PACSIN2 is a regulator of the metalloprotease/disintegrin ADAM13.

ADAM13 is a cell surface metalloprotease expressed in cephalic neural crest cells during early Xenopus development. The cytoplasmic domain of ADAM13 contains three potential SH3 (Src homology type 3) binding sites, suggesting that this region may support interactions with intracellular proteins. In this report we describe the identification, by a new strategy, of three proteins that bind the ADAM13 cytoplasmic domain in vitro: X-Src1, X-An4, and X-PACSIN2. We focused our study on X-PACSIN2 protein because it colocalizes with ADAM13 in migrating neural crest cells during embryonic development. Using pull-down experiments we show that X-PACSIN2 binds to ADAM13 in vitro. Using Xenopus XTC cells, we demonstrate that ADAM13 and X-PACSIN2 colocalize to membrane ruffles and cytoplasmic vesicles. We also show that X-PACSIN2 overexpression can rescue developmental alterations induced by overexpression of ADAM13, suggesting that both proteins interact in vivo. Finally, our results suggest that X-PACSIN2 overexpression reduces endogenous ADAM13 function while a truncated X-PACSIN2 (DeltaSH3) increases this activity in cephalic neural crest cells. We propose that X-PACSIN2 may regulate ADAM13 activity by influencing either its subcellular localization or its catalytic activity. In agreement with this model, elimination of the ADAM13 cytoplasmic domain increased developmental alterations attributable to ADAM13 proteolytic activity.

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

A mouse placental immunoregulatory factor different from transforming growth factor beta.

Of the growth-promoting factors, transforming growth factor beta (TGF-beta) has been most clearly shown to act as a potent regulator of inflammation and immunity. It is highly suppressive for T and B lymphocyte proliferation, cytotoxic T lymphocyte generation, and lymphokine-activated killer cell development, as well as natural killer cell activity. Moreover, there is accumulating evidence that TGF-beta also may contribute to impaired immune surveillance of tumor development. In previous work, we isolated and described a 40 kD glycoprotein extracted from mouse placenta. This placental factor (PF) is also a potent immune modulator in vivo: it is highly inhibitory of secondary antibody responses as well as cellular responses, such as local graft-versus-host reactions. Because placenta has been shown to be a major source of TGF-beta and several reports have indicated an important role for TGF-beta in the immunosuppressive mechanisms taking place during the course of mammalian gestation, we have looked for the presence of TGF-beta in our placental factor preparations. Our results clearly indicate that they do not contain TGF-beta or TGF-beta-like molecules by the following criteria: (1) no inhibition of Mv-1 Lu cell proliferation at any dose tested; (2) no band detected by immunoblotting using different polyclonal reagents specific for TGF-beta 1; and (3) no activity retained on or eluted from an affinity column made of immobilized monoclonal antibody against TGF-beta 2. Aliquots of the same preparations retained their full immune inhibitory capacity in vivo throughout the various assays.(ABSTRACT TRUNCATED AT 250 WORDS)

A mouse monoclonal antibody specific for the V beta 5.3 chain of the human TcR recognizes a subgroup of the mouse TcR V beta 8.2 chains.

We have analyzed the reactivity of a mouse monoclonal antibody directed against the human T cell receptor for antigen (TCR). This antibody (111-427) of immunoglobulin G1 isotype has been produced in a BALB/c mouse immunized with HPB-ALL cells and normal human peripheral blood leukocytes. It reacts specifically with the HPB-ALL lymphoma and 2 to 7% of normal human blood lymphocytes, on which it has a mitogenic effect in vitro. We have shown that it immunoprecipitates the alpha beta TCR of HPB-ALL and that it is specific for the V beta 5.3 chain of the human TCR. In addition, we have observed that this antibody stains a minor fraction of T lymphocytes in different strains of mice. We have screened a number of murine T cell clones or hybridomas and have found that the T cell hybrid line DO.11.10.S4.4 is positive. We have been unable to immunoprecipitate reproducibly the molecule recognized by 111-427 after 125I cell surface labeling and cell lysis in NP-40 or digitonin, probably because of low-affinity binding. On Western blotting, 111-427 revealed one band that has an apparent molecular mass of 89 kDa in nonreducing conditions and disappears after reduction. Similar results were obtained in parallel with the F23.1 and F23.2 antibodies. Thus, this antibody appears to recognize an epitope present primarily on the V beta 8.2 chain of the mouse TCR. We have assayed its capacity to stimulate splenic T lymphocytes in vitro. We have observed that it is capable of triggering, to a minor degree in soluble form and very effectively when coupled to Sepharose beads, the proliferation of spleen T lymphocytes from mice chronically infected with the blood parasite Trypanosoma cruzi.

