Low Serum Immunoglobulin G2 Levels in Infancy Can Be Transient

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ARTICLE

Low Serum Immunoglobulin G

₂

Levels in Infancy

Can Be Transient

Adelle R. Atkinson, MD, Chaim M. Roifman, MD

Division of Immunology and Allergy, Canadian Centre for Primary Immunodeﬁciency, Jeffrey Modell Research Laboratory for the diagnosis of Primary Immunodeﬁciency, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

The authors have indicated they have no ﬁnancial relationships relevant to this article to disclose.

ABSTRACT

OBJECTIVE.The immunoglobulin G2 subclasses contain predominantly

antipolysac-charide antibodies. It was therefore believed intuitively that low immunoglobulin G2 levels could predispose individuals to infections with encapsulated bacteria.

Although many reports initially supported this notion, more recent studies chal-lenged it. Regardless of the biological significance, the natural history of low immunoglobulin G2levels has not been carefully studied.

METHODS.We studied the outcome of low serum immunoglobulin G2subclass levels

in children. Thirteen patients who were referred because of recurrent infections were found to have low immunoglobulin G2 levels. Laboratory evaluation at

presentation and follow-up visits included total serum immunoglobulins, immu-noglobulin subclasses, and specific antibodies to protein antigens and to pneumo-coccal vaccine.

RESULTS.Low immunoglobulin G2levels resolved completely within 0.6 years to 6

years (median: 1.5 years) in all patients. All 13 patients responded adequately to vaccination with protein antigens such as tetanus toxoid and polio as well as to immunization with pneumococcal vaccine. Four of 13 patients had a previous history of transient hypogammaglobulinemia, raising the possibility that the other cases may simply represent the tail end of this condition.

CONCLUSION.We have demonstrated that low immunoglobulin G2detected in early

infancy and childhood is likely to resolve completely within several months and up to 6 years from the time of presentation.

www.pediatrics.org/cgi/doi/10.1542/ peds.2006-3613

doi:10.1542/peds.2006-3613

Key Words

primary immunodeﬁciency, immunoglobulin IgG subclasses, childhood, infections

Abbreviations IgG—immunoglobulin G IgA—immunoglobulin A IgM—immunoglobulin M PCP—pneumococcal capsular polysaccharide

Accepted for publication Mar 22, 2007

Address correspondence to Chaim M. Roifman, MD, Division of Immunology/Allergy and Infection, Immunity, Injury and Repair Program, Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8. E-mail: [email protected]

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I

MMUNOGLOBULIN G (IGG) comprises 4 distinct sub-classes that differ in structure and function.1,2_IgG

1is

better than IgG2and IgG3in fixing complement by the

classical pathway, whereas IgG4does not bind C1q and

therefore does not fix complement. IgG1 and IgG3also

bind Fc receptors at higher affinity than IgG2or IgG4.1–9

Antibodies to protein antigen are predominantly in the IgG1and IgG3subclasses, whereas antibodies to

poly-saccharide antigens are mostly of the IgG2 subclass.3,4

Unfortunately, IgG2antibodies are the least efficient in

crossing the placenta to the disadvantage of the new-born.5,6_{This disadvantage continues because production}

of IgG2 is sluggish, reaching adult levels only during

adolescence, whereas IgG1levels reach adult levels at 3

to 4 years of age.7–10 _{Acquisition of antipolysaccharide}

antibodies is also inefficient in the first 2 to 3 years of life. Beyond these physiologic variations, low levels of IgG2 with or without low IgG4 and in the presence or

absence of immunoglobulin A (IgA) deficiency have been linked to repeated infections.1,2 _{Patients with low}

IgG2have been initially reported to have infections with

organisms such asStreptococcus pneumoniae,Haemophilus influenzae, and other encapsulated bacteria.11–14_{Some of}

these patients were also reported to have impaired re-sponses to polysaccharide antigens.15,16_However,

subse-quent studies went on to identify asymptomatic individ-uals who had reduced IgG2 but normal responses to

vaccines that contained polysaccharide antigens.17

Fur-thermore, there have been descriptions of patients who experienced recurrent bacterial infections and had a re-duced ability to produce antipolysaccharide antibodies despite normal IgG subclasses.18,19_{Thus, the significance}

of low IgG2 levels in children and its natural history

remain unclear.20

We previously reported that low IgG2levels may

fol-low the resolution of total IgG levels in a patient with transient hypogammaglobulinemia. IgG2 levels in this

patient subsequently normalized.21_{Here we followed the}

outcome of patients with low IgG2levels in the face of

normal total IgG levels. We found that all cases that were diagnosed in infancy or early childhood with low IgG2

levels eventually resolved.

