Lectin-induced abnormalities of mouse blastocyst hatching and outgrowth in vitro

We have examined the role of cell surface glycoconjugates during mouse blastocyst maturation, hatching, attachment, and outgrowth by monitoring the influence of six lectins on blastocyst development in vitro. Two lectins, concanavalin A and wheat germ agglutinin were toxic to blastocysts at the concentrations used. Bandierea simplicifolia lectin 1 (BSL-1) induced abnormal growth, developmental arrest at the hatching stage, and some disruption of cell contacts. Culture with Lotus tetragonolobus lectin-1 (LTA-1) also disrupted cell contacts and caused developmental arrest. The remaining lectins, Dolichos biflorus agglutinin (DBA) and Ulex europaeus agglutinin (UEA), retarded blastocyst hatching and outgrowth but did not induce any major defects, although differentiation of the inner cell mass was limited by both. This study demonstrates that very low concentrations of lectins can disrupt blastocyst development, suggesting that exposed surface saccharide moieties may be involved in interactions between blastomeres and their environment.     

Lectin-induced differentiation of transformed neuroretinal cells in vitro.

The orderly course of chick neuroretinal cell differentiation was disrupted in vitro by infection with a temperature-sensitive strain of the Rous sarcoma virus (LA29). The resulting cell culture LA29NR remained mitotically active at 42 degrees C, yet rapidly adopted a transformed phenotype upon activation of the pp60v-src oncogene product at 37 degrees C. As a further indication of metabolic state, LA29NR cells expressed the protooncogene product c-Fos, as shown by Western blot analysis. Highly proliferative LA29NR cells proved refractory to standard differentiation agents such as cAMP, and prostaglandin E1. In our novel approach, succinylated concanavalin A (SCA), a nontoxic derivative of the lectin concanavalin A, induced dramatic, reversible morphological changes in LA29NR cells, including neurite outgrowth and increased cell-to-cell adhesion. Fluoresceinated SCA appeared to localize to Golgi and lysosomal structures. Cellular response to SCA treatment included decreased growth rate, reversible decrease in the phosphorylation state of a 41-kDa phosphoprotein, and induction of neuron-specific enolase. The glial marker vimentin was also evident in these cultures. These data suggest that SCA is an effective differentiation agent for cells of neuroectodermal origin, permitting neuronal as well as glial phenotypic expression within these cell populations.     

Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro

This study attempts to assess the developmental importance of cell surface glycoconjugates of preimplantation mouse embryos. This was done by incubating early embryos in various lectins and analyzing subsequent development. If specific cell surface glycoconjugates (lectin receptors) are linked to specific developmental processes, such as cell division, compaction, and blastocyst formation, then different lectins should block these different developmental processes. The results show that wheat-germ agglutinin (WGA; N-acetyl-D-glucosamine-specific) at 50 μg/ml prevents the cell division of four-cell embryos. However, this effect of WGA occurs only in embryos with intact zonae pellucidae. Concanavalin A (Con A; α-D-glucose and α-D-mannose-specific) treatment, 20 μg/ml, of four-cell or early eight-cell embryos prevents compaction, the first major change in cell shape in early mouse embryogenesis. Divalent succinly Con A does not affect development, suggesting that the Con A effect is due to crosslinking of cell surface glycoconjugates. Exposure of four-cell or early eight-cell embryos to 10 μg/ml Lotus Tetragonolobus puprureas agglutinin (LTA; α-L-fucose-specific) or 25 μg/ml Limulus polyphemus agglutinin (LPA; sialic acid-specific) allows compaction or development to the morula stage, but blocks blastocyst formation. All lectins tested retard cell division to some extent. Late morulae and early blastocysts are more resistant than earlier stages to all of the lectins studied. This study demonstrates that very low concentrations of these lectins affect different developmental processes, presumably based upon their sugar specificities.     

