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Home , Immunoglobulin D, Isotype (immunology)

Clin. exp. Immunol. (1985) 60, 654-660.

Light chain types of IgD in human and serum

J. A. VAN NIEUWKOOP * & J. RADLt *Department ofImmunohaematology and Bloodbank, University Hospital, Leiden and tTNO Institutefor Experimental Gerontology, Rijswyjk, The Netherlands (Acceptedfor publication 24 January 1985)

SUMMARY One of the unexplained features of human IgD is its preferential expression with either kappa or lambda light chains in different situations. While the membrane IgD on B shows a predominance of the kappa type, about 90% of all known IgD myeloma and 87% of normal IgD producing plasma cells in spleens of healthy individuals were shown to belong to the lambda type. Very little is known of the kappa/lambda light chain distribution of normal polyclonal IgD in the serum and in the bone marrow plasma cells. In this study, the kappa/lambda representation of IgD in bone marrow plasma cells and in the serum of 25 adult persons (two healthy and 23 suffering from various nonmalignant diseases) was investigated. The kappa/lambda ratio of IgD+ bone marrow plasma cells showed a large variation among the individuals ofthis group, in 84% of the cases being below 1 0. While about 1/3 of the investigated subjects had 80% or more of IgD of the lambda type (kappa/lambda ratio below 0 2), most showed a kappa/lambda ratio of IgD higher than that, with four persons exhibiting a clear cut predominance of IgD of the kappa type. A positive correlation (Spearman's correlation co-efficient, P=0 005) between the percentages of IgD+ plasma cells and their kappa/lambda ratio was found. Semiquantitative evaluation of the kappa/lambda composition within the serum IgD by immunoselection was in agreement with the kappa/lambda ratio of IgD+ plasma cells in all individual cases.

Keywords IgD light chain types serum IgD bone marrow IgD

INTRODUCTION

IgD, a predominant membrane immunoglobulin on B lymphocytes but a very minor component of the serum pool, represents one of the unexplained and controversial issues in . IgD is different from other immunoglobulins in structure, resistance to enzymatic treatment, biological site and behaviour in the ; its function as a membrane receptor as well as a secreted remains largely obscure. Another strange feature of IgD concerns its preferential expression of either kappa or lambda light chains in different situations. While most membrane IgD was found to be of the kappa type (Rowe et al., 1973; Johnston et al., 1982; Ligthart, Schuit & Hijmans, 1985), about 90% of all known IgD myeloma proteins have been shown to contain lambda light chains (Fine et al., 1974; Jancelewicz et al., 1975; Kyle and Bayrd, 1976; Fibbe & Jansen, 1984). Also, normal IgD producing plasma cells from spleens of healthy individuals showed a definite predominance of lambda light chains (Pernis, Governa & Rowe, 1969). Very little is known ofthe kappa/lambda light chain distribution ofnormal polyclonal IgD in the serum and in the bone marrow plasma cells. Correspondence: Mrs J. A. van Nieuwkoop, Department of Immunohaematology and Bloodbank, University Hospital, Leiden, The Netherlands. 654 IgD types in bone marrow and serum 655 In this investigation, we have demonstrated that most individuals with normal serum IgD levels and normal numbers of IgD positive plasma cells in the bone marrow produce IgD of predominantly lambda type, with, however, large variation. Persons with increased serum levels and increased numbers ofbone marrow plasma cells ofthe IgD often show a shift in the IgD kappa/lambda ratio towards the kappa type.

