoire d'Oncotogie et d'Immunohe- matologie du CNRS; Hrpital Saint-Louis, Paris, France.

ZTo whom correspondence should be addressed at INSERM U 108, H6pital Saint-Louis, 2 place Alfred Fournier, 75475 Paris Cedex 10, France.

178

METHODS

Donors

Sera from three male and four female HEIS patients were studied. Their ages ranged from 3 to 15 years at the time of study. Pertinent data on

0271-9142/83/0400-0178503.00/0 * 1983 Plenum Publishing Corporation

SERUM ANTIBODIES IN HYPER-IgE SYNDROME 179

Table I. Clinical Data, Total Serum IgE Levels, and Serum IgG and IgE Antibodies in Seven Patients with HEIS

Serum antitetanus Serum anti-Candida Time since last antibodies antibodies

Total antitetanus Age serum IgE immunization IgG (IU/ IgE (AU~/ IgG (x 103/ IgE

Donor Sex (years) (IU/ml) (years) ml) ml) AU/rnl) (AU/ml)

1 M 13 52,000 3 0.400 4.87 224.85 55.73 2 F 15 35,750 5 0.475 0.58 60,02 3,44 3 F 9 30,000 1 1 t .00 b 6.05 ND C 7.53 4 M 11 18,970 No previous 0.005 0.04 65.98 3.55

immunization 5 M 11 18,620 1 1.678 8.85 8.60 0.03 6 F 7 8,820 6 0.0t0 0.01 2.96 0.03 7 F 3 6,270 1 3.924 9.10 5.06 7.53

-Arbitrary units (see Methods), bMeasured by hemagglutination technique in cNot done.

this patient,

HEIS patients are summarized in Table I. All children had a history of recurrent staphylococcal lung infections. All but one (donor 6, Table I) had had previous transient or persistent dermatitis. In all children, serum IgE levels were over 6000 IU/ml. The dates (or in one case the absence) of previous immunization with TT were precisely recorded, and in all previously immunized patients the last immunization consisted in one booster injection of one dose of vaccine (30 IU).

Control sera studied in parallel were obtained from eight 3- to 15-year-old ambulatory children recovering from minor upper respiratory tract infections or minor surgery. Control sera from adults were obtained from volunteers with recorded previous antitetanus immunization or in whom the lack of previous immunization with TT was unambig- uously ascertained. Other patients studied consisted in three 3- to 15-year-old children with atopic respiratory manifestations, one child with recurrent atopic eczema, two children recovering from acute digestive Candida infection, one child with active schistosomiasis, and one 23-year-old male adult patient with chronic mucocutaneous candidiasis (CMCC) (see Table iI).

Antigens

Candidin (CDD; metabolic antigen) prepared from C. albicans was obtained from Institut Pas- teur, Paris. Two preparations of TT were used: (a) a purified toxoid (t050 Lf/mg N) obtained from Insti- tut Pasteur, Paris; and (b) a purified monomeric "7 S" toxoid (3150 Lf/mg N), a kind gift of Dr. B.

Bizzini, Laboratoire d'Immunochimie des Pro- teines, Institut Pasteur, Paris (10).

Determination o f Serum Antibody Levets

Anti-TT and anti-CDD serum antibody levels were determined using a solid-phase radioimmunoassay (11, 12). In each plastic microvial (Removeawell, Dynatech, Alexandria, USA), a 200-~zl aliquot of a 200 p.g/ml solution of antigen in 0.01 M phosphate buffer, pH 7.2, 0.5 M NaC1 (PBS) was incubated at 37°C for 1 hr. For each assay, control vials contained PBS instead of the antigen solution during this first incubation. The solution was aspirated, and the vials were filled with PBS containing 10% bovine serum albumin (BSA) (frac- tion V, Sigma, St. Louis, MO) and maintained for 1 hr at 37°C. The solution was decanted arid the vials were washed three times with cold 1% BSA PBS. Several dilutions of the asseyed sera were made in triplicate into the appropriate antigen-coated vials and incubated overnight at room temperature. Solu- tions were aspirated, and vials were washed three times with cold 1% BSA PBS. Bound antibodies were revealed using affinity chromatography-purified F(ab)'2 fragments from rabbit IgG specific for human ~/ chain. F(ab)2 fragments were iodonated (t25I) using the chloramine T method. The specific activity was I200 to 3520 cpm/ng depending on the preparation. Two hundred microliters of the labeled preparation diluted in PBS and containing about 1.5 tzg of antibody was distributed in each vial and incubated for 6 hr at room temperature. Vials were washed five times with 1% BSA PBS and counted in


