Development of Immunoglobulin A in Infancy and Childhood

Authors


Dr C. Weemaes, Department of Pediatrics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: c.weemaes@cukz.umcn.nl

Abstract

Serum and salivary concentrations of immunoglobulin A1 (IgA1) and IgA2 were studied in 105 Icelandic children aged 0–12 years. Serum concentrations of both IgA1 and IgA2 increased slightly (P < 0.001) during childhood. The salivary IgA1/IgA2 ratio tended to decrease during the same period; this trend is less apparent when omitting the youngest children. The salivary IgA1 and IgA2 output could be high, even in children with low levels of serum IgA. Only polymeric IgA was found in whole saliva. Interestingly, in serum, most IgA1 and IgA2 were polymeric during infancy. The proportion of polymeric IgA decreased, when the concentration of IgA increased. The polymeric form of IgA might provide the infant with better protection against invading microorganisms by activation of the innate immune mechanisms.

Introduction

Human immunoglobulin A (IgA) comprises monomeric and polymeric IgA of two subclasses, IgA1 and IgA2. In adulthood, IgA1 is the predominant subclass in serum. IgA2 production is relatively higher in the mucosa, especially in the lower intestinal tract [1]. Serum IgA (both IgA1 and IgA2) is mainly monomeric. Secretory IgA on the other hand is dimeric and is joined by the J chain and the secretory component.

Concentration of serum IgA is 20% of normal adult value at the age of 1 year and rises progressively through childhood and adolescence [2]. IgA does not cross the placenta, and serum concentration in cord blood is usually very low (0.004 g/l). Secretory IgA is undetectable at birth but can be detected by 1 week to 2 months of age in tears, nasopharyngeal secretions and saliva [3]. In infancy, a rapid increase of the salivary IgA level has been demonstrated, usually peaking before 2 months [4] and reaches adult values by age of 4–6 years, according to some authors [5], but rather late in childhood according to other investigators [4]. The development of the IgA κ/λ ratio differs from the light chain ratios of the other immunoglobulin classes, as the IgA κ/λ ratio decreases during childhood, whereas IgG and IgM κ/λ ratios increases [6].

Secretory IgA antibodies can neutralize viruses, bind toxins, agglutinate bacteria, prevent bacteria from binding to cells and bind various food antigens [7, 8]. At young age, the developing immune system is challenged with a variety of microorganisms as well as by protein antigens from the environment [8]. In addition, immunizations, even by oral route, may be given in this period, and allergic reactions often develop. The understanding of the mucosal and serum immune response in the infant is therefore of great interest.

The initial serum IgA response to antigenic challenges is often polymeric and subsequently shifts to the specific monomeric form over time [9]. In adults, approximately 90% of serum IgA is monomeric, but Delacroix and coworkers [10] found a high proportion of polymeric IgA in serum in infants (6–240 days, mean 83 days). This development could play a role in infant infections and the immune response in early childhood. Therefore, we studied the monomeric and polymeric IgA in both subclasses in serum and saliva in healthy infants and children.

Patients and methods

Patients.  Enrolled in the study were 105 Icelandic children, aged 0–12 years. They were admitted to the Children's Hospital Iceland for elective minor operation or medical examinations. Children with medical history of present or previous recurrent infections, other acute or chronic illnesses or syndromes were all excluded from the study. Children with history of asthma, allergy, atopic diseases, any suspected immunological disorder or gluten enteropathy were also excluded. The children enrolled were considered healthy. Informed consent was obtained from the parents before enrolment in the study.

Some of the infants were breastfed or on a combined formula breast regime. Saliva samples were obtained at least 2 h after a meal.

Methods.  Stimulated whole saliva was obtained from the mouth with a polyethylene suction catheter added to a sterile mucus extractor. Sampling started 3 min after local stimulation with citric acid. The saliva was aspirated during 10 min from the mouth. The saliva was, after centrifugation for 10 min at 2800 × g and 4 °C, stored in 10 ml tubes at −70 °C until measurement. Before measurement, it was again centrifugated under the same conditions as above. Data of salivary IgA are provided as secretion rates. In parotid saliva, excretion per minute is the best parameter to express the secretion rate of IgA [11, 12]. However, as we aspirated whole saliva from the mouth, the flow can not be so accurately measured as that of parotid secretion. Hence, salivary IgA is expressed in secretion rate per 10 min (the time of sampling). Blood was collected at the same time, and serum was stored at −70 °C until measured.

