Human T cell IgD receptors react with O-glycans on both human IgD and IgA1.

Previous studies on murine T cell IgD-R have shown that these receptors recognize N-glycans of murine IgD, and not of other Ig isotypes. We have now studied the specificity of IgD-R on human T cells. Human IgD digested with proteinase K to fragments of < 5 kDa inhibit the ability of T cells to form rosettes with IgD-coated ox erythrocytes. The same amount of digested IgG does not. We tested all the human Ig isotypes: IgG1, -2, -3, -4, IgA2, IgE and IgM fail to inhibit significantly at 20 microg/assay. However, IgA1 is as effective as IgD itself, showing approximately 60 % and 80 % inhibition at 5 microg and 10 microg/assay. Human IgA1 and IgD both contain Gal-1 --> 3-GalNac-rich O-linked glycans, and on this basis are both bound to ricin and jacalin. The O-linked glycans may therefore also represent the common moiety binding to IgD-R. Disaccharides Gal-1 --> 3-GalNac, and Gal-1 --> 4-Glc at 10 microg/assay blocked IgD rosetting while Gal-1 --> 6-Glc did not. We conclude that the human IgD-R is a lectin, differing from the murine IgD-R in that it has both IgA1 and IgD as ligands.

The effect of aging on the immune response: influence of phosphatidylcholine-containing lipid on IgD-receptor expression and antibody formation.

It was reported previously that IgD-receptors (IgD-R) are expressed on both CD4+ and CD8+ human T cells and CD4+ murine T cells after exposure to oligomeric IgD, certain cytokines, or various pharmacological agents, as shown by rosetting with IgD-coated erythrocytes. Enhancement of antibody production is observed in mice after injection of oligomeric IgD and is mediated by these IgD-R+ T cells, while injection of monomeric IgD inhibits both IgD-R upregulation and augmentation of antibody responses induced by simultaneously injected oligomeric IgD. The effects of oligomeric IgD on IgD-R upregulation are lacking in aged mice. However, the oligomeric IgD induced enhanced antibody production can be transferred to aged mice with IgD-R+ T cells from young donors suggesting that the environment of the aged mouse supports the effector function of IgD-R+ T cells. We now report, in addition, that exposure to phosphatidylcholine (PC) and a PC-containing lipid mixture, AL721, is effective in causing IgD-R upregulation on T cells from both young and aged mice, and young humans. This effect can also be demonstrated in mice in vivo after administration of AL721. Moreover, this agent causes a two-fold enhancement of antibody production, as measured by PFC/spleen, to 4-hydroxy-5-iodo-3-nitrophenyl(acetyl)-Brucella abortus (NIP-BA) and NIP-horse red blood cells (RBC) in young and aged mice. There is no difference in the baseline membrane fluidity of lymphocytes from aged and young mice. Although PC causes an increase in membrane fluidity of lymphocytes from both young and old mice, and from humans, this effect on fluidity is not prevented by a protein kinase inhibitor, while PC's effect on IgD-R upregulation is prevented by the inhibitor. Moreover, no correlation was observed between IgD-R upregulation and membrane fluidity changes induced by AL721 administered in vivo. To evaluate the role of IgD-R induction in the augmentation of antibody production by phospholipids, the effect of monomeric IgD was investigated. The augmenting effect of AL721 on antibody production was prevented by a single injection of monomeric IgD at the time of antigen administration. We conclude that (1) PC-containing lipid mixtures are effective in enhancing antibody production in aged mice, (2) induction of IgD-R is responsible for the augmenting effects of AL721 on antibody production, and (3) monomeric IgD not only blocks the upregulation of IgD-R, as shown previously, but also the augmenting effect of previously upregulated IgD-R on T cells by preventing their interaction with surface IgD+ B cells.

The effect of aging on IgD receptor expression by T cells and its functional implications.

Exposure to oligomeric or aggregated (a), but not to monomeric (m), IgD causes a rapid (within 1 h) upregulation of IgD-R expression on CD4+ T cells from young, but not from aged, mice and on both CD4+ and CD8+ T cells from all young and from approximately 65% of aged humans. In normal young (but not in IgD-/-) mice, this increase in IgD-R expression is associated with a marked increase in primary and secondary antibody responses, transferable to both aged and young mice with T cells from aIgD pretreated donors. In both species, immunization causes a rise in the IgD-R+ expression in vivo in the young. In mice, mIgD abolishes both the induction of IgD-R expression and augmentation of immune responses, suggesting that interaction between IgD-R+ T and IgD+ B cells is needed. In aged humans, the ability of peripheral blood lymphocytes to exhibit IgD-R expression in response to aIgD in vitro or to influenza vaccine in vivo is strongly correlated to the individual's ability to produce antibody. In T cells from aged mice, but not from aged IgD-non-responder humans, IgD-R are able to come to the cell surface if an additional signal has been supplied, such as by (ionomycin/thapsigargin + aIgD). Agents which induce IgD-R and augmentation of antibody production in aged and young mice include phosphatidylcholine and dehydroepiandrosterone sulfate. The immunoaugmenting effect of pretreatment with these agents appears indeed due to IgD-R+ T cells, because it is abolished by mIgD.