Selective effect of irradiation on responses to thymus-independent antigen

Low doses of ionizing radiation have a selective immunosuppressive effect on in vivo B cell responses to thymus-independent (TI) antigens. The B cell response, assayed as direct anti-trinitrophenyl (TNP)-specific plaque-forming cells (PFC), induced by type 2, TI antigens (TNP-Ficoll or TNP-Dextran), was reduced, on the average, by 10-fold in animals exposed to 200 rad of ionizing radiation 24 hr before antigen challenge. In contrast, PFC responses to type 1, TI antigens (TNP-lipopolysaccharide or TNP-Brucella abortus) are unaffected in mice exposed to the same dose of radiation. Adoptive transfers showed that this selective immunosuppression is a result of the specific inactivation of the B cell subpopulation responding to type 2, TI antigens. These experiments suggest that physiologic differences exist in the B cell subpopulations of normal mice which respond to type 1, or type 2, TI antigens.     

Functions of accessory cells in B cell responses to thymus-independent antigens

The functions of adherent accessory (A) cells in thymus-independent (TI) B cell activation were investigated using homogeneous A cell lines with distinct cell surface and functional characteristics, as well as inhibitors of antigen processing and interleukin 1 (IL 1) secretion. B cell responses to both type 1 and type 2 TI antigens were found to be strictly A cell dependent. Only A cells capable of IL 1 secretion could restore responsiveness in A cell-depleted spleen cells, regardless of Ia expression or antigen-processing capability. Moreover, recombinant IL 1 completely replaced A cell function in B cell responses to both TI 1 and TI 2 antigens. Finally, T cell depletion did not diminish the reconstitution by IL 1. Thus in contrast to T cell activation, IL 1 secretion is the only A cell function required in TI B cell activation, and the data are consistent with a direct role for IL 1 in B cell activation.     

Ontogeny of murine B-cell responses to thymus-independent trinitrophenyl antigens.

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP-Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP-Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP-Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic (nu/nu) BALB/c mice and their normal (nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.     

Temperature-mediated processes in teleost immunity: differential effects of temperature on catfish in vitro antibody responses to thymus-dependent and thymus-independent antigens

An in vitro culture system was employed to ascertain the effects of different temperatures on the anti-hapten antibody-secreting cell responses of channel catfish leucocytes to murine thymus-dependent (TD) and thymus-independent (TI) antigens. The magnitudes of primary responses to a TI antigen (TNP-LPS) and secondary responses to a TD antigen (DNP-KLH) were relatively independent of in vitro culture temperature. The kinetics of each of these responses as a function of temperature was characterized by a Q10 of 2. In contrast, the magnitudes of primary responses to TD antigens (DNP-KLH and DNP-HoSA) were suppressed at lower in vitro temperatures. Furthermore, it was observed that some of the low temperature suppression of primary responses to TD antigens could be abrogated by appropriate low temperature in vivo acclimation. These findings are interpreted as supporting the hypothesis that low environmental temperatures immunosuppress fish by virtue of differential inhibitory effects on the generation of carrier-specific helper cells.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Anaphylatoxin-mediated regulation of human and murine immune responses

C3a and C5a derived from the human complement components C3 and C5, respectively, were found to possess immunoregulatory activities. C3a was found to be capable of suppressing both antigen-specific and polyclonal antibody responses. In contrast, C3a was unable to suppress antigen- or mitogen-induced B or T cell proliferative responses. Helper T cells were found to be the target of C3a-mediated immunosuppression. Suppression occurred via the generation of suppressor T cells. In contrast to the results obtained with C3a, C5a was found to augment both antigen-specific and non-specific in vitro humoral immune responses. Moreover, C5a potentiated antigen- and alloantigen-induced T cell proliferative responses. As opposed to C3ades Arg-77, C5ades Arg retained all of the immunoregulatory activity associated with the intact molecule. Helper T cells are required for C5a-mediated potentiation of the Fc fragment-mediated polyclonal antibody response. Substitution for T cells by a soluble T cell-replacing factor rendered lymphocytes refractory to the enhancing properties of C5a.     

