Dextran-sulfate: a mitogen for human T lymphocytes

Dextran-sulfate (DxS) induced proliferation of human peripheral blood T lymphocytes but not of adult or neonatal B lymphocytes. The mitogenic activity on T cells by DxS required the presence of accessory cells because DxS was unable to trigger T cells to DNA synthesis in the absence of accessory cells. In addition, DxS stimulated OKT4+8- T cells to produce interleukin 2, a process that also occurred only in the presence of accessory cells. Cyclosporin-A strongly suppressed T cell proliferation induced by DxS by rendering T cells unresponsive to interleukin 2 and by inhibiting the synthesis of this T cell growth factor by OKT4+ T cells. These results indicate that DxS is a mitogen for human T lymphocytes but not for adult or neonatal B lymphocytes. The mechanism by which DxS triggers T cells is discussed.     

Responses of B cells with or without C3 receptors to polyclonal B cell activators

Responses of B cells with or without receptors for C3 (CR) to polyclonal B cell activators (PBA) were studied. Mouse spleen cells were incubated with sheep red blood cells (SRBC) coated with antibody and complement to form rosettes, and they were separated by Ficoll-Hypaque density sedimentation into populations depleted of and enriched with lymphocytes bearing CR (CRL). These 2 populations were cultured with lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), or dextran sulfate (DxS) and assayed for anti-TNP PFC. The CRL-depleted population responded well to LPS, poorly to PPD, and it showed practically no response to DxS, whereas the CRL-enriched population seemed to respond poorly to LPS but well to both PPD and DxS. The low responsiveness of the cRL-depleted population to PPD and DxS could not be explained by a shift of time-kinetics, by the dose-response profile of the responding cells, or by the depletion of adherent cells. Suppressor T cells did not take part in the reduced responses, since the treatment of the population with anti-Thy 1.2 plus complement could not restore the responses. These results indicate that B cells with CR [CR(+) B cells] respond well both to PPD and DxS, whereas the cells without CR [CR(-) B cells] respond poorly to PPD and DxS. It was difficult to evaluate the low responsiveness of CR(+) B cells to LPS because of the high background PFC of the cRL-enriched population.     

Responses of spleen cells from mice with X-linked B-cell defect to polyclonal B-cell activators, purified protein derivative of tuberculin, and dextran sulfate

Polyclonal responses to LPS, PPD, and DxS of spleen cells from mice expressing a X-linked B-cell defect were examined. Spleen cells from young (CBA/N × BALB/c)F₁ male mice responded slightly lower to LPS, significantly lower to PPD than the cells from age-matched F₁ female mice, and showed no response to DxS stimulation. This hypo- or unresponsiveness of F₁ male cells to PPD or DxS could not be explained by a shift in the dose-response or time kinetics of the responding cells, and also could not be due to the defect in the function of T cells or macrophages. Suppressor T cells to polyclonal response to PPD or DxS could not be shown in F₁ male spleen cells. The response of F₁ male cells to PPD was dramatically improved with age but not to DxS. These results suggest that B cells responsive to DxS may belong to a distinct subpopulation from the cells responsive to LPS or PPD.     

Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.     

The simultaneous antagonistic effects of a T cell hybridoma product on the growth and the maturation of activated lymphocytes

Lymphocyte maturation and growth are two antagonistic events; therefore, successful antibody synthesis accompanied by clonal expansion, as seen in most humoral immune responses, must be the result of a delicate quantitative balance between maturation and growth factors. On the basis of this hypothesis, it should be feasible to search for a T cell product that alters this equilibrium and favors either B cell growth or maturation. In an attempt to isolate T cell hybridomas producing these activities, we fused BW5147 thymoma cells with Con A-activated spleen cells. One of the hybridomas obtained constitutively produces a product (or products) that selectively inhibits mitogen-induced replication but not maturation of normal but not transformed B lymphocytes. More importantly, the same product(s) supports Ig synthesis but not growth of LPS blasts, which suggests that we are dealing with a B cell maturation factor. The effect of this supernatant can be completely abrogated by the B cell mitogen DxS. In addition, the proliferative response of B cells to this ligand is unaffected by the hybridoma product. The implications of our results for understanding the mechanism of B cell triggering are discussed.     