IgD-receptor up-regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination.

Aged individuals (more than 65 years) were classified as antibody (Ab) responders on the basis that they showed increases to more than or = 1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre-immunization samples of blood taken from seven Ab-responders and seven Ab-nonresponders were examined for their ability to exhibit up-regulation of IgD-receptor (IgD-R) after exposure for 2 h to immobilized cross-linked IgD, as shown by rosetting with IgD-coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD-R up-regulation was greater than 5% in all Ab-responders and less than 4% in all the Ab-nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD-R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894. Injection of influenza vaccine itself also induced IgD-R on PBMC in vivo. The percentage of IgD-R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre-injection levels by day 28 in young subjects and aged Ab-responders, but not in Ab-nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab-responders exhibited IgD-R upon immunization in vivo. These findings suggest that Ag injection causes rapid up-regulation of IgD-R by cross-linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD-R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab-responders than in Ab-nonresponders.

Relationship between humoral immunoaugmenting properties of DHEAS and IgD-receptor expression in young and aged mice.

IgD-receptors are associated with augmented antibody production in vivo and are induced on CD4+ T cells by aggregated IgD in young but not in aged mice. In the current study orally or intraperitoneally administered DHEAS was found to enhance antibody production, as measured in a plaque-forming cell assay, and also to cause an increased expression of IgD-R on T cells in both young and aged mice. IgD-R+ T cells are enumerated by rosette cell formation with IgD-coated SRBC. Since, as shown previously, the immunoaugmenting effect of IgD-R+ T cells is blocked by injection of monomeric IgD, the effect of monomeric IgD was also examined in DHEAS-pretreated mice. The inhibitory effect obtained with monomeric IgD in these studies indicates that upregulation of IgD-R by DHEAS plays an important role in the immunoenhancing effect of this hormone. In addition, no significant effect of DHEAS was obtained on contact hypersensitivity to DNCB or on proliferative responses of T cells from young or aged mice. Aged but not young mice showed increases in the numbers of Ia+ epidermal Langerhans cells after DHEAS treatment.

Oligomeric IgD augments and monomeric IgD inhibits the generation of IgG memory antibody responses in normal, but not in IgD-deficient, mice.

Dimeric or aggregated IgD causes augmentation of primary and secondary Ab responses in mice when injected a few days before or together with the primary dose of Ag. This effect is mediated by Th cells and seems to be linked to the up-regulation of receptors for IgD on CD4+ T cells. IgD-R cross-linking is needed for receptor up-regulation. Here we show that addition of monomeric IgD to dimeric or aggregated IgD blocks IgD-R up-regulation on T cells in vitro and in vivo, as well as their immunoaugmenting effect in vivo. More importantly, monomeric IgD injected 6 to 24 h before a primary Ag injection also inhibits 1) the up-regulation of IgD-R on T cells induced by Ag injection alone, and 2) the generation of IgG memory, as shown in the response to a second dose of Ag injected on day 10. These results suggest that IgD-R on T cells contribute to the T-B cell interaction involved in the priming for a secondary response. The augmenting effect of oligomeric IgD and the inhibiting effect of monomeric IgD on secondary Ab responses are not observed in IgD-/- (IgD-deficient) mice, although injection of oligomeric IgD leads to IgD-R up-regulation on T cells in these mice. These results indicate that IgD presented in the form of immune complexes, most likely on the surface of B cells, is a prerequisite for the immunoaugmenting effects exerted by IgD-R+ T cells. Thus, IgD is the only physiologic ligand for IgD-R on T cells.

Regulation of IgD-receptor expression on murine T cells. I. Characterization and metabolic requirements of the process leading to their expression.

