Cross-reactivity of human and a putative pig IgD. pig IgD-like molecules as serum and lymphocyte components

Proteins of pig serum and splenocytes which were isolated on immobilized guinea pig antibodies against human IgD were characterized by SDS-PAGE. In both cases the main isolated components possessed several properties nearly identical with those of human IgD: molar mass of the components and their chains, and susceptibility of the heavy chains, light chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains, the chymotryptic peptide maps of the chains, and susceptibility of the heavy chains in the native molecule to trypsin digestion. These IgD-like components not adsorbed on immobilized normal guinea-pig IgG. We suggest that the components of pig serum and splenocytes cross-reacting with highly specific antibodies against human IgD represent candidates for pig IgD. The percentage of the IgD-like molecules on the surface of pig splenocytes labeled with the antibody against human IgD was similar to the percentage of splenocytes bearing all immunoglobulins. Therefore, we suggest that IgD-like molecules were occurring on the surface of cells mostly together with molecules of another immunoglobulin class.     

Classes of rat lymphocyte surface immunoglobulins detected by enzymatic radioiodination]

By means of enzymatic radioiodination, immunopre-cipitation and acrylamide gel electrophoresis in sodium dodecyl sulfate it was found that on the surface of normal rat splenocytes there were two main immunoglobulin classes--monomeric IgM and Ig (H2L2) which had a heavy chain larger than they phi, yet smallerthan the micron chain, differing from them by the antigenic properties. It is possible that this Ig class corresponds to the human IgD and the mouse IgD-like cell surface molecules. Small amounts of thedetected cell surface IgG may indicate both its cytophilic properties and the immunological status of the experimental animals.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

The expression of the sIgD isotype in wild-derived mice

IgD and IgM are concomitantly expressed on the surface of most mouse B lymphocytes and both molecules serve as receptor for antigen. In this communication we report that in contrast to IgM, which is expressed in a constant manner on the surface of spleen B lymphocytes of different laboratory and wild-derived mice, IgD expression is variable among the spleen cells of wild-derived mice. SPE, SEI, and SFM mice belonging to the Mus 3 subgroup show a fluorescence profile characterized by a marked diminution in the population of B lymphocytes expressing the IgD isotype; in addition, these cells have a low sIgD density on their membranes. These findings were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the iodinated membrane proteins. Polyclonal in vitro activation with lipopolysaccharide increases the frequency of surface IgD (sIgD)-bearing spleen cells and sIgD density in the SPE strain but decreases both the frequency and the density of IgD bearing cells in the BALB/c strain. This result suggests that delta gene expression is regulated differently in SPE and BALB/c mice. In addition, genetic analysis of sIgD expression in (BALB/c X SPE)F1 hybrids suggests that the proportion of sIgD-bearing cells and sIgD density are independently regulated.     

Cell surface immunoglobulin XXI. appearance of IgD on murine lymphocytes during differentiation.

The differentiation of Ig-bearing lymphocytes in adult mice was studied by monitoring the appearance of IgD relative to IgM on the surface of splenocytes obtained from lethally irradiated animals reconstituted for various periods of time with adult bone marrow cells, neonatal splenocytes, or Ig- adult splenocytes. It was found that IgM appears before IgD on differentiating lymphocytes. Furthermore, the rate of appearance of IgD during differentiation of adult cells is similar to that observed with neonatal cells.     

Cell membrane IgD: demonstration of IgD on human lymphocytes by enzyme-catalyzed iodination and comparison with cell surface Ig of mouse, guinea pig, and rabbit.

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.     

Evidence for an IgD homologue on chicken lymphocytes

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.     

The ontogeny and distribution of B cells in normal and mutant immune-defective CBA/N mice: two-parameter analysis of surface IgM and IgD

A dual-laser fluorescence-activated cell sorter was utilized to study the distribution of the surface IgM and IgD on individual B cells of normal and immune-defective CBA/N mice. Cells from different lymphoid organs and from developing mice were studied. Two major populations of cells were seen. Those with low densities of surface IgM and intermediate-high densities of surface IgD were relatively or totally absent from the bone marrow, spleens, and lymph nodes of adult, immune-defective (CBA/N x DBA/2)F1 male mice, and developed late in ontogeny in the lymphoid organs of normal F1 female mice. By contrast, the second major population, with intermediate-high surface IgM and low surface IgD, was found in highest frequency in the lymphoid organs of immature mice, the bone marrow of adult mice, and the lymphoid organs of F1 male mice compared to F1 female mice at any age. These two major populations of B cells were further subdivided into five groups of cells to better define the surface IgM and IgD characteristics of developing B cells of immune-defective and normal mice. The relationship of these groups of cells to populations defined by other criteria are discussed.     