Polyclonal activation of primed rat B cells

In recent years, murine and human virgin B lymphocytes have been used to examine the steps necessary for polyclonal activation. In these models mitogens are used in conjunction with lymphokines to determine which signals are responsible for regulating B-cell triggering, proliferation, and differentiation. While progress has been made in understanding these events as they occur in virgin B cells, very little evidence exists to suggest whether these models of activation also apply to the memory B-cell population. In this report we have described an antigen-specific, secondary in vitro immune response using cells isolated from lymph nodes draining the site of antigen injection. Unfractionated cells, B cells, and size-fractionated cells from dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-primed rats were challenged in vitro with DNP-KLH, lipopolysaccharide plus dextran sulfate (LPS/DxS), and T-cell factors. We have consistently found, under all these conditions, that antigen challenge of primed cells results in the production of DNP-specific IgG antibody while stimulation with LPS/DxS plus T-cell factors results only in the polyclonal activation of virgin B cells; no antigen-specific IgG secretion is seen. This suggests that acquisition of memory status is associated with a loss in responsiveness to LPS/DxS-induced differentiation.     

Role of adherent cells in dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation in the rat and have found that the addition of the compound Dextran sulfate (DxS), which itself is not mitogenic, to LPS stimulated cultures results in significant enhancement of cell division. A "DxS-free" supernatant from DxS stimulated spleen cell cultures is able to substitute for DxS in stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity, however, the addition of purified recombinant IL-1 to LPS-stimulated cultures does not result in augmentation of proliferation. A DxS-free supernatant from DxS stimulated adherent cells is also able to substitute for DxS in stimulatory activity. The active molecule(s) present in the adherent cell-derived DxS-free culture supernatant appears to be distinct from classical IL-1.     

Distinction of B cell maturation factors from lymphokines affecting B cell growth and viability

Supernatants from S26.5 helper T cells, autoimmune viable motheaten (mev/mev) mouse spleen cells, EL4 lymphoma cells, and recombinant DNA-derived interferon gamma (IFN-gamma), all of which display B cell maturation factor (BMF) activity, were assayed for effects on B cell proliferation alone and with Dextran Sulfate (DxS) and anti-immunoglobulin antibodies (alpha-Ig). Both EL4 and S26.5 supernatants showed BCGF-II (DxS co-stimulator) activity, whereas only EL4 supernatant had BCGF-I (alpha-Ig co-stimulator or BSF-I) activity. Supernatants from mev/mev spleen cells and recombinant DNA-derived IFN-gamma showed no activity in either assay. Fractionation of S26.5 supernatant by chromatofocusing showed a divergence of BMF activity (BMF-T, pIa of 6.0) from BCGF-II activity (pIa of 5.4), providing evidence for their physical nonidentity. IFN-gamma, which decreases B cell viability in culture, was separable from BMF-T by phenyl-Sepharose chromatography. BMF-T from S26.5 supernatant was separated from IFN-gamma and BCGF-II and was shown to induce B cell maturation without affecting B cell proliferation. The molecular characteristics of the purified BMF-T were pIa 6.0, Mr 55,000 by G-75 gel filtration, and Mr 16,000 by SDS-PAGE. These data demonstrate that several lymphokines (BMF) exist that mediate the maturation of B cells to active Ig secretion without stimulating B cell proliferation.     

Dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation of rat lymphocytes and have found that a serum-free tissue culture medium supports significant cell division. When the compound dextran sulfate (DxS), which itself is not mitogenic, is added to LPS-stimulated cultures, significant augmentation of proliferation results. DxS will significantly enhance anti-immunoglobulin-induced proliferation as well. An intact T-cell compartment is not required for DxS-mediated enhancement of LPS-induced cell division, in that lymphocytes derived from athymic rats proliferate and respond to the influence of DxS to the same degree as euthymic derived lymphocytes. A "DxS-free" supernatant from DxS-stimulated spleen cell cultures is able to substitute for DxS in all stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity. However, purified recombinant IL-1 does not cause enhanced proliferation when added to LPS-stimulated cultures as is seen with DxS or the "DxS-free" supernatant.     

Activation/suppression of the immune response in vitro by antigen and dextran sulfate: 2. The role of T-cell subsets determined by cell separation and partition analysis

Culture of spleen cells with dextran sulfate (DxS) and antigen at various different cell densities revealed a T-cell-dependent regulatory pathway not observed in conventional culture. This finding can be explained by the frequent presence in the cultures of a helper cell and the less frequent presence of a suppressor cell, both activated by antigen and DxS. The classic, radioresistant, antigen-specific, helper T cell was not regulated by this newly revealed pathway. The highly frequent, DxS-dependent helper T cell is Lyt-1⁺2^?. The suppressive effect is mediated by a Lyt-1⁺2⁺ population consisting of helpers and latent suppressors that can be made active by DxS or Lyt-1⁺ cells. The specificity of the Lyt-1⁺ helper cells was not established, but the high frequency observed implies a nonspecific mechanism. The specificity of the suppressor effect was not determined by these experiments. This regulatory mechanism is similar to the phenomena exhibited by polyclonally activated T-cell populations.     