METHODS

Patients

All patients who were referred to the immunology clinic at the Hospital for Sick Children for evaluation between 1987 and 2005 were considered. Patients with normal quantitative IgG and below-normal IgG2in the presence

or absence of low IgG4 subclass were included in the

study when they were available for appropriate follow-up. Patients with a diagnosis of a well-defined primary immunodeficiency such as chronic mucocutaneous can-didiasis, combined immunodeficiency, Wiskott Aldrich

syndrome, or ataxia telangiectasia were excluded from the evaluation.

The following was collected on patients who met the inclusion criteria: gender; age at presentation; reason for first referral; duration of follow-up; and serum IgG, IgA, and immunoglobulin M (IgM) and IgG subclasses at presentations and at each subsequent visit. Titers to po-lio, tetanus, measles, mumps, and rubella were deter-mined at presentation and periodically thereafter. Re-sponses to pneumococcal vaccine were also measured. The study protocol was approved by the Hospital for Sick Children Research Ethics Board.

Lymphocyte Markers and T Cell Proliferative Responses The surface phenotypes of blood mononuclear cells ob-tained by Ficoll-Hypaque density gradient centrifugation were determined by direct immunofluorescence with fluorescein isothiocyanate– conjugated goat anti-human immunoglobulin antibody (Tago, Burlingame, CA) or fluorescein isothiocyanate– conjugated monoclonal anti-bodies anti-CD3, CD4, CD8, CD20, and CD56 (Coulter Instruments, Mississauga, Ontario, Canada). Analysis was performed on a Coulter EPICS V flow cytometer. Lymphocyte proliferative responses to mitogens includ-ing phytohemagglutinin were determined by tritiated thymidine incorporation using the microtiter plate tech-nique. All assays were performed in triplicate and were compared with those simultaneously performed on nor-mal controls.22

Serum concentration of Immunoglobulins and IgG Subclasses Serum concentrations of immunoglobulins were mea-sured by nephelometry. Quantification of IgG subclasses was done by using the BINDARIO kit (The Binding Site Ltd, Birmingham, United Kingdom). The kit is based on single radial immunodiffusion methods that were de-rived from the work of Fahey et al23_{and Mancini et al.}24

In short, the method detects a protein diffusing radially from a cylindrical well through an agarose gel that con-tains the appropriate antibody. The resulting immune complexes form a precipitin ring. Once the ring size reaches equilibrium, a linear relationship exists between the square of the ring diameter and the concentration of the IgG subclass. The concentration of an IgG subclass in an unknown sample may be determined by measure-ment of the ring diameter and reading off the calibration curve. Normal range was determined according to eval-uation of 40 to 50 normal infants and children for each age group: 0 to 2 years, 2 to 4 years, 4 to 6 years, 6 to 8 years, 8 to 10 years, 10 to 12 years, 12 to 14 years, and 14 to 18 years. Mean and SDs were determined using least-squares regression analysis.23,24

Determination of Speciﬁc Antibodies

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immu-nosorbent assay (Vacczyme anti–tetanus toxoid IgG en-zyme immunoassay kit, Euroimmune Mumps EIA kit, Euroimmune Measles kit, and Euroimmune rubella kit; Euroimmune, Gross-Groenau, Germany). Polio anti-body titers were determined by complement fixation according to the manufacturer’s instructions and as pre-viously described.21,22 _{Antipneumococcal} _antibodies

were assessed using 2 kits, according to the manufac-turer’s instructions. Anti–pneumococcal capsular poly-saccharide (PCP) IgG enzyme immunoassay kit and anti-PCP IgG2enzyme immunoassay kit both were

man-ufactured by The Binding Site Ltd. The assays are de-signed for the in vitro measurement of specific IgG antibodies against PCP present in human serum. The kits are designed to measure antibody responses to pneumo-coccal vaccines incorporating 23 polysaccharides that are isolated fromStreptococcus pneumonia.25_{The PCP antigens}

used include 1 to 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F accord-ing to Danish nomenclatures.