Interspecies cross-reactivity of bovine lymphocytotoxic sera with pig lymphocytes

Forty-three bovine BoLA antisera were tested on pig lymphocytes by a microlymphocytotoxicity test. Twenty-five were found to be cytolytic. Fifteen sera detected the A blood group antigen on porcine lymphocytes but showed no reaction with the J antigen on bovine lymphocytes. Six BoLA reagents reacted with all pig cells tested. Cross-reactions with SLA antigens were observed in only four sera, the highest correlation being recorded with SLA-W7 (r = 0.87). Bovine alloantisera are not of value for SLA typing.     

Studies on non-H-2-linked lymphocyte-activating determinants. IV. Ontogeny of the Mls product on murine B cells.

The minor lymphocyte stimulating (Mls) locus codes for lymphocyte activating determinants (LADs) on murine B lymphocytes, but not T lymphocytes. This observation was strengthened by a series of techniques which allow deletion and addition of T and B cells. These included the use of cytotoxic antisera such as anti-Thy 1.2, anti-MTLA, anti-MBLA, and complement, and the use of a goat anti-μ antisera, and finally the use of a fluorescence activated cell sorter (FACS).The studies in this report document the organ distribution and the ontogenetic appearance of the surface LADs on the surface of B lymphocytes from DBA/2N (H-2^d, Mls^a) and CBA/J (H-2^k, Mls^d) mice. Adult-like ability to stimulate H-2 identical BALB/c (H-2^d, Mls^b) and C3H/He (H-2^k, Mls^c) responder cells appeared at about 4–5 weeks of age. Inability of neonatal cells to induce an Mls-defined MLC was found not to be due to a low frequency of B lymphocytes or to the presence of suppressor cells, but due to the absence of the Mls-coded LADs on their surface. These data support the concept that the Mls-coded LADs are present on adult B lymphocytes and are specific markers of B-cell differentiation, which is preceded by membrane IgM and the δ homologue of human IgD, Ia, and the receptor for the third component of complement.     

Terminal differentiation of PWM-stimulated human B lymphocytes.

Patterns of surface and cytoplasmic immunoglobulins were simultaneously studied on human B blast cells induced by pokeweed stimulation of peripheral blood lymphocytes. A double-staining immunofluorescent technique was used. After 4 and 7 days of culture, a gradual loss of surface IgD was observed on blast cells whereas numbers of plasmablasts with cytoplasmic immunoglobulin showed a marked increase. After 7 days, 92% of surface IgA positive blasts had passed terminal differentiation to cytoplasmic IgA-producing plasma-blasts. At the same time, 73% of surface IgM positive blasts were found to contain cytoplasmic IgM, and 30% of surface IgG positive cells had cytoplasmic IgG. Only a small fraction of blast cells with surface IgD was able to mature to IgD producing plasmablasts. In general, the class of surface and cytoplasmic immunoglobulin coincided in single blast cells, with the exception of surface IgD which was present on 10% of the cytoplasmic IgM-containing blasts.     

Regulation of IgM and IgD synthesis in B lymphocytes. II. Translational and post-translational events

Studies investigating the relative rates of biosynthesis of mu- and delta-polypeptide chains in normal resting B lymphocytes have shown that the translation rate of mu m is about sevenfold higher than that of delta m, thus reflecting the relative abundance of mRNA encoding these two chains. The turnover rate of cell surface IgM is faster, however, than that of cell surface IgD, resulting in higher expression of cell surface IgD relative to IgM under steady state conditions. LPS stimulation of B lymphocytes induces the complete cessation of synthesis of the delta-chain, thus accounting for the gradual disappearance of IgD from the cell surface of activated cells.     

Presentation of myelin basic protein by murine cerebral vascular endothelial cells

Guinea pig basic protein (GPBP)-sensitized murine lymph node cell cultures depleted of macrophages-monocytes (MO) are no longer able to proliferate in response to specific antigen in vitro. The addition of MO reconstitutes the response that can be blocked by the addition of syngeneic anti-I-A antisera. Freshly isolated murine central nervous system endothelial cells (CNS-EC) cannot replace MO for the presentation of GPBP antigen to specifically sensitized lymph node lymphocytes. The pretreatment of CNS-EC with concanavalin A-conditioned media resulted in the expression of Ia molecules and the consequent ability to present GPBP. Antigen presentation by CNS-EC could be blocked by anti-I-A antisera for the CNS-EC donor haplotype.     

Isotachophoretic isolation of IgD and IgE and production of monospecific antisera against them]

The method of preparative isotachophoresis in acrylamide gel ensuring a high yield of IgD and IgE with insignificant admixtures of IgG, etc. was used for the isolation of IgD and IgE from the blood sera of myeloma patients. As a result of immunization with these antigens, monospecific IgD and IgE antisera were obtained. These antisera, alongside with specific antibodies, contained antibodies to admixtures; the latter were eliminated by the method of immune absorbtion carried out with the use of a sorbent based on Sepharose activated with bromo-cyanogen and conjugated with normal human blood serum. Ig D antisera were also shown to contain antibodies to idiotypical IgD determinants located in the Fab fragment of this immunoglobulin.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Structure and biologic functions of human IgD. XI. Identification and ontogeny of a rat lymphocyte immunoglobulin having antigenic cross-reactivity with human IgD.