MATERIALS AND METHODS

Persons under investigation. Twenty-five adult patients ofthe University Hospital in Leiden were selected for this study because ofthe fact that their bone marrow and serum samples were available and because the bone marrow preparations contained enough IgD positive plasma cells to be investigated (Table 1). Their clinical diagnoses were classified arbitrarily into the following four groups. (1) Hyperimmunoglobulinaemia D and periodic fever syndrome (van der Meer et al., 1984) (three patients). (2) Diseases in which the is presumably not involved, such as diabetes mellitus, von Willebrand's disease, malabsorption syndrome, polyneuropathy and ischaemic heart disease (14 patients). (3) Non-malignant diseases in which the immune system might potentially be involved, such as rheumatoid arthritis and vasculitis (six patients). (4) Healthy persons selected as bone marrow donors for a family member suffering from aplastic anaemia or acute myeloid leukaemia (two persons). Malignancies of the immune system such as , Waldenstr6m's macroglobulinaemia and non-Hodgkin lymphoma were excluded from this study. Serum samples. All serum samples were processed at less than 1 h after venipuncture; because of the high susceptibility of IgD to degradation during storage by proteolytic enzymes present in the serum (Skvaril & Radl, 1967), 8-aminocaproic acid was added to a final concentration of 0.5% (Skvaril & Griinberger, 1962) before storage at - 20'C. All sera were tested by agar electrophoresis according to Wieme (1965) and by immunoelectrophoresis (IEP) using an oligospecific antiserum to IgG, IgA, IgM and IgD classes and both kappa and lambda light chain types (Nordic Immunological Laboratories). An immunoselection technique (IS) for IgD (Radl, 1970, 1972) was carried out in order to exclude IgD paraproteins and to assess the kappa/lambda light chain representation within the heterogeneous IgD. Quantification of IgD was done by single radial immunodiffusion according to Mancini, Carbonara & Heremans (1965) in Behringwerke Partigen Plates (Hoechst-Behringwerke, Marburg/Lahn, FRG). The IgD levels were expressed in iu/ml and in mg/dl (Rowe, Anderson & Tackett, 1970) and values exceeding 15 mg/dl were considered as increased (Buckley & Fiscus, 1975). If a monoclonal component of any isotype was detected or suspected by employing the above techniques, the serum was also tested by (IFIX) in a modification described elsewhere (Radl, 1981). Bone marrow cells. Cytocentrifuge slides were prepared according to Hijmans, Schuit & Klein (1969). These preparations contain comparable numbers of nucleated cells equally distributed over the same area and are therefore, suitable for comparative studies. After fixation for 15 min in 5% acetic acid in ethanol at -20°C and washing, a set of five slides was stained with FITC labelled antisera against each of the five immunoglobulin classes in order to calculate the percentage of plasma cells positive for these isotypes. Two more slides were stained with TRITC antisera to kappa and lambda light chains, respectively, and counterstained with FITC labelled anti-IgD serum for the calculation of the light chain type distribution within the IgD isotype. At least 50 positive cells were scored in most of the specimens in both the kappa IgD and in the lambda IgD slides. Only in five patients with a low number of IgD containing cells was less than 50 but more than 25 cells per each light chain type counted. Because of insufficient data on the normal frequency of IgD+ bone marrow plasma cells among those of all isotypes, a provisional upper limit was set at 10% on the basis of the mean value plus two standard deviations as determined in a group of 66 adult patients without an overt immune system disease. All conjugated antisera were shown to be specific by performance testing according to Schuit, Moree van der Linde & Hijmans (1981). The slides were evaluated by direct immunofluorescence (IF) with a Zeiss standard microscope equipped for selective visualization of TRITC and FITC. 656 J. A. van Nieuwkoop & J. Radl Antisera used in the different techniques. Goat anti-IgD-FITC (IF): Cappel Laboratories Inc., Downington, Pennsylvania, USA. Sheep anti-IgE-FITC (IF): Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam, The Netherlands. Rabbit anti-kappa and anti-lambda light chains (IFIX), rabbit anti-IgM-FITC (IF): Dakopatts, A/S, Copenhagen, Denmark. Sheep anti-fab fragments of IgG and goat anti-IgM (IFIX), rabbit anti-kappa and lambda light chains, and rabbit anti-IgD (IS): TNO Institute for Experimental Gerontology, Rijswijk, The Netherlands. Burro anti-kappa and anti-lambda TRITC and goat anti-kappa FITC (IF): Kallestad Laboratories Inc., Chaska, Minnesota, USA. Rabbit anti-Ig (GAMD, kappa and lambda) (IEP), goat anti-IgG, goat anti-IgA (IFIX), goat anti-IgG-FITC, goat anti-IgA-FITC (IF): Nordic Immunological Laboratories, Tilburg, The Netherlands. Statistics. The Spearman rank order correlation coefficient was used to assess the significance of differences between the percentages of IgD positive plasma cells in the bone marrow and their kappa/lambda ratios.

