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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Serum immunoglobulinD (IgD) concentration is usually low in healthy individuals as compared to other immunoglobulin classes. Most studies on serum IgD are concerned with serum levels in healthy adults but reference values for young children and infants are not easily available. In order to establish age specific reference values we measured IgD levels in serum of 184 healthy Icelandic children, age 0–14 years and 60 healthy blood donors age 18–63, using the ELISA technique. Special attention was paid to the youngest age groups. Results showed low IgD values in infants and young children, gradually increasing until the age of 10 but then decreasing with age. We conclude that IgD gradually increases with age in childhood as other immunoglobulin classes but later declines. These findings can be of importance in revealing the function of IgD in the immune system as well as in the diagnosis of the hyper-IgD syndrome.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The IgD was first described in 1965 [1] and later determined on the lymphocyte surface [2, 3]. The delta heavy chain was completely sequenced in 1985 [4]; the molecule is 184 kDa and has a serum half-life of 3 days.

The role of IgD in the immune function is still obscure. Regulation of the synthesis still remains uncertain but it has been argued that an independent mechanism, possibly genetically determined, is important in the basal production [5]. Correlation has also been found between serum IgD levels and concentrations of IgA and IgE in serum [5] indicating that the same factors may influence the production of these immunoglobulins [5]. Little is known about individual variation in the serum levels of IgD. It has been suggested that IgD play a role in immunological memory [6[7]–8] and in immune regulation [9–10]. This molecule is therefore of special interest in children when the immune system is developing as well as the immunological memory.

In 1984 the hyperimmunoglobulinaemia D and periodic fever syndrome was first described [11]. This interesting syndrome is now usually referred to as the hyper-IgD syndrome. The clinical findings of hyper-IgD syndrome are recurrent attacks of fever, enlarged lymph nodes, abdominal pain and diarrhoea with frequent skin lesions and joint involvement [11[12]–13]. The serum level of polyclonal IgD is elevated in the hyper-IgD syndrome [11[12]–13]. However, the role of the elevated serum IgD in the syndrome remains uncertain. A correlation has not been found between the elevated serum IgD and the severity or frequency of attacks [13[14]–15] and clinical symptoms of hyper-IgD syndrome may herald the rise in serum IgD [14–15]. The serum levels of other immunoglobulins are sometimes also affected, especially IgA and IgG3 [13[14]–15] as well as the immunoglobulin light chain ratios [14]. This may indicate a more complex pathophysiology. Recently, mutations in MVK, encoding mevalonate kinase, has been reported to cause hyperimmunoglobulinaema D and periodic fever [16–17].

Until now, age specific reference values, especially for infants and young children have not been easily available [18]. It has therefore been difficult to establish the diagnosis of hyper-IgD syndrome in children and infants. The aim of the current study was to establish reference values of serum IgD with special emphasis on infants and children for further studies of IgD.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Patients

One-hundred-and-eighty-four children, aged 0–14 years, were enrolled in the study. They were admitted to the pediatric departments of the University Hospital and St. Joseph's Hospital in Iceland for elective minor operation or medical examinations. Only when blood was drawn for other reasons were the children included in the study. Children with a medical history of present or previous recurrent infections, other acute or chronic illnesses or syndromes, with suspected or proven other disorders were all excluded from the study. Children with a history of asthma, allergy, atopic diseases, any suspected immunological disorders or gluten enteropathy were also excluded. The children enrolled were considered healthy. Informed consent was obtained from the parents for enrolment in the study. In addition serum samples were collected at the Icelandic Blood bank from 60 healthy blood donors age 18–63 years and measured anonymously.

Methods

Serum was drawn from each individual at the time of enrolment. The serum samples were kept frozen until measured. The samples were measured at the University of Nijmegen. The enzyme linked immunosorbent assay for the measurement has been published earlier [19]. Briefly, microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4 °C with rabbit antihuman IgD (200 mul, Dako, Copenhagen, Denmark; 1 : 5000 dilution in bicarbonate buffer, pH 9.5). After automatic washing of the plates with phosphate buffered saline (PBS) containing 0.01% Tween 20 (washing buffer), serum samples or standard serum dilutions (100 mul) diluted in washing buffer were added. These were also incubated overnight at 4 °C. After washing, the plates were incubated for 90 min at 37 °C with mouse monoclonal antihuman IgD recognizing the Fc-part of the IgD molecule (Dako; 100 mul 1 : 200 in washing buffer). After washing, Horse-Radish-Peroxidase labelled rabbit antimouse immunoglobulins were added (Dako; 100 mul 1 : 4000 in washing buffer) and incubated for 90 min at 37 °C. The plates were washed again and incubated with substrate containing orthophenylenediamine (Sigma, St Louis, MO, USA; 2 mg/ml) and H2O2. After stopping the reaction with 2 m H2SO4 absorbance was read at 492 nm, using a Titertek Multiskan ELISA reader (Eflab, Oy, Helsinki, Finland). As the standard serum, Behring OTRD 02/03 (Marburg, Germany) calibrated against the British Research Standard 67/37, was used. This standard serum contains IgD-Fc fragments. In this ELISA, only Fc-regions are measured and standardization is also performed on the Fc-regions. Presumably therefore, no influence of the notorious splitting of IgD molecules has been found in this assay. Even after storage for 24 h at room temperature still the same values were measured. The lower limit of detection of this ELISA was 1 IU/ml (1.4 mg/l). Special effort was made to read values as low as 0.5 IU/ml. Statistical methods. The nonlinear regression model a(1-e−b age)/(age + c) was adapted to the log of the IgD data against age by the SPSS-statistical package. The parameters a and b were evaluated for a few values of c. The value c = 20 gave the best fit with the model. The corresponding estimates of a and b with their 95% CI were a = 138.7 (122.4–155.0), b = 0.17 (0.13–0.21), c = 20. A change of c by, e.g. 2 units (± 2 units) gives estimates for a and b which are well within the respective confidence intervals given. The variation in the log of IgD explained by this model is 53% of the total variation. According to this model the predicted means of the IgD concentration, at each age, and its 95% percentile was calculated. IgD values below the detection limit, 0.5 IU/ml, of our method were given the value 0.25 IU/ml.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

One-hundred-and-eighty-four children aged 0–14 years were enrolled in the study and 60 healthy adults.

