Distribution of substance P receptors on murine spleen and Peyer's patch T and B cells

Previous studies have demonstrated that the sensory neuropeptide substance P (SP) can modulate immune responses in vitro. Work from this laboratory has shown that SP enhances immunoglobulin synthesis by murine splenic and Peyer's patch lymphocytes stimulated with concanavalin A. One mechanism underlying these effects is the binding of SP to specific receptors on lymphocytes. We examined the distribution of SP receptors on murine T and B lymphocytes and their subsets by one and two color fluorescence-activated cell sorter analysis. The specificity and nature of binding was examined using radiolabeled SP, and competitive inhibition experiments were performed with cold SP. In cytofluorimetry experiments, both T and B lymphocytes from Peyer's patches and spleen were bound to SP, with those from Peyer's patches having a higher proportion than lymphocytes from the spleen. The majority of T cells from both organs bound SP with binding being evenly distributed between Lyt-1+ and Lyt-2+ cells. Similarly, the majority of B lymphocytes from spleen and Peyer's patches showed SP binding. There were no significant isotype-specific differences within any organ. Studies using 125I-labeled SP showed specific binding to all lymphocyte subpopulations examined. SP receptors were fewer in number on cells isolated from spleen than on cells from Peyer's patches although the dissociation constants were similar for all populations examined. These studies demonstrated that SP receptors are present both on murine T and B lymphocytes from Peyer's patches and spleen. There is no evidence for differential SP receptor expression on distinct lymphocyte subsets in spleen or Peyer's patches.     

Recognition of antigens by T lymphocytes

Summary The present review briefly summarizes our knowledge of antigen-specific B and T lymphocyte receptors. Antigen-specific receptors on mammalian B lymphocytes are mainly monomeric IgM and IgD consisting of conventional immunoglobulin heavy and light chains. The nature of the T lymphocyte receptor which can specifically recognize antigens is not yet fully defined. However, it seems that conventional light chains do not participate in the build up of this receptor, and that the receptor is made up of heavy chains of a new immunoglobulin class which has to be further characterized and which we call Tau-chain. The variable region of the T lymphocyte receptors share idiotypic determinants with the corresponding B lymphocyte receptors. The possible linkage between the T cell idiotypes present on the Tau-chains and molecules which are under the control of genes of the Major Histocompatibility Complex of the species are discussed.In the last part of the review two methods for the induction of specific transplantation tolerance in adult animals are described. These methods are based on the concept that T lymphocytes reactive against alloantigens bear idiotypic determinants against which a specific auto-immune response can be initiated.     

The BLyS Family: Toward a Molecular Understanding of B Cell Homeostasis

The B Lymphocyte Stimulator (BLyS) family of ligands and receptors regulates humoral immunity by controlling B lymphocyte survival and differentiation. Herein, we review the ligands and receptors of this family, their biological functions, and the biochemical processes through which they operate. Pre-immune B lymphocytes rely on BLyS signaling for their survival, whereas antigen experienced B lymphocytes generally interact more avidly with a homologous cytokine, A Proliferation Inducing Ligand (APRIL). The molecular basis for signaling via the three BLyS family receptors reveals complex interplay with other B lymphocyte signaling systems, affording the integration of selective and homeostatic processes. As our understanding of this system advances, molecular targets for manipulating humoral immunity in both health and disease should be revealed.     

Interactions between lymphocyte membrane molecules. II. Characterization of an interaction between B lymphocyte surface IgD and Fc IgG receptors that differs from the surface IgM-Fc IgG receptor interaction