Biochemical and functional characterization of an avian homolog of the integrin GPIIb-IIIa present on chicken thrombocytes.

We have analyzed the reactivity of a new mouse monoclonal antibody (mAb), 11C3, which identifies a cell marker detected on the surface of chicken thrombocytes. Tissue distribution studies have shown that only cells of the thrombocytic lineage in blood, spleen, and bone marrow are stained by 11C3. However, it does not react with other species such as quail, mouse, and man. The 11C3 mAb immunoprecipitates an heterodimeric molecule made of two bands with an apparent molecular weight of 112 and 90 kDa under nonreducing conditions and 112 and 26 kDa following reduction. This pattern of migration is similar to the one observed for members of the integrin family of cell adhesion molecules. We have used the previously described mAb AP-2, which is specific for the human platelet integrin GPIIb-IIIa and cross-reacts with chicken thrombocytes. We have shown that it immunoprecipitates two bands with an identical electrophoretic mobility. Cross-inhibition and immunodepletion studies reveal that the two antibodies recognize two different isoforms or two conformational variants of the same molecule. Moreover, our data demonstrate that in contrast with AP-2, 11C3 is a potent inducer of thrombocyte activation measured by cell aggregation, chemiluminescence, or release of [3H]serotonin. It also inhibits the adhesion of thrombin-activated thrombocytes to fibrinogen and, to a lesser degree, to fibronectin, in a dose-dependent manner. Altogether, these results indicate that this antibody identifies the avian homolog of the mammalian platelet integrin and fibrinogen receptor GPIIb-IIIa.

Functional and biochemical properties of a mouse placental immunoregulatory factor.

The authors have a long standing interest in immune regulations which control the absence of rejection of a semi-allogeneic fetus by the mother. A previous work described a soluble 40 kDa factor extracted from mouse placenta and capable of inhibiting secondary immune responses in vitro. The present paper reports the following on its mode of action in vivo: (1) it is active even in a fully allogeneic host; (2) it can be administered i.v. or i.p. along with antigen; and (3) the injections of factor and antigen must not be more than 2 days apart for maximum efficacy. Moreover, the results of the study described here indicate also that this factor is a concanavalin A-binding glycoprotein, sensitive to heat and pronase, and different from interleukin 10 (IL-10). Thus, this placental factor appears to be different from previously described immune regulators such as IL-10 and could contribute significantly to immune regulations at the level of the placenta.

A new marker on chicken hematopoietic cells is defined by a monoclonal antibody raised against a V beta chain of the human TCR.

In this paper, we show that a mouse monoclonal antibody, 111-427, specific for the V beta 5.3 chain of the human T-cell receptor (TCR) for antigen, also reacts with chicken hematopoietic cells. Our data indicate that the majority of 111-427 positive cells among peripheral blood leucocytes (PBL) are thrombocytes. This antibody also recognizes two in vitro cell lines, III-C5, and IL-2-dependent T-cell line and HD11, a macrophage cell line. In addition, erythrocytes and a minor subpopulation of thymus and spleen cells are also stained by the monoclonal antibody (mAb). No specific immunoprecipitation could be detected from 125I radiolabeled cell lysates. By Western blotting techniques, the 111-427 mAb identifies a single band of apparent molecular weight 91 kD, unaffected by reduction, from III-C5 and HD11 cell lysates. This band is absent in negative cell control lysates. On thrombocytes, the apparent molecular weight of the band is shifted to 87 kD. These results indicate that the mAb does not recognize the chicken T-cell receptor for antigen, but a cell surface marker shared primarily between thrombocytes and erythrocytes. This new chicken cell marker is compared to other cell surface markers in avian or mammalian species that present some analogies in their tissue distribution.

Comparison between autoantibodies arising during Trypanosoma cruzi infection in mice and natural autoantibodies.