Immunoglobulin quantification in serum.  Immunoglobulin concentrations were determined nephelometrically on a Cobas Fara analyzer (Roche Diagnostics, Mijdrecht, The Netherlands). In this assay, rabbit Ig fractions of antihuman IgG, IgA or IgM diluted in 0.012 m phosphate-buffered saline (PBS) with 4% polyethylene glycol 4000 and 0.1% sodiumazide were used (Q-series, Dakopatts, Copenhagen, Denmark). As a calibrator, a pooled serum of 500 healthy blood bank donors (5 ml each), stored at −70 °C and calibrated against the internationally accepted standard for serum proteins CRM 470 (described by Whicher [13] and Baudner [14]) was used. Measuring ranges were 59.9–958.2 mg/l for IgG, 12.4–198.7 mg/l for IgA and 5.6–89.2 mg/l for IgM. %CV of the methods were 4.6, 4.2 and 4.7% for IgG, IgA and IgM, respectively.

Immunoglobulin quantification in saliva.  Measurements of Igs in saliva were performed by sandwich enzyme-linked immunosorbent assay (ELISA). In these assays, polystyrene microtitre plates (maxisorp F96, NUNC, Roskilde, Denmark) were coated overnight at 4 °C with 0.2 µg/well of affinity purified goat antibodies from Cappel (ICN Laboratories, Zoetermeer, The Netherlands) in 0.05 m NaHCO3, pH 9.5. The same calibrator as for nephelometry was used. Dilutions were made in the range of 5–180 µg/l, in 0.05 phosphate buffer containing 0.01% Tween and 0.2% bovine serum albumin (BSA), using a Microlab 2200 pipetting robot (Hamilton, Bonaduz, Switzerland). After sample incubation for 120 min at 37 °C, goat horseradish peroxidase (HRP)-conjugated antibodies (Cappel) were added to the plates and incubated for 90 min at 37 °C. The secondary antibodies used are affinity purified for anti-IgG and anti-IgM, whereas anti-IgA is an IgG fraction. Colour development was performed by (orthophylene diamine) OPD in 0.1 m citrate-phosphate buffer, pH 5.0. Incubation was stopped when the highest standard reached 1.0–1.5OD. OD was measured at 492 nm on a Titertek Multiskan MC photometer. %CV for these ELISAs were 5.4% for IgG, 3.5% for IgA and 5.3% for IgM.

Quantification of IgA subclasses in serum and saliva.  IgA subclasses were measured by sandwich ELISA, mainly as described before [15]. Microtitre plates, buffers (except for the coating buffer) and the readout system were as described above. The ELISA plates were coated overnight at 4 °C with mouse monoclonal anti-IgA1 or anti-IgA2 [16] (Nordic, Tilburg, The Netherlands), anti-IgA1 (69–11.4) diluted 1 : 4000, anti-IgA2 (16–512-H5) diluted 1 : 8000 both in PBS. After washing, the plates were incubated with the serum or saliva samples (in three different dilutions in duplicate) or standard serum dilutions in wash buffer with 0.01% Tween and 0.2% BSA. Detection of IgA in these samples was by HRP-labelled goat antihuman IgA (Cappel, Organon Teknika, Turnhout, Belgium) (dilution for IgA1 detection is 1 : 20,000 and for IgA2 detection 1 : 10.000). As the standard serum, the KIK-21 serum was used. This is a pool of normal human serum that was a kind gift of Dr J. Radl (Department of Immunology and Infectious Diseases, TNO, Leiden, The Netherlands). The serum pool was repeatedly tested by various techniques, always with the result of 2 g/l of IgA1 and 0.2 g/l of IgA2. A secondary standard calibrated against this standard was produced, which was used in the study presented here. The lower limit of detection of IgA1 was 5 mg/l, of IgA2 2.25 mg/l. Interassay %CV were 5.0 and 6.5% for IgA1 and IgA2, respectively.