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.     

Characterization of embryonic liver suppressor cells by peanut agglutinin.

Liver cells of 19-day-old mouse embryos were separated by peanut agglutinin (PNA) into two fractions. The fraction agglutinated with the PNA was found to be enriched for cells capable of suppressing the MLC reaction and the response to the mitogens Con A, PHA, and LPS. The fraction not agglutinated by PNA was significantly less suppressive. The response to DxS was not suppressed by any of these fractions. On the other hand, the response to LPS and DxS, but not to Con A or PHA, was expressed by the nonagglutinated fraction. It is thus inferred that the suppressor cells in the embryonic liver are separable from the potentially reactive cells.     

Sex hormones and immune dysregulation in multiple myeloma

A group of 49 multiple myeloma patients, 20 men and 29 women, were evaluated. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), 17-oestradiol (E) and testosterone (T) serum concentrations have been detected by radioimmunoassay. Peripheral blood lymphocyte proliferation in response to phytohaemagglutinin (PHA), concanavalin A (ConA), recombinant interleukin-2 (rIL-2) and dextran sulphate (DxS) was investigated. Our findings provide evidence for two different patterns of sex hormone changes and immune dysfunctions presented differently by male and female multiple myeloma patients. In men increased FSH, LH and E concentrations and an augmented E to T ratio were associated with decreased lymphocyte blastogenic response to PHA, ConA and increased proliferation to rIL-2 and DxS. Female patients with multiple myeloma demonstrated normal values of FSH, LH and T, but a diminished E level and decreased E to T ratio correlated with a lymphocyte normal response to PHA and ConA and augmented blastogenesis to IL-2 and DxS. Our data, while admittedly preliminary, suffice to provide an indication of sex hormone changes in multiple myeloma patients, which could be responsible, at least in part, for the immune dysfunction observed in multiple myeloma.     

Ultrathin antibiotic walled microcapsules

Ultrathin microcapsules comprised of anionic polyelectrolytes (PE) and a polycationic aminoglycoside (AmG) antibiotic drug were prepared by depositing PE/AmG multilayers on zinc oxide (ZnO) colloid particles using the layer-by-layer self-assembly technique and subsequently dissolving the ZnO templated cores. The polyelectrolytes, dextran sulfate sodium (DxS) and poly(styrenesulfonate) (PSS), were selected owing to their different backbone structure. An aminoglycoside, tobramycin sulfate (TbS), was used for studying DxS/TbS or PSS/TbS multilayer films. The multilayer growth on ZnO cores was characterized by alternating zeta potential values that were different for the DxS/TbS and PSS/TbS multilayers due to the PE chemistry and its interaction with Zn(2+) ions. Transmission and scanning electron microscopy provide evidence of PE/TbS multilayer coating on ZnO core particles. The slow acid-decomposition of the ZnO cores using weak organic acids and the presence of sufficient quantity of Zn(2+) in the dispersion were required to produce antibiotic multilayer capsules. There was no difference in the morphological characteristics of the two types of capsules; although, the yield for [PSS/TbS](5) capsules was significantly higher than for [DxS/TbS](5) capsules which was related to the physicochemical properties of DxS/TbS/Zn(2+) and PSS/TbS/Zn(2+) complexes forming the capsule wall. The TbS quantity in the multilayer films was determined using a quartz crystal microbalance and high performance liquid chromatography techniques which showed less TbS loading in both, capsules and multilayers on planar gold substrate, than the theoretical DxS:TbS or PSS:TbS stoichiometric ratio. The decomposition of the [PE/TbS](6) multilayers was fastest in physiological buffer followed by mannitol and water. The decomposition rate of the [PSS/TbS](6) multilayers was slower than [DxS/TbS](6) monolayers. The incomplete decomposition of DxS/TbS under saline conditions suggests the major role of hydrogen bonding for stability of DxS/TbS multilayers. A combination of hydrogen bonding and hydrophobic interaction between phenyl rings in PSS was responsible for PSS/TbS multilayer stability. In vivo studies in rabbits highlight the safety and sustained drug delivery potential of the PE/AmG microcapsules. The antibiotic walled ultrathin capsules presented here are suitable for sustained ophthalmic antibiotic delivery.     