The autoantibodies induced in (C57BL/6 x BALB/c)F1 mice during Trypanosoma cruzi (CL strain) infection were analyzed and compared with natural autoantibodies present in healthy mice. Mice were killed at intervals after infection and their sera were tested by enzyme immunoassay against a panel of self- and non-self-Ag: actin, myoglobin, myosin, tubulin, DNA, and TNP-OVA. The level of IgM and IgG autoantibodies against all Ag started to increase from day 15 until 6 wk after the parasite infection. The high level of all autoantibodies persisted 3 mo postinfection, and 1 yr later, half of the mice still had elevated levels of IgM and IgG autoantibodies, particularly antitubulin IgG antibodies. IgM and IgG were isolated from pools of normal and infected mouse sera and their binding capacity to all Ag was compared. The titers of infected mouse sera were increased and the slopes of both IgM and IgG binding curves of autoantibodies to actin, myosin, and tubulin were greater than those of control mouse sera, indicating higher affinities. The average dissociation constant of the IgG2a autoantibody to mouse tubulin was 5 times lower than that of natural antitubulin IgG2a antibodies. Furthermore, absorption of the IgG from infected mouse sera onto a tubulin immunoadsorbent removed half the reactivity with tubulin and also with myosin, actin and parasite extracts. The eluted antibodies bound the same Ag. When IgG were further analyzed by Western blot on proteolytic fragments of tubulin, we found that antibodies from both groups bound to the same broad spectrum of polypeptide bands. However, additional fragments were recognized by antibodies from infected mice. All these results indicate that the autoantibodies naturally present in mice are significantly affected after infection with T. cruzi, in quantity as well as in specificity and affinity.

Antigen-specific modulation of graft-versus-host reactions by two distinct placental factors.

We have previously shown that two distinct mouse placental fractions (PF) are potent immunomodulators in vivo. A 40 kDa PF induces a marked decrease of plaque forming cell (PFC) responses, while a 60 kDa PF increases them. Both effects are specific for the priming antigen. In the present study, these two PF are assayed on a cell-mediated response to allogeneic cells, i.e. in a local graft-versus-host reaction (LGVHR). Mice were primed with allogeneic cells in the presence of various amounts of the 40 kDa or 60 kDa PF, or liver extract (LE) as control. Six days later, their spleen cells were injected into the footpads of F1 recipients. Precise dose-response curves were established and the kinetics of the GVH response were carefully followed. Parallel with the modulation of PFC responses, the 40 kDa PF caused a potent inhibition of the LGVHR, while the 60 kDa PF greatly enhanced it. Both effects were specific for the alloantigens injected with the PF. Furthermore, we showed that these modulations were observed whatever the intensity of the GVH reaction, which varied according to the number of primed spleen cells transferred. This report also demonstrates that these PF can be greatly enriched by passage over affinity columns made of insolubilized lectins. The 40 kDa PF is retained on and can be eluted from columns of insolubilized concanavalin A (Con A) or wheat germ agglutinin (WGA), which indicates that it is a glycoprotein. Conversely, the 60 kDa PF does not bind to any of the above lectins and is probably not a glycoprotein. This biochemical purification step is also a good procedure for obtaining an even cleaner separation of the two fractions from each other. Thus, this paper demonstrates that both PF have important regulatory properties on specific cellular immune responses.

T cell tolerization in vitro: modulation of helper and suppressor cell activities.

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.

Simultaneous responses to TD, TI-1 and TI-2 antigens originate from both specific and multipotent precursors.

The classification of antigens into TD, TI-1 and TI-2 varieties raises the question of whether responses to these antigens are produced by distinct or identical subpopulations of B cells. In the present study we have examined the extent of intraclonal specificity variation in the progeny of PFC appearing after stimulation with two unrelated antigens. Mouse lymphoid cells were stimulated with pairs of TD and TI antigens, PFC were individually cultured and daughter PFC examined for their specificity. In all combinations used, PFC responding to TD antigen engendered, after 48 h of culture, a high frequency of PFC daughters expressing one or the other antibody specificity, notwithstanding the specificity of parental PFC. However, PFC responding to TI antigens seemed less subject to variation in specificity, and PFC daughters engendered after a 48 h culture period were, in the majority, of the parental specificity. These results are analysed in relation to different subpopulations of B cells.