Determination of monomeric and polymeric IgA.  Sera or saliva were separated by gelfiltration (FPLC-system, Pharmacia Biotech, Roosendaal, The Netherlands) using a 10/30 superose 6 column and analysed by ELISA as described above [15]. The column was calibrated with a mix of purified human secretory IgA and IgG (Nordic) in a 0.05 m PBS containing 0.005% sodium azide, pH 7.4. Sera or saliva were diluted with PBS, and a maximum of 15 µg of IgA was applied to the column by a 200 µl loop. Gelfiltration was performed at room temperature and at a flow rate of 0.2 ml/min. Fraction volumes of 500 µl were collected in polystyrene cups containing 50 µl of 0.05% Tween-20 in PBS. The fractions were then, within 1 h after elution, analysed by ELISA as mentioned above.

Standardization.  One has to be aware of the difference in behaviour of the IgA in serum or saliva in the assays because of the difference in molecular size, as Brandtzaeg and coworkers [11] already discussed in 1970. Because a salivary calibrator was not available, we used a serum calibrator in the ELISAs for serum as well as saliva. We assume this was legitimate for two reasons. Firstly, the trends in maturation of the salivary maturation and the correlation to the maturation of serum IgA that we wished to examine will remain the same. Secondly, we expect that the ELISAs used are less sensitive for the molecular size than radial immunodiffusion assays used by Brandtzaeg [11]. Parallelism of the serum and saliva samples in the ELISAs was alright.

Statistics.  For a description of the mean age trend in the concentration of IgA values, a linear regression model was adapted to the logarithm of the IgA values for both IgA1 and IgA2 in serum and saliva (Table 2). The model is given for each of these cases together with the proportion of the total variation in log(IgA) explained by the model (R2). Similarly, the mean age trend in the ratio of the geometric mean of IgA1 and IgA2 was estimated. Association between the variables was assessed by Pearson and Kendall correlation.

Table 2.  Linear regression model for immunoglobulin A1 (IgA1) and IgA2 in serum and saliva with relative increase in geometric mean per year
Age trend in IgAnLinear regression modelR2 (%) (P value)Relative increase in geometric mean per year (%)
  • As there is no significant age trend in the ratios of the predicted geometric means of IgA1 and IgA2, the ratio is taken as independent of the age in both cases, and its evaluation is given.

  • *

    Values given for 95% CI.

Saliva92log(IgA1) = 3.814 + 0.163 age35 (<0.001)18
Saliva92log(IgA2) = 3.078 + 0.212 age44 (<0.001)24
Serum99log(IgA1) = 1.276 + 0.196 age41 (<0.001)22
Serum99log(IgA2) = 3.803 + 0.166 age41 (<0.001)18
Saliva91log(IgA1/IgA2) = 0.695 – 0.042 age3 (0.07) 
Serum99log(IgA1/IgA2) = 2.527 + 0.030 age1 (0.16) 
Age independent
SalivaIgA1/IgA2 = exp(0.695) = 2.01.6–2.6* 
SerumgA1/IgA2 = exp(2.527) = 12.59.9–15.8* 

The level of significance was set at 0.05. IgA values below 0.1 mg/l were given the value of 0.05 mg/l.

Results

The IgA1 level in serum and the salivary IgA1 secretion rate increased with age, and the same was true for IgA2 (Table 1) and (Fig. 1). However, IgA2 levels remained very low in serum. There was a temporary rise in the salivary secretion rate of IgA1 at the age of 3–6 months.

Table 1.  Levels of immunoglobulin A (IgA) subclasses in serum and saliva according to age
  SerumSaliva
  IgA1 (g/l)IgA2 (g/l)IgA1 (µg per 10 min)IgA2 (µg per 10 min)
ChildrennMeanRangeMeanRangeMeanRangeMeanRange
0–3 months61.40.2–2.90.150.1–0.23310–78170.5–43
3–6 months43.40.9–6.60.30.1–0.7209135–3495736–78
6–12 months84.50.8–10.00.30.1–0.96717–259607–271
1–2 years94.42.7–5.70.30.1–0.64622–803113–52
2–5 years227.50.8–15.10.60.1–1.19516–2616417–225
5–8 years3613.02.7–40.30.80.2–2.119862–18119236–202
8–10 years1514.83.6–28.41.00.4–2.126635–90217251–468
Figure 1.

Concentrations of immunoglobulin A (IgA) subclasses in serum and saliva according to age. Correlation coefficients: serum IgA1 R2 = 0.33, IgA2 R2 = 0.32; saliva IgA1 R2 = 0.28, IgA2 R2 = 0.38.