The B lymphocyte surface membrane receptors IgD (sigD) and Fc IgG receptors (Fc gamma R) were evaluated for interactions by means of immunofluorescence. Ligand-(F(ab')2 anti-delta) induced capping of sIgD resulted in co-capping of Fc gamma R if the latter were occupied during the capping process by soluble antigen-antibody complexes (which themselves provided insufficient cross-linking to result in capping), but not if the Fc gamma R were occupied by monomeric IgG or unoccupied. Capping of Fc gamma R by highly cross-linked complexes did not cause co-capping of sIgD occupied by monomeric F(ab') anti-delta. The interaction between sIgD and Fc gamma R was specific in that cross-reactions between ligands were excluded and ligand-induced capping of sIgD did not cause co-capping of ligand-occupied sIgM or I-A antigens. The sIgD-Fc gamma R interaction occurred on only approximately 60% of B lymphocytes, and this B cell subpopulation did not correlate with other B cell subpopulations (CBA/N strain B cells and B cells bearing either large or small amounts of sIgD). The sIgD-Fc gamma R interaction differed from the sIgM-Fc gamma R interaction in that co-redistribution of the Fc gamma R was occupied by monomeric IgG and involved nearly all B lymphocytes. The qualitative and quantitative differences between the sIgD-Fc gamma R and sIgM-Fc gamma R interactions suggest a mechanism whereby the two antigen receptors could provide different signals to the B lymphocyte.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Adherence of bacteria to human lymphocyte subpopulation and the role of monosaccharides in bacterial binding

In the present investigation lymphocyte bacterial binding, receptors for bacteria on lymphocytes, and the relationships between bacterial binding and lymphocyte activation were studied. Of the strains used Escherichia coli, Bacillus subtilis, and Corynebacterium xerosis bound to both purified T and B cells. Staphylococcus aureus, Staphylococcus albus, and Brucella melitensis bound chiefly to B lymphocytes. Monosaccharides and treatment of lymphocytes with lectins, enzymes, or sodium metaperiodate affected bacterial adherence. Thus the lymphocyte receptors for bacteria appeared to contain carbohydrate moieties. There was no clear-cut correlation between lymphocyte binding of bacteria and bacterium-induced leukocyte-inhibitory factor (LIF) synthesis and proliferation.     

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.     

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.     

Cytokines as regulators of proliferation and survival of healthy and malignant peripheral B cells

Adaptive immunity depends on the production and maintenance of a pool of mature peripheral lymphocytes throughout life. The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Lasting B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. Cytokines have been shown to play a critical role in maintaining lymphocyte homeostasis. This review focuses on the role of cytokines and their receptors in the regulation of peripheral B cell survival, with an emphasis on those that have received relatively less attention in the literature.     

Receptor editing in lymphocyte development and central tolerance

The specificities of lymphocytes for antigen are generated by a quasi-random process of gene rearrangement that often results in non-functional or autoreactive antigen receptors. Regulation of lymphocyte specificities involves not only the elimination of cells that display 'unsuitable' receptors for antigen but also the active genetic correction of these receptors by secondary recombination of the DNA. As I discuss here, an important mechanism for the genetic correction of antigen receptors is ongoing recombination, which leads to receptor editing. Receptor editing is probably an adaptation that is necessitated by the high probability of receptor autoreactivity. In both B cells and T cells, the genes that encode the two chains of the antigen receptor seem to be specialized to promote, on the one hand, the generation of diverse specificities and, on the other hand, the regulation of these specificities through efficient editing.     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

Binding of bacteria in lymphocyte subpopulations: role of lectin-carbohydrate interactions

Bacteria have been found to bind to lymphocyte subpopulations in a highly reproducible manner. Some of these bacteria such as B. melitensis and a strain of E. coli binds to mammalian B. cells. The binding of B. melitensis and other bacteria is due, at least in part, to lectins on lymphocytes interacting with the carbohydrates on the LPS or LTA of the bacteria. These receptors for bacteria give some indications regarding the functional potential of the cells, suggesting the possibility that the receptors identified by bacteria are used in cellular interactions with normal or malignant cells.     

Fc receptor heterogeneity: immunofluorescent studies of B, T, and "third population" lymphocytes in human blood with rabbit IgG b4/anti-b4 complexes.