Modulation of mouse anti-SRBC antibody responses by placental extracts. II. Antigen specificity and regulatory role of B and T cell populations affected by two distinct placental fractions.

Previous results from our group had shown that when CBA mice are primed to sheep red blood cells (SRBC) in the presence of various doses of placental extract (PE) (or liver extract [LE] as control), their spleen cells injected into normal syngeneic recipients have important immunoregulatory properties. Low doses of PE (0.25 to 4 mg per mouse) induce a marked decrease of the PFC response against SRBC in recipient animals. In contrast, higher doses of PE (8 to 13 mg per mouse) have a potentiating effect on the same response. LE is not different from a saline injection, at any dose. The suppressive and enhancing effects can be reproduced with two distinct placental fractions (PF) of 40 KD and 60 KD, respectively. In the present report, we have studied the requirement for an antigenic stimulation at the same time as the injection of PE, and the antigenic specificity of the subsequent immunoregulatory effects. In addition, we have analyzed the functional properties of the spleen cell populations affected by PE or placental fractions: surface Ig- cells mediate the suppressive effect due to low doses of PE or the 40-KD fraction, whereas surface Ig+ cells are responsible for the enhancing effect due to high doses of PE or the 60-KD fraction. These immunoregulatory activities do not appear to be related to the presence of Ig fragments in PF, because our results have shown that no Ig fragments can be detected in either PF. Surface Ig- T cell populations from spleen cells treated with the 40-KD fraction and antigen have been further separated into Lyt-2- and Lyt-2+ subpopulations. Our results showed that Lyt-2+ cells alone suppress the PFC response anti-SRBC in both normal and irradiated syngeneic recipients. Thus, the injection of a 40-KD PF in the presence of antigen activates splenic T suppressor cells capable of specifically regulating a secondary antibody response in vivo.

[Cells secreting antibodies originate from cells secreting immunoglobulins without a detectable antibody function for the antigen injected]. / Des cellules sécrétrices d'anticorps onto pour origine des cellules sécrétrices d'immunoglobulines sans fonction anticorps décelable pour l'antigène injecté.

Cells secreting immunoglobulins without detectable antibody function arising after an injection of horseradish peroxidase were micromanipulated from the center of haemolytic plaques of Sheep red blood cells coated with anti-Ig antibodies. These cells were cultured individually for 48 hrs, with irradiated cells as feeder layer and in the presence of the immunogen and of LPS. It was shown that after this time 22% of the immunoglobulin-secreting cells had generated antiperoxidase antibody-secreting cells or were transformed into antibody-secreting cells.

Evidence for multipotentiality of antibody synthesizing cells.

Immunization of mice with two unrelated antigens or antigenic determinants regularly results in the appearance of hemolytic plaque forming cells (PFC) of each specificity and of some PFC (1-4%) reacting to both determinants. Micromanipulation of individual double PFC appearing after immunization with trinitrophenyl conjugated sheep erythrocytes (TNP-SRBC) into media containing indicator erythrocytes (native SRBC and TNP-horse RBC) and a soluble specific inhibitor (TNP-BSA or soluble SRBC antigen), showed that the specific inhibitor suppressed the lysis of the corresponding indicator but did not interfered with the lysis of the unrelated indicator. Persistance of one specific activity in spite of complete inhibition of the other indicated that these double PFC synthesize two different antibody molecules. The destiny of double cells was studied in individual cell cultures by micromanipulating them into wells containing heavily irradiated normal mouse spleen cells and both determinants (TNP-SRBC). Those double cells which divided they generated in 24 hours monospecific daughter PFC (either anti-TNP or anti-native SRBC). Individually cultured monospecific PFC from mice immunized with one antigen (native SRBC) generated daughter PFC of the same specificity. By contrast, monospecific PFC from mice immunized with TNP-SRBC generated PFC of either the same specificity of (more frequently) of the one the other specificity. Thus, immunization with substituted erythrocytes resulted in the development of three clonotypes: two clones each composed only of cells of either specificity (amphispecific clonotype). For the maintenance of this amphispecific clone, continuous presence in the culture media of both antigenic determinants was necessary. Removal of the one for 48 hours (but not for 24 hours) abolished the generation of daughter cells of the same specificity.