In short, the principle of assay includes microdisks precoated with PCP antigens. Calibrators, controls, and diluted patient samples are added to the wells, and an-tibodies that recognize the PCP antigens are allowed to bind during the incubation. Purified peroxidase sheep-labeled anti-human IgG or IgG2conjugate is added. The

bound conjugate is visualized with tetramethylbenzidine substrate, which gives a blue reaction.

RESULTS

Patients

Thirteen patients who met the study inclusion criteria were 1.0 to 4.5 years of age at presentation (mean: 4 years; median: 3.7 years) and were followed for a period that ranged from 0.6 to 6.0 years (mean: 2.5 years; median: 1.5 years; Table 1).

Clinical Features

Patients were referred to the immunology clinic because of repeated infections (Table 1). Most common, recur-rent otitis media (7 patients) and repeated upper respi-ratory infections (4 patients) were reported. Two pa-tients had ⬎1 episode of pneumonia, 3 patients had a history compatible with recurrent sinusitis, and 4 were referred because of recurrent diarrhea. One patient pre-sented with recurrent varicella infections. In addition to the history of infections, 2 patients had asthma and 1 of them also had eczema. During follow-up, no major in-vasive infection was registered in the patients. Specifi-cally, no episodes of pneumonia, acute sinusitis, ab-scesses, or meningitis were recorded. However, normal frequency of upper respiratory infection or otitis media were reported in 5 patients during the follow-up period. In addition, patient 4 continued to have repeated epi-sodes of sinusitis even after IgG2levels normalized. He

experienced 3 episodes in a period of 4.5 years of follow-up.

Evaluation of the Immune System

Serum IgG and IgM levels were normal in all studied patients. IgA levels that were below normal but still detectable were found in 1 of 13 patients at presentation. Specific antibody response to tetanus vaccine was normal in 12 of 13 patients tested at presentation. The remaining patients responded normally after reimmuni-zation. Responses to polio vaccine were also normal in all patients. Isohemagglutinin levels when examined at the age of 2 years or older were also normal in 12 of 13 patients (1 patient had an AB blood group). Responses to vaccination with Pneumovax was also normal in all 13 patients.

Four patients had additional laboratory evaluation, including lymphocyte markers as well as in vitro re-sponses to mitogens and antigens. All 4 patients had normal markers and mitogenic responses.

TABLE 1 Patients Features

Patient Gender Presentation

Age, y

Resolution Age, y

Reasons for Referral

Recurrent Infections Other

1 F 4.5 10.5 Diarrhea

2 M 3.7 6.9 Sinusitis and URI

3 M 2.5 5.6 Diarrhea

4 M 4.4 5.3 Sinusitis, OM, and tonsillitis

5 F 1.0 2.5 Pneumonia, OM

6 M 1.7 2.3 Diarrhea Asthma, eczema

7 M 3.8 4.5 OM and sinusitis

8 M 1.2 2.0 OM and URI Asthma

9 M 4.4 5.2 Sinusitis and OM

10 M 1.1 1.8 URI, OM, and pneumonia

11 M 1.4 3.0 OM

12 M 1.5 5.5 Varicella

13 M 2.8 8.0 URI

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IgG Subclasses

All patients had low IgG2level, and 3 also had a reduced

IgG4 level. For the purpose of this study, only patients

with extremely low IgG2 levels were included. All

pa-tients had IgG2levels⬍95th percentile range (Fig 1); 3 of

13 had undetectable IgG2levels at presentation.