IgG Fc receptors on human peripheral blood lymphocytes (PBL) were characterized by immunofluorescence studies with defined rabbit IgG b4 allotype/anti-allotype complexes. Three discrete types of Fc receptor-bearing cells, totaling approximately 33% of PBL, were identified. Fc receptors of the three types differed in their sensitivity to trypsin and in either absolute or localized density (topography) as determined by variable requirements for anti-IgC cross-linking in order to visualize bound complexes microscopically. The question of additional heterogeneity related to differences in individual Fc receptor affinity for complexed IgG was not approached in this study. Ten to 15% of PBL had pronase-sensitive, trypsin-resistant Fc receptors readily detected by direct immunofluorescence by using large fluorescein-conjugated complexes prepared near equivalence. Double label and lymphocyte fractionation experiments established this population to be largely distinct from suface IgM+ B cells and T cells, and identical to EA Ripley rosette-forming cells. Approximately 50% of surface IgM+ B cells and approximately 10% of T cells had lower density Fc receptors identified by indirect immunofluorescence with small complexes prepared in antigen excess or by cross-linking fluorescein-conjugated complexes with anti-rabbit IgG anti-serum. An additional approximately 15% peripheral T and B cells had very low density Fc receptors detectable by complexing the IgG on the cell surface by sequential incubations of cells with b4 IgG and anti-b4. Fc receptors on B and T cells were sensitive to both pronase and trypsin digestion. The heterogeneity of IgG Fc receptors on different lymphocyte subpopulations as defined by these these experiments may be of relevance for further analysis of normal and abnormal immune function.     

Immunogenicity of human B cell antigens solubilized from cultured human lymphoid cells.

Antigens solubilized from culured human lymphoid cells WI-L2 and RPMI 1788 were partially purified by ultracentrifugation on a KBr gradient. These antigens injected into rabbits produced xenoantibodies which after absorption with melanoma cells became specific to B cell antigens. Three such xenoantisera were submitted to the Second Histocompatibility Workshop of the Americas and reacted much like alloantisera to B cell antigens against a large panel of B peripheral lymphocytes and cells from patients with chronic lymphocytic leukemia. Xenoantisera to B cell antigens inhibited the mixed lymphocyte reaction, but did not affect the mitogenic activity of phytohemagglutinin or the functional properties of C3 receptors, monkey red blood cell receptors, or T cell receptors.     

Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation

Cell surface receptors belonging to the CD2 subset of the Ig superfamily of molecules include CD2, CD48, CD58, 2B4, signaling lymphocytic activation molecule (SLAM), Ly9, CD84, and the recently identified molecules NTB-A/Ly108/SLAM family (SF) 2000, CD84H-1/SF2001, B lymphocyte activator macrophage expressed (BLAME), and CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1. Some of these receptors, such as CD2, SLAM, 2B4, CRACC, and NTB-A, contribute to the activation and effector function of T cells and NK cells. Signaling pathways elicited via some of these receptors are believed to involve the Src homology 2 (SH2) domain-containing cytoplasmic adaptor protein SLAM-associated protein (SAP), as it is recruited to SLAM, 2B4, CD84, NTB-A, and Ly-9. Importantly, mutations in SAP cause the inherited human immunodeficiency X-linked lymphoproliferative syndrome (XLP), suggesting that XLP may result from perturbed signaling via one or more of these SAP-associating receptors. We have now studied the requirements for SAP recruitment to CD84 and lymphocyte activation elicited following ligation of CD84 on primary and transformed human T cells. CD84 was found to be rapidly tyrosine phosphorylated following receptor ligation on activated T cells, an event that involved the Src kinase Lck. Phosphorylation of CD84 was indispensable for the recruitment of SAP, which was mediated by Y(262) within the cytoplasmic domain of CD84 and by R(32) within the SH2 domain of SAP. Furthermore, ligating CD84 enhanced the proliferation of anti-CD3 mAb-stimulated human T cells. Strikingly, this effect was also apparent in SAP-deficient T cells obtained from patients with XLP. These results reveal a novel function of CD84 on human lymphocytes and suggest that CD84 can activate human T cells via a SAP-independent mechanism.     

Change of genetic expression by physical compression forces

Despite intense investigation of transmembrane signalling mediated by lymphocyte surface molecules, the subcellular mechanisms responsible for these phenomena remain poorly understood (Kishimoto, T. et al., 1983). In the present study, an answer for the mechanism is demonstrated by using human B blastoid cell line (CESS) which has IgG and Interleukin 6 (IL-6) receptors on the cell surface and produces IgG in response to IL-6 without any requirement for proliferation (Muraguchi, A. et al., 1981).     