180 SCHMITT AND BALLET

Table II. Status, Total Serum IgE Level, and Serum IgG and IgE Antibodies in 17 Normal Donors or Patients Studied in Parallel with HEIS Donors

Serum antitetanus Serum anti-Candida Time since last antibodies antibodies

antitetanus Age Total serum immunization IgG (IU/ IgE (AUa/ IgG (x 103/ IgE

Status of donors Sex (years) IgE (IU/ml) (years) ml) ml) AU/ml) (AU/ml)

Control children 4 M 3-15 11.5 -+ 22.10 2-4 0.92 -+ 1.58 0.01 11.70 -+ 7.36 0.01 ( iV= 6) 2 F

Children with 1 M 3-15 1470 -+ 319 2-4 2.02 -+ 1.03 0.01 53.16 -+ 21.70 0.01 chronic respira- 2 F tory atopy (N = 3)

Child with chronic M 6 4550 5 0.055 0.04 5.62 9.39 atopic eczema ( N = 1)

Normal adult M 35 200 0.4 1,629 0.01 6.58 0.01 (N = i)

Normal adult M 25 15 5 0.012 0,03 ND ° ND (N= 1)

Normal adult F 22 110 1 13.130 0.32 ND ND ( N = 1)

Acute digestive 1 M 0.5-3 ND ND ND ND ND 0.01 Candida infection 1 F (N = 2)

Active schistomiasis M 5 5625 1 0.129 0.93 2,07 0,34 (N = I)

CMCC (N = 1) M 23 130 ND ND ND 255.61 0.03

aArbitrary units. bNot done.

a gamma counter. Specifically bound radioactivity was calculated by subtracting the counts measured in antigen-coated vials incubated in the absence of antigen.

For each series of anti-TT serum antibody determination, a standard curve was established using serial dilutions of a standard human anti-tetanus IgG preparation (110 IU/ml, CNTS, Orsay, France). The standardization curve indicated that the binding of 125I-labeled rabbit anti-human IgG antibody to the vial was linear between 0.05 x 10 -3 and 6 x 10 -3 IU/ml of anti-TT human IgG. The highest value of the standardization curve (6 x 10 -3 IU/ml of standard anti-TT antibody, corresponding to 68 ng of 125I-labeled anfi-IgG calculated from the specific activity) suggests that the 125I-!abeled antibody was in excess. The estimated sensitivity of the test was below 0.5 x 10 -3 IU, i.e., 2 ng.

The maximal anti-CDD IgG antibody binding capacity of the CDD-coated vials was calculated from the specific activity of the radioiodinated anti- human IgG and corresponded to approximately 60 ng of rabbit anti-human F(ab)'2 per vial. The amount of radioactivity bound to the CDD-coated

vials was linear between 6 and 42 ng of rabbit antihuman F(ab)'2. Serum anti-CDD IgG antibody levels were experimentally determined with serum dilutions resulting in the binding of a quantity of anti-human IgG antibody included within these lim- its. Results of anti-CDD IgG antibody levels are expressed as arbitrary units (AU) per milliliters of undiluted serum. One AU corresponds to 1 ng of iodinated rabbit anti-human IgG antibody bound to the vial. In control experiments, the antigen specificity was verified by adding antigen solutions during the incubation with the first antibody.

For each serum, determinations were performed at three dilutions chosen in the linear portion of the standardization curve, and results are expressed as the mean + 1 SD, reported to 1 ml of undiluted serum. For anti-TT antibodies, the reproducibility of the assay was tested in sextuplicates of the same samples: in a typical experiment, the mean value was 0.242 -+ 0.028 IU/ml. The chain specificity of anti--/chain F(ab)'2 fragments was shown by their lack of reactivity with human IgM absorbed on anti- human immunoglobulin antibody-coated vials in the same assay.


SERUM ANTIBODIES IN HYPER-IgE SYNDROME ~,8|

Determination of Total Serum IgE Levels

Total serum IgE levels were measured using Phadebas IgE PRIST kits (purchased from Pharma- cia France, 78390 Bois d'Arcy, France), following the instructions of the manufacturer.