Table 1. Clinical and laboratory features of the individuals investigated

IgD in bone marrow IgD in serum Number Sex Age Diagnosist %IgD§ %K-%O) Ratio iu/ml mg/dl 1 F 20 1 81 76-24 3 17 5,300 757 2 F 34 4 43-3 61-39 1 56 383 55 3 F 31 1 36 59-41 1 44 1,383 198 4 M 64 2 33.8 44-56 0-79 240 34 5 M 33 2 28 9 31-69 0 45 NS NS 6 M 18 1 28 77-23 3-35 674 96 7 M 29 4 27-8 30-70 0-43 238 34 8 F 48 3* 20-4 40-60 0-67 188 27 9 F 48 2* 14 5 8-92 0-09 225 32 10 M 43 2 8-9 10-90 0 11 240 34 11 F 41 2 7.8 48-52 0 92 159 23 12 M 53 2* 7-2 28-72 0 39 31 4 13 F 57 3 6-6 9-91 0 10 NS NS 14 M 37 3* 50 13-87 015 40 6 15 M 78 2 4-8 48-52 0-92 < 20 < 3 16 M 80 2 4-7 13-87 0-15 <40 <6 17 F 60 2 4 0 28-72 0 39 NS NS 18 F 68 2* 34 16-84 0.19 61 9 19 F 54 2* 3-2 33-67 0-49 < 20 < 3 20 M 63 2* 3-2 6-94 0-06 44 6 21 M 43 2 30 10-90 011 NS NS 22 F 56 2* 2-9 41-59 0 69 27 4 23 F 67 3 1.9 21-79 0 27 NS NS 24 F 53 3* 1.9 10-90 0 11 28 4 25 M 55 3* 1-4 29-71 0 41 51 7

t See text. * Individuals with a serum H-Ig component (IgM or IgG) of a very low concentration. § Percentage of all immunoglobulin isotypes. NS = Not shown because of long intervals between serum and bone marrow sampling. IgD types in bone marrow and serum 657 RESULTS

Serum agar electrophoresis showed no abnormalities except for single or multiple homogeneous Ig components at very low concentrations in the sera of 10 patients (four ofthem in diagnostic group 3, six in group 2). Immunofixation demonstrated those components to belong to the IgM (two times) or the IgG (eight times) isotypes; none of these small homogeneous Ig were of the IgA or IgD isotypes. Of these 10 patients, eight had a normal Ig serum level and two showed a moderate hyperimmunoglobulinaemia. All 10 patients had a normal isotype distribution of the bone marrow plasma cells. The results of the determination of the serum IgD level, the percentage of IgD positive plasma cells in the bone marrow and the kappa/lambda light chain distribution within these IgD+ plasma

+ K-SP - L-SP

...... ~ ~~~~~~~~~~~Ril ...... ii'.-. /IgD

Fig. 1. Example of the immunoselection technique (IS) performed in sera ofpatients No. 1 (2) and No. 3 (1) (see Table 1). In the kappa selection plate (K-SP), an antiserum to human kappa light chains is incorporated into the agar. During electrophoresis, all immunoglobulins of the kappa type from the tested serum are precipitated within the central cigar like precipitate. The anti-IgD serum applied in the throughs will therefore form a line only with IgD of the lambda type. The same principle applies to the lambda selection plate (L-SP). The IS pattern in the two patients proves a normal heterogeneity ofthe IgD and indicates a slight (1) or pronounced (2) predominance of the kappa type IgD.

100 _ 90 _ 80 _ o 70 ai) a- 606 0 o 50 o 40 ac) c 30 _ 20 S 10 50 02 06 1 0 4 8 22 26 30 34 KIA ratio of intracytoplasmic IgD Fig. 2. The kappa/lambda ratio of IgD positive plasma cells in the bone marrow of25 persons as determined by immunofluorescence. 658 J. A. van Nieuwkoop & J. Radl cells from the entire group are given in Table 1. The first nine persons had increased numbers of IgD+ plasma cells in the bone marrow. All ofthese as well as two others also had an increased serum IgD level. In all cases, the serum IgD was normally heterogeneous and contained both light chain types as demonstrated by immunoselection (Fig. 1). The kappa/lambda ratio within the IgD+ bone marrow plasma cells showed a large variation among the different individuals. While most ofthe persons showed a predominance oflambda light chain type, there was a clear cut shift towards the kappa type in some of the patients, especially in those having high numbers of IgD+ plasma cells and hyper IgD immunoglobulinaemia (Fig. 2 & Table 1). The Spearman rank order correlation coefficient showed a significant correlation between the increase in the IgD+ bone marrow plasma cells and the increase in their kappa/lambda ratio (P=0 005). Semi-quantitative evaluation by immunoselection of the kappa/lambda light chain representation within the serum IgD was in agreement with the kappa/lambda ratio determinations of IgD+ bone marrow plasma cells in all individual cases.