Stress-induced responses of human skin fibroblasts in vitro reflect human longevity

Unlike various model organisms, cellular responses to stress have not been related to human longevity. We investigated cellular responses to stress in skin fibroblasts that were isolated from young and very old subjects, and from offspring of nonagenarian siblings and their partners, representatives of the general population. Fibroblasts were exposed to rotenone and hyperglycemia and assessed for senescence‐associated β‐galactosidase (SA‐β‐gal) activity by flow cytometry. Apoptosis/cell death was measured with the Annexin‐V/PI assay and cell‐cycle analysis (Sub‐G1 content) and growth potential was determined by the colony formation assay. Compared with fibroblasts from young subjects, baseline SA‐β‐gal activity was higher in fibroblasts from old subjects (P = 0.004) as were stress‐induced increases (rotenone: P < 0.001, hyperglycemia: P = 0.027). For measures of apoptosis/cell death, fibroblasts from old subjects showed higher baseline levels (Annexin V+/PI+ cells: P = 0.040, Sub‐G1: P = 0.014) and lower stress‐induced increases (Sub‐G1: P = 0.018) than fibroblasts from young subjects. Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from young subjects (P = 0.017 and 0.006, respectively). Baseline levels of SA‐β‐gal activity and apoptosis/cell death were not different between fibroblasts from offspring and partner. Stress‐induced increases were lower for SA‐β‐gal activity (rotenone: P = 0.064, hyperglycemia: P < 0.001) and higher for apoptosis/cell death (Annexin V+/PI? cells: P = 0.041, Annexin V+/PI+ cells: P = 0.008). Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from offspring (P = 0.001 and 0.024, respectively) whereas rotenone‐induced decreases were lower (P = 0.008 and 0.004, respectively). These data provide strong support for the hypothesis that in vitro cellular responses to stress reflect the propensity for human longevity.     

Lectin-induced actin polymerization in human lymphocytes: A possible signal for mitogenesis

Employing the DNase I inhibition assay, a decrease in G-actin is demonstrated in human mononuclear cells following stimulation with mitogenic lectins concanavalin A (Con A) and phytohemagglutinin (PHA), as well as a nonmitogenic lectin, wheat germ agglutinin (WGA). The decrease in G-actin can be prevented by pretreatment of cells with cytochalasin E, indicating that the decrease is likely due to conversion to F-actin. Thus, the receptor-mediated actin polymerization is common to both the mitogenic as well as the nonmitogenic lectins. The maximal decrease in G-actin with Con A and PHA occurs at the same concentrations of the lectins that give optimal mitogenic responses. It is a distinct possibility that actin polymerization could be one of the signals necessary for the initiation of mitogenesis. The difference between a mitogenic and a nonmitogenic lectin may lie in the fact that a second signal (or signals), derived from macrophages, may not be generated by a nonmitogenic lectin such as WGA.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions.     

Lectin-induced alterations on the proliferation of three human prostatic cancer cell lines

Summary While lectins are known to influence the cell growth of several types of normal and neoplastic tissues, their roles in the case of prostatic cancer cells remain relatively unexplored. In the present work, we report thein vitro influence of five lectins, namely peanut (PNA), wheat germ (WGA), Concanavalin A (Con A),Griffonia simplicifolia (GSA-IA₄), andPhaseolus vulgaris (PHA-L) agglutinins, on the cell proliferation of one androgen-sensitive (LNCaP) and two androgen-insensitive (PC-3 and DU 145) human prostatic cancer cell lines cultured in either 10% or 1% fetal bovine serum (FBS)-supplemented media. The cell proliferation was assessed by means of the colorimetric 3-(4,5-dimethythiazol-2-yle)2,5-diphenyltetrazolium bromide. (MTT) assay. Four lectin concentrations were tested (i.e., 0.1, 1, 10, and 100 μg/ml) at five experimental states (i.e., 2, 3, 5, 7, and 9 d following the addition of each lectin to the culture media). Our results demonstrated that the five lectins under study had a globally significant dose-dependent toxic effect on prostatic cancer cell proliferation. Nevertheless, low doses of GSA-IA₄ and PHA-L significantly (P<0.05 toP<0.001) increased the cell proliferation of confluent PC-3 cells. Increasing the FBS from 1% to 10% in the culture media significantly antagonized lectin-induced toxicity in the three prostatic cell lines. In conclusion, the present data strongly suggest that some lectins might influence the proliferation of prostatic carcinoma cells. In addition, because lectins are present in our diet, and are able to pass into the systemic circulation and thus reach the prostate, the present results suggest that some lectins might exert an influence on prostate cancer growth under clinical conditions.     