Determination of Serum IgE Antibody Levels

Anti-TT and anti-CDD IgE antibody levels were determined using the radioimmunoassay previously described for serum IgG antibody level determination. One hundred microliters of ~25I-labeled rabbit anti-human IgE (Phadebas PRIST reagent) was used for the detection of IgE antibodies. Results are expressed as arbitrary units (AU), in order to standardize data obtained with different preparations of ~25i-labeled anti-IgE antibodies. One AU consisted in the radioactivity (cpm) of ~25I-labeled anti-human IgE antibody bound to 20 IU/ml IgE standard solution in a PRIST test performed in parallel to each series of experiments. In addition, the class specificity of IgE antibody detection was ascertained by verifying that no significant (i.e, less than 0.01 AU) IgE antibody was detected in the standard anti-tetanus IgG preparation.

R E S U L T S

Antitetanus Serum IgG and lgE Antibody Levels in Patients w#h the Hyper-IgE Syndrome

In six patients with recorded previous immunization with TT, antitetanus IgG antibody levels were over 0.005 IU/ml (Table I). They were lower in one case in which the absence of previous immunization was ascertained. A correlation was found between the anti-TT antibody levels and the time since last immunization (P < 0.01). In five recently immunized HEIS children, the mean antibody level (1 °29 to 1.59 IU/ml) was within a range similar to that of the levels observed in an age-paired control group of nonimmunodeficient children who received immunization at the same ages (0.92 to 1.58 IU/ml) (Table II).

Significant antitetanus IgE antibodies were found in five of seven HEIS patients. The lowest values were observed in patients with remote (6 years) or no previous immunization. Again, a relationship was found between the anti-TT IgE antibodies and the time since last immunization (P < 0.01). No correlation was found between total IgE and IgE

anti-TT antibodies (P < 0.05). These observations contrast with the undetectable or low level of anti- TT antibody IgE in control children and adults, whatever their IgG anti-TT antibody level (0.012 to 13.1 IU/ml). A very low or low anti-TT IgE level was observed in control children with elevated total IgE, in which anti-TT IgG levels ranged from 0.055 to more than 2.02 IU/mt (Table II).

We have compared the radioimmunological detection of antibodies to a 1050 Lf/mg N TT preparation and to a highly purified TT "7 S" monomer (3150 Lf/mg), free of polymers and aggregates. As shown in Fig. 1, a good direct correlation was found for both IgG and IgE in the sera of HEIS patients and of other donors with hyper-IgE.

Anti-Candida Serum IgG and IgE Antibody Levels in Patients with the Hyper-IgE Syndrome

Anti-CDD IgG antibodies were detected in sera from all HEIS patients. They ranged from low values comparable to those of normal or hyper-IgE individuals without active Candida infection to high levels similar to that of patients with CMCC (Tables I and II). In all HEIS patients with dermatitis, anti- CDD IgG antibodies were detected. Significant levels of IgE antibodies to CDD were found in five of seven HEIS patients and were correlated with total IgE levels (P < 0.001). Values were high by comparison to those of controls and similar to values found in one eczema-prone child. A good positive correlation was found between IgG and IgE anti-CDD antibody levels (P < 0.01).

D I S C U S S I O N

In this report, data on serum anti-TT and anti- CDD antibodies in seven patients with the HEIS are presented. The same purified antigens, TT and CDD, were used in a solid-phase radioimmunoassay for both IgG and IgE. Controls established the sensitivity and specificity of these assays. In particular, it is of note that in sera from control donors with IgG antibodies against TT or CDD (such as the children and adults presented in Table II), IgE antibody levels were low or at the lower limit of detection (0.01 AU/ml). In only one adult control were high anti-TT IgG antibodies (13.130 IU/ml) associated with the detection of significant anti-TT IgE antibodies (0.32 AU/ml), a fact in agreement with previous reports of the detection of

Journal o f Clinical Immunology, Vol. 3, No. 2, 1983

182 SCHMITT AND BALLET

100

E

~- 1.0

%

E

L

0.0~ • ( r = 0,99)

E I0

<

E

c~

%

I0 J L~

A 1

• &

(r=0,96)

m , i

o.oo o. o 1.'o o,o lo-'

Serum Ig6 onfi TTIUI/mt) Serum lqE onh TT (AU /mt l

Fig. 1. Correlations between antibodies to tetanus toxoid (1050 Lf/mg N; TT) and antibodies to its monomeric form (3150 Lf/mg N; TTm). (@) Serum from HEIS patient; (A) serum from non- HEIS individual.