DISCUSSION

Human IgD immunoglobulin was discovered in 1965 (Rowe & Fahey) as a myeloma protein and several IgD myelomas have since been described (Fine et al., 1974; Jancelewicz et al., 1975; Kyle & Bayrd, 1976; Fibbe & Jansen, 1984). In these reports, a pronounced predominance (about 90%) of the lambda light chain type of the IgD protein was a main feature. Also, normal IgD producing plasma cells in six human spleens were shown to contain IgD consisting of 87% of the lambda light chain type (Pernis et al., 1969). Membrane bound IgD (m-IgD) is associated with IgM in the vast majority of B cells (Rowe et al., 1973; Knapp et al., 1973); less often, it is found in association with IgA or rarely with other isotypes (Vessiere-Louveaux, Hijmans & Schuit, 1980). Only about 5% of B cells have m-IgD as the only membrane immunoglobulin. For this reason, exact typing of the light chain type of cells bearing m-IgD alone is very difficult and most data therefore concern B cells that carry IgD along with other immunoglobulins, mainly IgM. The investigators agree, however, that all multiple m-Ig isotypes on the same cell have the same light chain type and that the kappa/lambda ratio is always greater than 10, as a result of the predominance of kappa over lambda light chains (Rowe et al., 1973; Johnston et al., 1982; Ligthart et al., 1985). On the basis of clinical and laboratory observations showing that the frequencies of malignancies of the IgG, IgA and IgM isotypes and their kappa/lambda representation reflect a similar distribution within the normal serum immunoglobulin pool, it was assumed that normal IgD would consist of about 90% of the lambda type. The differences in the kappa/lambda ratio between the m-IgD and the cytoplasmic IgD were suggested to be possibly due to the existence of different subclasses of IgD (Spiegelberg, 1977). In our investigation, we found that the kappa/lambda ratio of IgD in bone marrow plasma cells and accordingly also in serum showed a large variation among 25 adult persons who were either healthy or were suffering from various diseases. In the entire group, only about 1/3 of the individuals had 80% or more of IgD of the lambda type. Most of the investigated showed a kappa/lambda ratio of IgD higher than 0-2, with four persons having a clear cut predominance of the kappa type within their IgD. Interestingly, the higher the percentage of IgD+ bone marrow plasma cells found, the higher was usually the kappa/lambda ratio. None ofthe increased IgD levels were due to monoclonal IgD production, as shown by the immunoselection technique; in all cases, the IgD of either kappa or lambda type was heterogeneous. The question, whether the kappa/lambda ratio within the IgD as found in our group reflects its normal distribution is difficult to answer. There is too little available information on the function of IgD as an antibody or on its potentially specific involvement in a disease. On the newly described Hyperimmunoglobulinaemia D and periodic fever syndrome (van der Meer et al., 1984) is suggestive for a special role ofIgD in the pathogenesis ofthis disorder. It is interesting in this respect that all three patients suffering from this disease showed a high kappa/lambda ratio. All the other persons represent a heterogeneous group, healthy or with various diseases with no clear cut IgD types in bone marrow and serum 659 relationship with the immune system. In some individuals, one may suspect a very mild form of . This may be the case in those patients having small homogeneous immunoglo- bulin components (H-Ig) of IgM and IgG isotypes. It has been shown that these kinds of monoclonal gammapathies often represent the first symptoms of an age related immunodeficiency (Radl, 1982, 1985). It has also been observed that hyper-IgD immunoglobulinaemia accompanies some forms of (Buckley et al., 1975; Radl, Masopust & Lackova, 1967; unpublished observations). However, except for the two patients with an increased IgD and a H-Ig component of, respectively, IgM and IgG isotypes, all other patients with H-Ig had serum IgD concentrations within normal limits and their kappa/lambda ratio of IgD did not differ substantially from that ofindividuals in the same diagnostic groups but without a H-Ig component. No parameters (such as age, sex, diagnosis, presence ofH-Ig) other than an increased number of IgD+ bone marrow plasma cells and hyper-IgD immunoglobulinaemia showed any clear cut correlation with either a high or low kappa/lambda ratio of IgD. Therefore, we assume that the lambda type preponderance with a large variation within the kappa/lambda ratio of IgD found among the individuals in our group reflects more or less also an actual wide range in which normal polyclonal IgD can be combined with either of the two light chain isotypes. We have no plausible explanation for the increasing trend in the kappa/lambda ratio in cases with increased IgD production. This may reflect differences in biological regulation or a selective proliferative advantage due to preferential combination of IgD kappa with certain . Much deeper knowledge of the biology of IgD and its function as an antibody is necessary to reveal the significance of its different light chain isotype combinations in different situations.

Thanks are due to Mrs H. R. E. Schuit for the specificity testing on the conjugates, Dr J. d'Amaro and Dr Th. Stijnen for advise in the statistical analysis, Dr A. Brand, Dr G. J. Ligthart and Mrs H. R. E. Schuit for critical reading of the paper and Ms M. Roggenkamp for typing the manuscript.

REFERENCES

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