Functional differentiation of B lymphocytes in congenital agammaglobulinemia. I. Generation of hemolytic plaque-forming cells.

Despite the absence of B lymphocytes, peripheral blood lymphocytes (PBL) from four of five patients with congenital agammaglobulinemia (cAgamma) generated a specific hemolytic plaque-forming cell (HcPFC) response in vitro to sheep red blood cells and ovalbumin. The kinetics, antigenic, and cellular requirements were similar to normals, but significantly less HcPFC were found in patient cultures. Normal but not patient HcPFC-precursor cells were inactivated by treatment with anti-mu antisera whereas generated HcPFC in both controls and patients were sensitive to treatment with anti-mu. Pokeweed mitogen (PWM) and dextran sulfate (DXS) enhanced the HcPFC-response of normal PBL; cAgamma-cells were unresponsive to DxS and, in the presence of PWM, the development of HcPFC was inhibited. These findings indicate the presence of B lymphocyte precursors in the majority of patients with cAgamma investigated.     

Analysis of the activation of lymphoid cells by mitogens in vitro with limiting dilution methods: adherent peritoneal cells suppress B-cell activation and synergize with WEHI-3 in the activation of T cells by Con A

Adherent peritoneal cells (APC) have often been used as a pure and effective macrophage population. Using partition analysis and small numbers of lymphoid cells activated by mitogens (concanavalin A for T cells (in the presence of TCGF) and LPS + DxS for B cells) we found that APC were accessory cells for T cell activation and growth but were not effective for B cells. Although APC were effective in assisting T-cell mitogenesis, they were not especially efficient. However, when APC were mixed with irradiated WEHI-3 cells (a tissue culture line previously shown to exhibit accessory cell activity in vitro for mitogenic activation T and B cells), the APC and WEHI-3 showed apparent synergy. One reason for failure of APC to assist B-cell mitogenesis was traced to the presence of a suppressor cell population which overcame the accessory cell help given by irradiated WEHI-3 cells to LPS-DxS stimulated murine B cells. It is thus possible to find "helper" effects (synergy of APC and WEHI-3 assisting the mitogenesis of T cells), as well as suppressor effects within the range of cells found in adherent accessory cells.     

Activation of rat B lymphocytes. II. Functional and structural changes in "aged" rat B lymphocytes

Anti-mu, anti-gamma, and anti-delta antibodies induce proliferation of splenic B lymphocytes from young Lewis rats, measured by 3H-TdR uptake. In contrast, splenic B cells of aged Lewis rats respond poorly or not at all to these reagents. T lymphocytes or interleukin 2 (IL-2) of young or aged rats augment the uptake of 3H-TdR in cultures of "young" B cells responding to anti-Ig reagents or LPS and DxS, but have no significant effect on the responses of "old" B cells. Analysis of spleen cells of young and aged rats in a fluorescence-activated cell sorter indicates the density of mu, gamma, and delta isotypes is reduced in "old" B cells, and that B cells of aged rats are significantly larger than those of young rats. These results delineate anatomic and structural changes in B lymphocytes of aged rats.     

Role of BCGFII in the differentiation to antibody secretion normal and tumor B cells

This report describes the effects of B cell growth factor (BCGFII) and other lymphokines in the differentiation of normal and tumor B cells. We compared BCL1 tumor B cells, normal B cells giving rise to a polyclonal response without the intentional addition of antigen, and an antigen-driven, SRBC-specific response. BCL1 tumor B cells gave maximum PFC responses when partially purified BCGFII was added or when suboptimal doses of BCGFII were mixed with one of several putative terminal differentiation factors we call B cell differentiation factors BCDF. IFN-gamma was not active as any of these factors. Maximum polyclonal responses of B cells were seen when either IL 2 or BCGFII were mixed with BCDF. In contrast, SRBC-specific responses showed a strict requirement for IL 2, and BCGFII and BCDF synergized with IL 2 to give a maximum response. The involvement of BCGFII in all of these responses suggests that BCGFII acts as a growth factor for a population of B cells that has differentiated much of the way towards Ig secretion, and that many B cells become responsive to this growth factor. In addition, the fact that different lymphokine requirements were seen in the different experimental systems raises the possibility that there are multiple pathways to Ig secretion, and suggests that different subpopulations of B cells defined either by different lineages or by different stages of development within a single lineage have requirements for distinct lymphokines that regulate their growth and differentiation.