IgE antibodies in some normal adults (13). In other cases such as the patient with active schistosomiasis, comparatively low IgG antibodies were associated with elevated IgE antibody levels. This find- ing, previously described controls, and the experi- mental conditions of our assay establish the class specificity of IgE antibody detection. Antigens coated to microvials were present in excess with regard to the total antibody concentration present at the serum dilutions used, thus minimizing the effect of competition between immunoglobulin classes. All determinations were performed at serum dilutions corresponding to the linear portion of the standardization curves.

Seven 3- to 15-year-old HEIS patients were studied. From early infancy, they had suffered severe bouts of staphylococcal pneumonia. In most of them, previous or recent dermatitis was recorded. Their current serum IgE levels ranged from 6200 to 52,000 IU/ml. These features led us to consider these patients as closely resembling those described previously by several authors and referred to in this report as "hyper-IgE syndrome" (I-4).

The date, or lack, of previous antitetanus immunization was known in these children. In five patients, a booster immunization with TT was admin- istered less than 6 years before our study. In previously immunized patients, anti-TT IgG antibody levels were found in ranges which were observed in age-matched normal children with a similar immunization status (Table II). In two other

patients, the very low or undetectable IgG level was consistent with a more remote, or the lack of, previous immunization. Similarly, whereas the na- ture of previous sensitization to Candida could not be defined, IgG antibodies to Candida were demonstrated in most patients.

A significant IgE anti-TT antibody level was observed in previously immunized HEIS patients. Their levels were high compared to those of age- matched normal, atopic, and parasite-infected individuals. These findings extend to TT the capacity of HEIS patients to produce antistaphylococcal (5, 6) and anti-Candida (5; present data) serum antibodies. In HEIS patients, we found a weak correlation between IgG and IgE antibody levels, as well as between the total IgE level and the level of IgE antibodies to TT and CDD. In most normal individuals, IgE antibodies were found at the lower limit of detection. This was in agreement with previous reports (13, 14), whereas in three atopic children in the present series, IgE antibodies were .very low. Our observation of anti-Candida IgE antibodies in a Schistosoma-infected patient is also in agreement with others (5).

Our data and previous reports support an abnormal regulation of IgE class in HEIS. Rodents ap- pear to have separate regulation for IgG and IgE antibodies, suggesting the presence of distinct iso- type-specific T cells or, alternatively, of two sets of T cells, one specific for antigen and the other controlling the distribution of antibodies among

Journal of Clinical Immunology, VoL 3, No. 2, 1983

SERUM ANTIBODIES IN HYPER-IgE SYNDROME 183

different isotypes (15). Selective potentiation of IgE antibody responses to unrelated antigens was obtained with a variey of stimuli, including helminthic infection (16, 17). In humans, the suppressive activity of T cells on IgE production was investigated in

vitro, and evidence was obtained in favor of an abnormal suppressor activity in conditions with hyper-IgE production, including HEIS (4, 17, 18). However, the significance of in vi tro IgE detection in lymphocyte cultures is still a matter of controver- sy, and it is of note that no relation between serum IgE levels and numbers of circulating T cells with Fc receptors for IgE could be demonstrated (21).

The mechanism of repeated infections in HEIS patients remains so far poorly understood. Our data and other reports suggest that HEIS patients are able to make an antibody response to a variety of antigens, including TT, CDD, and S. aureus . A

predominant rote of an antibody defect appears unlikely in the immunodeficient state, whereas S. aureus infection does not seem to play a unique rote in the elevation of IgE production.

ACKNOWLEDGMENTS

We thank Drs. D. Buriot, C. GrisceUi, and P. Perraudin for providing us serum samples of their patients. We thank Dr. J .C . Brouet for critical reading of the manuscript. This study was support- ed in part by INSERM Grants 59.78.91 and 80.10.21.