In vitro effects of retinoids on murine thymus-dependent and thymus-independent mitogenesis

The effects of three retinoids: all-trans-retinoic acid (RA), 13-cis-retinoic acid (13-cis-RA), and N-(4-hydroxyphenyl)retinamide (4-HPR) on murine splenic lymphocyte proliferative responses to mitogens were evaluated. The responses to T-cell mitogens, PHA and Con A, and a T-cell-dependent B-cell mitogen, PWM were significantly potentiated by these retinoids. However proliferative responses to a B-cell mitogen, Escherichia coli LPS were unaffected or inhibited. All three retinoids at concentrations ranging from 10(-6) to 10(-15) M significantly potentiated Con A-induced proliferative responses. In response to PWM, 10(-13) M RA, 10(-12) M 13-cis RA, and 10(-11) M 4-HPR were the lowest concentrations producing significant potentiation. Endpoint concentrations of retinoids significantly potentiating responses to PHA were; 10(-9) M RA, 10(-8) M 13-cis RA, and 10(-6) M 4-HPR. These responses were independent of retinol contained in fetal calf serum supplemented medium since responses were reproduced in serum-free medium devoid of retinol. Optimal potentiation by retinoids of responses to these T-cell-dependent mitogens were found at superoptimal concentrations of mitogen suggesting a selective inhibition of T-suppressor cells. Thus, potentiation of T-cell-dependent mitogen responses provides the most sensitive biological assay yet described for detection of retinoid activity and is a reproducible system to explore the cellular and molecular mechanisms of retinoid-mediated immunopotentiation.     

Interferon inhibition of the primary in vitro antibody response to a thymus-independent antigen

Partially purified and crude mouse L cell interferon preparations inhibited the in vitro plaque-forming cell (PFC) response of mouse C57B1/6 spleen cells to the T-cell independent lipopolysaccharide antigen of Escherichia coli 0127. PFC responses of 5-day cultures were inhibited approximately 70–90% by 100–200 NIH reference units of interferon/culture. A similar inhibitory effect was obtained with spleen cells from athymic (nude) mice homozygous for the nu/nu allele. Spleen cultures depleted of adherent cells were also inhibited in their anti-0127 PFC response by interferon. Interferon, then, appears capable of inhibiting the PFC response to E. coli 0127 via direct action on B cells. Heating experiments along with the use of interferon preparations of different specific activities suggest that the inhibition was due to the interferon in the preparations.     

Hormonal modulation of responses to thymus-independent and thymus-dependent antigens in autoimmune NZB/W mice

Previous work suggested that gonadal steroids influence immunity through the thymus, but the mechanisms were unclear. To investigate the effects of these hormones on immune responses to T1 and TD antigens in autoimmune mice, we studied hybrid NZB/W mice and the nonautoimmune DBA/2 strain. Mice castrated at 14 days of age were implanted with Silastic capsules releasing, in adults, physiologic levels of E2 in males or Te in females. Sham-operated controls received empty capsules. Splenic PFC were quantified 4 to 5 days after challenge with the TI2 antigen TNP-Ficoll, the TI1 antigen TNP-LPS, or the TD antigen SRBC. Young castrated NZB/W males implanted with E2 had striking enhancement of IgM responses to TNP-Ficoll when compared to castrated Te-treated females and comparable sham-operated controls of both sexes. E2 also stimulated responses to TNP-LPS. In response to challenge with SRBC, young E2-treated NZB/W males had a consistent trend to increased IgM PFC, and the stimulatory effect of E2 on IgG plaques was variable. Physiologic doses of Te had no consistent effect on responses in young mice. In old female NZB/W mice, Te caused PFC response after immunization with TNP-Ficoll to resemble age-matched NZB/W males. As sham-operated NZB/W females grew older, PFC responses to SRBC fell. This age-related phenomenon was delayed, however, in female castrates implanted with Te. In contrast, Te clearly suppressed responses to TNP-LPS. Implantation of E2 did not alter responses to TNP-Ficoll, TNP-LPS, or SRBC in nonautoimmune DBA/2 males. This finding suggested that exogenous E2 given in physiologic doses did not influence immunologic responsiveness in a normal strain to the degree seen in hormone-sensitive NZB/W mice. It was concluded that E2 enhanced responses to a variety of exogenous antigens in autoimmune NZB/W mice. The most consistent E2-induced increase in PFC response was observed with TI antigens, suggesting that E2 exerted its effects on B cells or Ts.