Twenty-three children were less than 6 months of age including five cord blood samples. Twenty-one children were aged 6–12 months and 16 children 1–2 years. The age distribution is shown in Figs 1 and 2.

image

Figure 1. 4 years and 60 healthy adults. Graph indicate geometric mean and 95% percentile at each age.

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image

Figure 2. . Serum IgD values of 91 children less than five years of age (closed marks) and serum IgD values of four different patients with hyper IgD syndrome, followed up for several years (open marks). Some patient values fall above the upper margins of the figure.

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The serum IgD levels were low or not detectable in the youngest children and only three children under the year of five had levels above 50 IU (Fig. 2). The concentration increased gradually with age in the population studied during the first years of life (Figs 1 and 2). At the age of 10, the serum concentration was highest. After that age, the serum values gradually decreased (Fig. 1).

The IgD concentrations according to age groups are given in Table 1.

Table 1.  . Geometric mean and 95% percentile for serum IgD-levels according to different age groups (calculated only from the IgD values from the respective age groups) * The age intervals do not include their right margins.Thumbnail image of

Two children had serum IgD levels above the 95th percentile. These children had no clinical symptoms of hyper-IgD syndrome. Five adults had serum levels above these reference values. No clinical data are available on these individuals but they are considered healthy blood donors.

Five patients with the hyper-IgD syndrome who have been published earlier [14–15] showed IgD values above the new reference values in early infancy or childhood (Fig. 2).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

We have studied the serum levels of IgD in healthy children and adults. We maintain that age matched reference values for infants, children and adults are needed to evaluate the IgD and IgD function in children as well as to diagnose the hyper–IgD syndrome in children.

The serum levels of IgD have been described [21[22][23]–24]. Joseph's et al. described mean values of 0.22 IU/ml in premature infants, 0.14 IU/ml in neonates, 13.65 IU/ml in the age group 1–20 years and at last 11.78 IU/ml in healthy adults [21]. The study was done by using the double-antibody RIA. No further discrimination between different age groups was made. Zegers et al. described IgD values in patients with immune disorders and in healthy children [24]. Their results are in context with our findings, however, they only described children older than 4 years of age. Other authors have also described low IgD values in children without any age distribution or in patients with other immune deficiencies [22[23]–24].

Our results of increasing IgD concentration with age are in context with those of Siwinska-Golebiowska et al [18]. In that study [18], the Mancini method was used whereas we used the ELISA technique. Storage of sera and repeated freeze-thawing did not alter the IgD levels in our samples as was shown by storage experiments (data not shown). Theoretically, the IgD ELISA used is unlikely to be influenced by the reported degradation of IgD in Fc and Fab fragments [20] as only Fc-regions are being measured and standardization is performed on IgD-Fc fragments. Studies using the Mancini technique are possibly more sensitive to storage and sample handling than the ELISA method. Differences between our results and those of Siwinska-Golebiowska [20] are small and might be owing to methodological differences. In the study by Siwinska-Golebiowska the variance was greater as compared to our results.

The function of IgD in the immune system still remains uncertain. The low serum IgD values in children are of special interest in this context. The receptor for IgD has been demonstrated on T cells [9, 10] suggesting a role for IgD in the immune regulation. In vitro studies have also revealed that additional IgD influences the immune response [6]. It has also been pointed out that serum levels of IgD are higher in healthy adults with the HLA-DR1 phenotype [25]. A number of studies have suggested a function for the IgD in immunological memory and an immunological regulation [7[8][9]–10]. This, of course, is of particular interest in children when the immunological memory is developing. In contrast to this, animal studies have shown that mice lose the ability to express the IgD receptor on their cell surface after stimulation with increasing age [26]. These findings are of particular interest in the context of earlier reported data on decreasing levels of IgD concentration with age in humans [27]. Although our study did not concentrate on older age groups, a trend towards decreasing values in older individuals was noted.

The hyper-IgD syndrome awards special attention. The onset of this syndrome is usually at a young age [13[14]–15]. It has also been pointed out that clinical symptoms may precede the rise in serum IgD-levels and children may have symptoms for years before an elevation in the serum levels [14–15]. Moreover, the frequency or severity of attacks are not related to the serum IgD levels [13[14]–15]. In addition, the serum levels of other immunoglobulins are sometimes also affected in the syndrome, especially that of IgA and IgG3 [13[14]–15] as well immunoglobulin light chain ratios [14].

To diagnose the hyper-IgD syndrome in children, reference values are necessary and our results may be very helpful. We have earlier published data on children with the hyper-IgD syndrome, presenting in early childhood or infancy with clinical symptoms of the disorder. The serum IgD values of these patients are clearly raised from early childhood. The diagnosis in these children could have been confirmed earlier if age-matched reference values had been available at that time, confirming the need for such values.

Substantial research has been devoted to IgD and it's function in the immune system. However, the functions of this interesting molecule still remain uncertain. Future studies will hopefully reveal the exact function for IgD in children as well as in adults.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

A grant was received from the University Hospital Research Fund. Serum samples from adults were collected anonymously by the Icelandic Blood Bank.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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