REFERENCES

1. Buckley RH, Wray BB, Belmaker EZ: Extreme hyperim- munoglobulinemia E and undue susceptibility to infection. Pediatrics 49:59-70, 1972

2. Clark RA, Root RK, Kimball HR, Kirkpatrick CH: Defec- tive neutrophil chemotaxis and cellular immunity in a child with recurrent infections. Ann Intern Med 78:515-519, 1973

3. Hill HR, Quie PG: Raised serum IgE levels and defective neutrophil chemotaxis in three children with eczema and recurrent bacterial infections. Lancet 1:183-197, 1974

4. Buckley RH, Becker WG: Abnormalities in the regulation of human IgE synthesis. Immunol Rev 41:288-314, 1978

5. Berger M, Kirkpatrick CH, Goldsmith PK, Gallin JI: IgE antibodies to Staphylococcus aureus and Candida albicans

in patients with the syndrome of hyper-immunogtobulin E and recurrent infections. J Immunol 125:2437-2443, 1980

6. Schopfer K, Baerlocher K, Price P, Krech U, Quie PG, Douglas SD: Staphylococcal IgE antibodies, hyperimmuno- gtobulinemia E and staphylococcus aureus infections. N Engl J Med 300:835-838, 1979

7. Church JA, Frenkel LD, Wright DG, BeUanti JA: T lymphocyte dysfunction, hyper-immunoglobulinemia E, recurrent bacterial infection and defective neutrophil chemotaxis in a Negro child. J Pediatr 88:982-985, 1976

8. Gallin JM, Wright DG, Malech HL, Davis JM, Klempner MS, Kirkpatrick CH: Disorders of phagocyte chemotaxis. Ann Intern Med 92:520-525, 1980

9. Buckley RH: Augmented immunoglobulin E synthesis in primary immunodeficiency. In Cellular, Molecular and Clini- cal Aspect of Allergic Disorders, S Gupta, RA Good (eds). New York, Plenum, 1979, pp 513-535

10. Bizzini B: Tetanus toxin. Microbiol Rev 43:224-240, t979 11~ Zollinger WD, Datrymple JM, Artenstein MS: Analysis of

parameters affecting the solid phase radio immunoassay quantitation of antibody to meningococcat antigens. J Imrnu- not 117:1788-1798, 1976

12. Stevens RH, Saxon A: Reduced in vitro production of antitetanus toxoid antibody after repeated in vivo immunization with tetanus toxoid. J Immunol 122:592-598, 1979

13. Nagel J, Svec D, Waters T, Fireman P: IgE synthesis in man. I. Development of specific IgE antibodies after immunization with tetanus-diphtheria (TD) toxoids. J Immunot 118:334-341, 1977

14. Mathur S, Goust JM, Horger EO, Fudenberg HH: Immuno- globulin E anti-Candida antibodies and candidiasis. Infect Immun 18:257-260, 1977

15. Ishizaka K, Ishizaka T: Mechanisms of reagininic hypersen- sitivity and IgE antibody response, Immunol Rev 41:109- 148, 1978

16. Jarret EEE: Stimuli for the production and control of IgE in rats. Immunol Rev 41:52-76, 1978

17. Patterson R, Suszko IM, Hsu CCS, Roberts M, Oh SH: In vitro production of IgE by lymphocytes from a patient with hypergammaglobulinemia E, eosinophilial and increased lymphocytes carrying surface IgE. Clin Exp Immunol 20:265-276, 1975

18. Saxon A, Stevens RH: Subpopulations of circulating B-cells and regulating T-cells involved in in vitro IgE production in atopic patients with elevated serum IgE. J Clin Invest 65:1457-1468, 1980

19. Sampson HA, Buckley RH: Human IgE synthesis in vitro: A reassessment. J Immunol 127:829-834, 1981

20. Romagnani S, Maggi GF, Delprete F, Maggi E, Bargellesi A, Ricci M: In vitro production of IgE by human peripheral blood mononuclear cells. III. Demonstration of circulating IgE bearing cells involved in the spontaneous IgE synthesis. Clin Exp Immunol 49:176-184, 1982

21. Gupta S: Subpopulation of human T lymphocytes. XVIII. T lymphocytes with receptors for IgE (T epsilon) in patients with primary immunodeficiency and hyperimmunoglobuline- mia E states. Clin Exp Immunol 45:113-117, 1981


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