Cutting edge: B and T lymphocyte attenuator and programmed death receptor-1 inhibitory receptors are required for termination of acute allergic airway inflammation

T cell activation is regulated by coordinate interaction of the T cell Ag receptor and costimulatory signals. Although there is considerable insight into processes that regulate the initiation of inflammation, less is known about the signals that terminate immune responses. We have examined the role of the inhibitory receptors programmed death receptor-1 and B and T lymphocyte attenuator in the regulation of allergic airway inflammation. Our results demonstrate that there is a temporally regulated expression of both the receptors and their ligands during the course of allergic airway inflammation. Following a single inhaled challenge, sensitized wild-type mice exhibit peak inflammation on day 3, which resolves by day 10. In contrast, mice deficient in the expression of programmed death receptor-1 or B and T lymphocyte attenuator have persistent inflammation out to 15 days following challenge. Thus, these receptors are critical determinants of the duration of allergic airway inflammation.     

RGS molecule expression in murine B lymphocytes and ability to down-regulate chemotaxis to lymphoid chemokines

Ag-mediated changes in B lymphocyte migration are important for normal immune function, yet the mechanisms by which these changes occur are poorly defined. Because chemokines direct many lymphocyte movements, molecules that regulate signaling by G protein-coupled chemokine receptors are likely to participate in Ag receptor-induced changes in cell migration. In this study, we have investigated the expression pattern and activity in murine B cells of members of the regulators of G protein signaling (RGS) family of molecules. We present the sequence of mouse RGS1 and describe a novel short isoform of RGS3 that we term RGS3s. Following in vivo activation by Ag, B cells rapidly up-regulate expression of RGS1 and RGS2 while simultaneously decreasing expression of RGS3 and RGS14. Anergic hen egg lysozyme autoantigen-binding B cells are also shown to have slightly elevated RGS1 and RGS2 expression. CD40 signaling, by contrast, fails to cause rapid up-regulation of RGS1 or RGS2. Using a transient transfection approach in a mature B cell line, 2PK3, we demonstrate that RGS1 and RGS3s are effective inhibitors of chemotaxis toward the lymphoid tissue chemokines stromal cell-derived factor-1, B lymphocyte chemoattractant, and EBV-induced molecule 1 ligand chemokine, whereas RGS2 has a minimal effect on migration to these chemokines. Together these findings support the conclusion that Ag-mediated changes in RGS molecule expression are part of the mechanism by which Ag receptor signaling regulates B cell migration within lymphoid tissues. The findings also suggest important roles for additional G protein-mediated events in B cell activation and tolerance.     

Activation of lymphocytes by a thiol-derivatized nucleoside: characterization of cellular parameters and responsive subpopulations

Lymphocyte activation, whether specific or nonspecific, is generally conceptualized as initiated by the binding of an activating ligand to a surface membrane receptor, followed by transduction of the signal across the cell membrane. In many situations several qualitatively distinct signals are required. We have recently described a new class of lymphocyte activator, the C8 bromine substituted guanine ribonucleosides, that traverse the cell membrane, bypassing classical triggering mechanism(s), apparently activating the lymphocyte at an intracellular site. However, the identity of the lymphocyte population(s) activated, as well as the nature of any cellular interactions involved in activation, has not been studied heretofore. The present experiments describe the cellular parameters of lymphocyte activation by a thiol substituted member of this class of activators, 8-mercaptoguanosine (8MGuo). Upon addition of this nucleoside derivative to cultures of murine spleen cells, a marked increase in [3H]TdR uptake and blast transformation ensues. Normal splenic B cells and spleen cells from congenitally athymic (nu/nu) mice are responsive to 8MGuo, whereas thymocytes and splenic T cells are not. Two subpopulations of B cells appear to be involved in the response to this nucleoside. The predominant one is a mature population that bears surface delta-chains, la antigens, C receptors, and (by indirect evidence) the Lyb3, 5, and 7 antigens. These cells also bear mu-chain and Fc receptors. In addition, a second, minor subpopulation of less mature cells that bear only mu-chain and Fc receptors also appears to be reactive to 8MGuo. The existence of this immature, reactive B cell subset was confirmed by observation of 8MGuo responsiveness in lymphocytes from 4-day-old mice whose cells do not yet exhibit these later-appearing markers. Accessory cells appear to play a minimal, if any, role in the 8MGuo response. These results establish two distinct B cell subpopulations as the major and minor cellular targets of C8-derivatized nucleosides, and suggest that the activation process results from a direct interaction between the nucleoside and target cell.