Clin. exp. Immunol. (1985) 60, 654-660. Light chain types of IgD in human bone marrow 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 lymphocytes shows a predominance of the kappa type, about 90% of all known IgD myeloma proteins 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 antibody pool, represents one of the unexplained and controversial issues in immunology. IgD is different from other immunoglobulins in structure, resistance to enzymatic treatment, biological site and behaviour in the immune response; its function as a membrane receptor as well as a secreted protein 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 isotype 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 immune system 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 multiple myeloma, 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 immunofixation (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.