The age trend in IgA1 and IgA2 in serum and saliva is summarized in Table 2. The P value was lower than 0.001 for all the regression coefficients. The proportional increase in the estimated geometric mean of the IgA1 values of saliva by 1 year increase in age was estimated as exp(0.163) = 1.18 or 18%. Similarly, for the other models, we obtained 24, 22 and 18% increase, respectively (Table 2). The mean age trend in the ratio of IgA1 and IgA2 was estimated in the same way (Table 2). The P values for the age coefficients were 0.07 for saliva and 0.16 for serum, respectively. Thus, the proportion was taken as independent of the age in both cases and was then evaluated accordingly. Hence, the geometric means of IgA1 and IgA2 in saliva increased by the same rate by increasing age, and, therefore, the difference between the 18 and 24% increase described by equation 1 and 2 was not significant (P > 0.05). Similarly, the difference between the 22 and 18% increase in serum, described by equation 3 and 4, was not significant. To conclude, IgA1 and IgA2 had the same average rate of increase by increasing age both in saliva and serum in the age interval considered (0.1–10.4 years) and the ratio of IgA1 and IgA2 was constant on average in that age interval.

Nevertheless, there was a trend for the salivary IgA1/IgA2 ratio to decrease slightly during this period, most apperent when omitting the youngest children.

For children aged 0–2 years, the increase in the serum concentrations of IgA1 and IgA2 was independent of the increase in the saliva, especially in IgA2 (Fig. 2). No correlation was found in the individuals between the levels of IgA1 in serum and saliva. The same held true for IgA2.

Figure 2.

Correlations between the serum and saliva concentrations of immunoglobulin A (IgA) subclasses in children aged 0–2 years. Correlation coefficients: IgA1 saliva versus serum R2 = 0.001; IgA2 saliva versus serum R2 = 0.016; serum ratio IgA1/IgA2 R2 = 0.01; saliva ratio IgA1/IgA2 R2 = 0.13.

In saliva, both IgM and IgG were present at very low levels (Fig. 3). These Igs were found in saliva of the youngest children throughout childhood but remained at lower concentrations compared to IgA.

Figure 3.

Levels of immunoglobulins (Igs) in saliva according to age. Correlation coefficients: IgM R2 = 0.04, IgG R2 = 0.07 and IgA R2 = 0.26.

In the first months of life, when the IgA production is starting, the proportion of polymeric IgA was high in serum for both IgA1 and IgA2 (Fig. 4). When the children are older, the percentage of polymeric IgA in serum decreases for both IgA1 and IgA2.

Figure 4.

Percentages of polymeric immunoglobulin A1 (IgA1) and IgA2 in serum in infancy. Normal values for children (age 2–9 years): pIgA1, 21% (SD 8.9); pIgA2, 41% (SD 11.2). Correlation coefficients: IgA1 polymers R2 = 0.29 and IgA2 polymers R2 = 0.47.

In saliva, nearly all IgA was polymeric, both in IgA1 and IgA2, from the start and remained so during infancy and childhood (data not shown).

Discussion

The serum concentrations of IgA1 and IgA2 as well as the salivary output of these isotypes were, not surprisingly, found to increase during childhood. IgA synthesis started shortly after birth, as revealed both in serum and saliva, according to previous reports [1, 17]. The production of both IgA subclasses appeared to be initiated simultaneously. A change from IgM to IgA started shortly after birth in serum for both IgA1 and IgA2. This is in contrast with IgG subclasses, where the serum levels of IgG1 and IgG3 develop first but IgG2 levels remain very low during the first year of life. In individual saliva samples, the secretion rate of IgA1 or IgA2 could be high, even in children with low serum IgA1 or IgA2, especially in young children (Fig. 2).

The serum IgA1/IgA2 ratio increased only slightly during childhood. The variability was seen in the first year of life, when IgA values are very low. If the youngest children, i.e. under 1 year of age were excluded, this ratio remained more or less constant. The salivary IgA1/IgA2 ratio, however, decreased slightly during this period. In a study of IgA-producing cells in parotid glands in fetal and postnatal material from children aged 1 day to 3 months, a rapid increase of IgA2-positive cells was demonstrated during this period [18]. In our study, the trend of the salivary IgA1/IgA2 to decrease is less obvious when omitting the youngest children. We can therefore conclude that no clear changes in IgA1/IgA2 ratios can be found during childhood after one year of age, for neither serum nor saliva.