Chicken anti-human IgD antiserum (anti-delta) has demonstrated an antigenically cross-reactive homologue on rat lymphocytes. IgD and IgM are the only cell surface immunoglobulins detectable by the lactoperoxidase radiolabeling technique employed. The results indicate that, although rat surface IgD is antigenically distinct from rat IgM, the respective H chains co-electrophorese in 10% polyacrylamide-SDS gels. Rat delta-chain has an apparent m.w. of 73,000 daltons and exhibits a minor 65,000 dalton component which probably represents a partially degraded delta-chain. The ontogenic emergence of rat IgD occurs approximately 3.5 weeks after birth whereas IgM, in contrast, is apparent by 6 days of age. Thus, as in the human, IgM develops before IgD. IgD receptors are undetectable in the thymus but are present in increasing levels in spleen, blood, lymph nodes, and Peyer's patches.     

Differences in glycoprotein complexes associated with IgM and IgD on normal murine B cells potentially enable transduction of different signals.

Studies presented here demonstrate that IgM and IgD molecules on normal murine B lymphocytes exist in different, noncovalently associated molecular complexes containing distinct but potentially related glycoproteins. The glycoproteins in these complexes, particularly those associated with IgD, show striking differences in various lymphoid organs and in X-linked immunodeficient (Xid) mice. These differences are due in part to post-translational processing. They apparently reflect the differential expression of the Ig-associated glycoproteins in the various B cell subpopulations and lineages and the differential distribution of the subpopulations and lineages in the various lymphoid organs. In addition, they reflect structural differences in the IgM and IgD complexes which, we suggest, permit differential signal transduction by IgM and IgD on the same B cell.     

Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes.

A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.     

Stimulation of guinea pig lymphocytes by Lens culinaris lectin-A. Stimulation by insolubilized LcL-A and effect of erythrocytes and peritoneal macrophages on stimulation by soluble LcL-A. Presence of a lymphokine in culture fluids of LcL-A-stimulated cells

Guinea pig lymphocytes in culture are mitogenically stimulated by either soluble LcL-A or by LcL-A covalently attached to Sepharose beads. The degree of stimulation by LcL-A-Sepharose is directly dependent upon the density of LcL-A molecules per bead ranging from essentially zero with low density to a better stimulation than by soluble LcL-A with high density. Addition of small numbers of either erythrocytes or peritoneal macrophages to lymphocytes cultured with suboptimal doses of soluble LcL-A increases stimulation significantly. With optimal or supraoptimal doses the potentiation effect disappears. These observations support the proposal that a stimulating lectin presented to stimulatable lymphocytes in high local concentrations is more effective than the same concentration of lectin molecules binding more randomly on the lymphocyte surface. During stimulation of human and guinea pig lymphocytes by LcL-A or LcL-A-Sepharose a soluble lymphokine is released which is either itself a lymphocyte stimulant or one which makes lymphocytes more stimulatable by low doses of LcL-A.     

Abnormal expression of surface immunoglobulin isotypes on antigen-binding B lymphocytes from mice tolerant to trinitrophenyl determinants

Temporary B-cell tolerance to the trinitrophenyl (TNP) hapten can be produced in BDF1 mice by intraperitoneal injection of trinitrobenzene sulfonic acid (TNBS). Antigen-binding cells (ABC) specific to TNP, measured as TNP donkey erythrocyte rosettes, are found in tolerant mice as well as in immune mice. We have studied the surface immunoglobulin isotype profile of these TNP-binding lymphocytes (TNP-ABC) in four groups of animals: nonimmune, immune, tolerant, and tolerant-challenged. Immune mice received intravenous TNP sheep erythrocytes (TNP-SRC), whereas tolerant-challenged mice received TNP-SRC and TNBS on Day 0. TNP-ABC from mice immunized with TNP-SRC exhibit increased expression of surface IgG and decreased expression of surface IgD, compared to the ABC from nonimmune mice. Tolerant mice have a higher proportion of ABC with surface IgG, and a lower proportion with surface IgD, than nonimmune mice. Tolerant-challenged mice have a lower proportion of ABC with surface IgG, and a higher proportion with surface IgD, than immune mice. Thus, B-cell tolerance in this model entails an attenuation of the surface immunoglobulin isotype switch (loss of IgD and gain of IgG) on ABC seen in the normal immune response. For most TNP-ABC, tolerogen exposure prevents the switch in surface isotypes normally induced by exposure to TNP antigen; i.e., the tolerance lesion precedes the surface isotype switch. However, a minority of the TNP-ABC appear to switch surface isotypes in response to the tolerogen itself.