Receptors for IgD (IgD-R) are found on murine CD4+ T cells and T cell clones. Previous work has shown that incubation with aggregated (but not monomeric) IgD causes the rapid upregulation of IgD-R and enables the T cells to respond with augmented helper function in antibody production. In the present study, IgD-R upregulation is shown to be (a) rapid, reaching plateau levels by 60 min, (b) independent of de novo protein or RNA synthesis, and (c) only slightly reduced at 4 degrees C. The IgD-R+ T cells present both before and after upregulation of IgD-R expression are predominantly resting T cells, whose ability to rosette with IgD-SRBC is inhibited by soluble IgD. The upregulation of IgD-R, even after overnight exposure to IgD, does not cause any detectable change in the expression of other T cell surface markers. Also characteristic of resting T cells is that they exhibit IgD-R in response to IL-2 and IL-4 only after overnight incubation with these cytokines, and fail to respond at all to IL-1. In contrast, cloned Th2 cells, expressing IL-1 and IL-2 receptors, show IgD-R upregulation after a 2-hr exposure to IL-1 or IL-2. GM-CSF, TNF-alpha, IL-6, and IL-10 do not modulate IgD-R expression. T hybridoma cells constitutively express much higher IgD-R levels than resting splenic T cells and can be stained with aggregated IgD followed by FITC-anti-IgD. Their levels of IgD-R expression decrease, as assayed both by rosetting and by staining, on 4-14 hr of incubation with tunicamycin or deoxynojirimycin, suggesting that N-linked glycosylation and oligosaccharide processing, respectively, are needed for continued expression of IgD-R. Tunicamycin-treated cells without detectable IgD-R on their surface still show IgD-binding protein in the cell extracts, suggesting that surface expression is more dependent on glycosylation of the IgD-R molecules than on the ability to bind IgD. Ca2+ ions are needed for optimal binding of IgD to IgD-R, in line with previous findings showing IgD-R to be lectin-like in binding carbohydrate rather than peptide regions of the IgD molecule.

Regulation of IgD-receptor expression on murine T cells. II. Upregulation of IgD receptors is obtained after activation of various intracellular second-messenger systems; tyrosine kinase activity is required for the effect of IgD.

The presence of IgD receptors (IgD-R) on T cells during a primary response to antigen causes augmented antibody production and facilitates priming for a secondary response. Cross-linked, but not monomeric IgD leads to a rapid upregulation of these receptors on T cells. As shown in the present study, the rapid upregulation of IgD-specific receptors is also induced by cross-linking of T cell surface molecules known to mediate triggering of T cell activation, such as CD3, CD2, and Thy 1. Furthermore, IgD-R are also upregulated by pharmacologically active compounds that increase intracellular cAMP and by PMA/DiOG plus ionomycin, but not by either PMA or ionomycin alone. The upregulation of IgD-R by anti-CD3 is inhibited by both calphostin C and herbimycin A, while that due to DiOG plus ionomycin is only inhibited by calphostin C. Upregulation of IgD-R by increased cAMP is blocked by HA1004, but not by low concentrations of staurosporine or herbimycin A. IgD itself does not cause an increase in intracellular cAMP, protein kinase C translocation, influx of extracellular Ca2+, or a change in membrane potential. Relatively specific inhibitors of these activation pathways, HA1004, calphostin C, and neomycin, also fail to interfere with IgD-receptor upregulation by IgD itself. However, tyrosine kinase inhibitors, including herbimycin A, tyrphostin C11, and genistein, completely prevent the effect of IgD on IgD-R expression. Although an influx of Ca2+ is apparently not involved, a role for intracellular Ca2+ in the upregulation of IgD-R by IgD on T cells is indicated by the susceptibility to inhibition by BAPTA, W7, and FK520. We conclude that activation of at least three different second-messenger systems can cause IgD-R upregulation, but that the effect of IgD itself requires tyrosine kinase activity, perhaps in an intracellular Ca(2+)-dependent manner.

The immunoaugmenting properties of murine IgD reside in its C delta 1 and C delta 3 regions: potential role for IgD-associated glycans.

IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C delta 1 domain (Fd delta) or the C delta 3 domain (Fc delta). Neoglycoproteins (D-galactose--BSA and N-acetyl-D-glucosamine--BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C delta 1), KWD6 (which lacks C delta 1 plus C delta-hinge), and Fab delta can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD. Moreover, the immunoaugmenting effect of intact oligomeric IgD molecules in primary antibody responses is competitively blocked by simultaneous injection of monomeric forms of KWD6 and Fab delta. These results suggest that the binding of IgD to IgD-R, previously shown to be dependent on N-glycans present on Fd delta and Fc delta regions, also contributes to the upregulation of IgD-R and immunoagumentation.