In our study, IgA1 predominated in saliva, but the range was wide (Table 1). The secretion rate of both IgA1 and IgA2 increased with age. However, there was a temporary increase in secretion rate of IgA1 at the age of 3–6 months (Table 1). This might also be true for IgA2, and there was a peak for IgM as well. It is of interest that, during this period, infections are common. These infections are mainly of the respiratory and gastrointestinal tract. In addition, breast feeding is frequently decreasing in this time and new nutrition and proteins are being introduced. Moreover, immunizations usually start in this period. It is tempting to connect the rise in IgA in saliva with these alterations. Wan and coworkers [19] described an increase in IgA in unstimulated whole saliva associated with introduction of solid food as well as tooth eruption. Solid food did not stimulate a rise in IgM or IgG. In his study, mean IgA increased on eruption of the primary first incisors and the primary first molars. IgM increased after the eruption of the primary first molars. Tooth eruption leads to the development of a gingival crevice through which plasma-derived Igs may emerge and then mix with those secreted by the salivary glands. In our study, we investigated stimulated whole saliva. The peak in IgA and IgM in the present study was also found at month 3–6. At that time, eruption of the first teeth is possible but not of the first molars. The first solid food is usually not introduced in this period.

A higher secretion rate of IgM, IgG and IgA in saliva after the age of 6 years was noted in some children (Fig. 3). This might be connected to a mix of salivary and plasma-derived Igs due to gingival crevice [11, 19].

In a previous study of the group of Cripps [17], 72% of infants aged 6 weeks to 2.5 years had secretory IgA in their unstimulated salivary secretions, whereas 28% had monomeric IgA. In our study and in the study of Smith [20], only polymeric IgA was found in saliva; the difference between the results may be due to technical differences [20] and difficulties in standardization discussed in the Materials and methods.

During infancy, most IgA is polymeric in serum. A considerable variation in levels of IgA subclasses was demonstrated between the children, but all children below the age of 6 months had high percentages of polymers in both subclasses. A high proportion of polymeric IgA in serum in infants (6–240 days, mean 83 days) has earlier been described by Delacroix et al. [10]. Absorption of IgA dimers from the intestines has not been demonstrated in infants [21]. The polymeric IgA in serum must therefore be synthesized by the child and not be due to resorption of breast milk IgA.

The predominance of polymeric IgA antibodies specific for several microbial antigens in sera of systemically immunized or infected individuals has been reported [9]. In the initial response to rubella, measles and varicella-zoster virus-specific IgA first appears as polymeric IgA and later becomes or is replaced by monomeric IgA [22]. In infection with Campylobacter jejuni, polymeric IgA antibodies accounted for most of the anti-C. jejuni activity at the peak of the IgA response but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time [23]. After parenteral immunization with tetanus toxoid (TT), 5–20 years after a boost, both monomeric and polymeric components of IgA were observed. At the peak response, polymeric IgA accounted for approximately half of the anti-TT activity. However, polymeric IgA antibodies rapidly disappeared from the serum over a few weeks, whereas the serum monomeric IgA antibody response was maintained over a prolonged period of time [24].

Although, typically, the form of IgA in antigen-specific systemic responses to protein antigens is predominantly polymeric in sera of patients shortly after exposure and shifts to monomeric form in sera obtained several weeks later, the form of IgA in response to each pneumococcal polysaccharide remained predominantly polymeric 1 month after natural infection and up to 1 year following vaccination [25].

Altogether, these findings suggested that the predominance of polymeric serum IgA in infants may reflect frequent exposure to new antigens. Our study showed that the proportion of polymeric IgA decreased, when the concentration of IgA increased (Fig. 4). As in infants, with only small amounts of IgA, a high percentage of polymeric IgA antibodies might enhance protection against microorganisms. Activation of mannan-binding lectin (MBL) pathway by IgA is most prominent for polymeric IgA [26]. Binding of MBL to IgA induces complement activation and activation of innate immunity.

The development of the IgA system in infants and children can be of major importance with regard to infections, development of allergy and immunizations, in particular, when considering mucosal vaccination. The development of subclasses of IgA, both in serum and in secretions, as well as the development of polymer and monomer productions of IgA needs more attention in order to understand the immune response in children better.

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