Within 0.6 years to 6.0 years (mean: 2.24 years; me-dian: 1.5 years), IgG2 levels returned to the reference

range in all 13 patients who were available for follow-up (Fig 1). All 3 patients who had low IgG4 levels also

normalized by the end of follow-up.

Transient Hypogammaglobulinemia and Low IgG2Levels

Three of 13 patients had a history of transient hypo-gammaglobulinemia. Although IgG levels normalized, the IgG2fraction remained low for a period that ranged

from 1.0 to 4.5 years. This finding raises the possibility that the other patients with low IgG2in our study may

have gone through a similar course.

DISCUSSION

We studied here the outcome of low IgG2in 13 infants

and children. The pathogenesis of “IgG2deficiency”

re-mains unknown. A delay in IgG2production in infants

seems physiologic, because IgG1 but not IgG2 reaches

near adult normal levels by 3 to 4 years of age.7–10_{It is}

possible that in the cases reported here, there may be an exaggeration of this physiologic process.

Alternatively, the low IgG2levels observed in these

patients may be part of and perhaps the end of transient hypogammaglobulinemia. Thus, despite normalizing

to-tal IgG levels, IgG2subclass levels remain low for a while

until they reach normal values.21 _{Indeed, 3 patients in}

this series had a history of transient hypogammaglobu-linemia. It is conceivable that in the rest of the patients, transient low IgG levels may have been missed before the identification of IgG2 subclass deficiency. Such a

scenario is plausible because both transient hypogam-maglobulinemia and low IgG2subclass levels have been

detected in asymptomatic individuals.17

Throughout the 1980s, a variety of publications cited the association of low subclasses with recurrent sino-pulmonary infections.11–16 _{The link was based on the}

assumption that reduced IgG2, which contains

predom-inantly polysaccharide antibodies,1,2 _{should intuitively}

be associated with an increased susceptibility to infection caused by encapsulated bacteria. Subsequent studies se-verely challenged this theory. First, asymptomatic chil-dren with low IgG2levels were found to have normal

responses to polysaccharide antigens after vaccination.17

Furthermore, children who have some inability to pro-duce antipolysaccharide antibody have been found to have normal serum levels of IgG2in isolation18,19or as a

feature of Wiskott-Aldrich syndrome.26 _Finally,

heavy-chain deletion of various IgG subclasses was identified in asymptomatic individuals.27,28 _{Together, these findings}

question the causative link between IgG2deficiency and

susceptibility to infections.

Patients in this study were referred to us because of a history of recurrent infection. Assessment of responses to polysaccharide antigens was limited to children who were older than 2.5 years because studies in younger children are inconsistent and lack reliability. Evaluation that was performed in patients who were older than 2.5 years at presentation or later during follow-up showed normal responses to unconjugated polyvalent pneumo-coccal vaccine. These results are consistent with the observation that during follow-up, patients did not re-port severe invasive infections. One patient continued to have repeated episodes of sinusitis after IgG2normalized,

suggesting that infections in these patients may not have been related to the level of serum IgG2 subclass. It is

possible that these patients had a history of infections because of a previous condition of transient hypogam-maglobulinemia, or, alternatively, the clinical history was part of a normal pattern of infections in this age group.

CONCLUSIONS

This unique follow-up of patients with low IgG2subclass

levels demonstrates that this phenomenon could be transient and is therefore of limited biological signifi-cance. In most cases, it may reflect an exaggeration of an already existing maturational delay in producing IgG2

subclass, possibly as the last stage in resolution of tran-sient hypogammaglobulinemia.

FIGURE 1

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ACKNOWLEDGMENTS

This work was supported by the Canadian Immunode-ficiency Society, the Jeffrey Modell Foundation, and the Donald and Audrey Campbell Chair in Immunology.

We thank Ms Rena Roifman for collecting the data.

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DOI: 10.1542/peds.2006-3613 originally published online August 6, 2007;

2007;120;e543

Pediatrics

Adelle R. Atkinson and Chaim M. Roifman

Levels in Infancy Can Be Transient

2 Low Serum Immunoglobulin G

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Low Serum Immunoglobulin G2 Levels in Infancy Can Be Transient