Serum IgD levels in mice: effect of strain, age and autoimmune disease.

Serum IgD levels were studied in mice. Strain-related variability of serum IgD levels was noted, and advanced age was associated with markedly increased IgD levels in a large percentage of mice from all strains. Strains prone to spontaneously arising autoimmune disease had elevated IgD levels; in NZB mice this was already present very early after birth (one week), whereas in MRL mice the elevated serum IgD levels were first seen somewhat later (3 months). In contrast, mice with collagen type II (CII)-induced autoimmune arthritis had no increase in serum IgD. Injection of the immunomodulating agents LPS and complete Freund's adjuvant (CFA) did not have a significant effect on serum IgD levels, but IL-1 induced a significant decrease in IgD.

Exposure to crosslinked IgD induces receptors for IgD on T cells in vivo and in vitro.

IgD is a surface immunoglobulin, which is coexpressed with IgM on greater than 90% of mature B cells, but its levels in serum are extremely low compared to those of IgM. It role as a surface receptor has been reemphasized by our recent findings that IgD receptors are induced on helper T cells by exposure to IgD and that such cells have immunoaugmenting properties. The present study shows that crosslinking of soluble IgD or of monomeric cell surface IgD is required and sufficient for the induction of T cells bearing receptors for IgD, both in vivo and in vitro. Effective IgD crosslinking in this respect can be obtained with antigen or with heterologous and immunogenic as well as nonimmunogenic allotype-specific anti-IgD. These results reinforce the concept that the induction of T cells bearing receptors for IgD is an integral component of the normal immune response.

Physiology of IgD. IX. Effect of IgD on immunoglobulin production in young and old mice.

Weekly i.p. injections of IgD from birth in (SJL X BALB/c)F1 mice were found to accelerate the development of IgG- and IgA-secreting cells and to increase the numbers of Ig-secreting cells of all isotypes in 17-28-day-old mice, but not in 7-10-day-old mice. Similarly, repeated weekly injections of IgD in normal adult BALB/c mice increased the numbers of reverse plaque-forming cells/spleen for all isotypes studied, including IgM, IgG1, IgG2, and IgA, but not for IgD itself. No such effect was observed in IgD-treated aged (20 months old) BALB/c mice. The absence of an effect of IgD on Ig secretion appeared to correlate with a lack of induction of receptors for IgD on T cells of the host, both in 7-10-day-old and in aged mice. In 7-10-day-old mice this lack of induction appeared due to their very low numbers of L3T4+ T cells. A comparison was made between the effect of a single injection of IgD or lipopolysaccharide (LPS) on numbers of Ig-secreting cells in the spleen determined 1-7 days after injection. Both agents caused increases, but the increase in IgM-producing cells was much greater after LPS (day 4), while IgD caused a relatively greater increase in IgG2 and IgA (days 4-7). Increases in IgG1 and IgG3-producing cells induced by LPS and IgD were of similar magnitude (days 6-7). IgD production, however, was not increased. The number of cells producing antibody of anti-trinitrophenyl (TNP) specificity was enhanced by LPS (day 4), but not by a single injection of IgD, although more than one injection of IgD caused a significant increase in anti-TNP-producing cells above background. LPS, but not IgD, caused B cell proliferation in vitro in the presence or absence of gamma-irradiated T delta cells. However, in vivo, IgD injections caused a significant increase in the percentage of lymphoid follicles with germinal centers in lymph nodes from 17-21-day-old and normal adult mice, but not in 7-10-day-old or aged mice. Such an effect was also absent in 24-28-day-old mice, where germinal center development, even in untreated mice, was very high.

Characterization of chloramphenicol-resistant Haemophilus influenzae.

We examined nine chloramphenicol-resistant (minimal inhibitory concentration, greater than or equal to 15 micrograms/ml) Haemophilus influenzae strains isolated in various parts of the world to characterize the genetic and biochemical bases of the resistance; four were type b. All nine contained conjugative plasmids, ranging in molecular weight from 34 x 10(6) to 46 x 10(6), which encoded for resistance to chloramphenicol and tetracycline or chloramphenicol, tetracycline, and ampicillin. Deoxyribonucleic acid homology studies showed that these plasmids were closely related to a previously described ampicillin-resistant plasmid, RSF007, and to each other. All nine isolates and their chloramphenicol-resistant transconjugants produced chloramphenicol acetyltransferase. We conclude that chloramphenicol resistance in these strains of H. influenzae is via plasmid-mediated production of chloramphenicol acetyltransferase.