Signaling complex formation of CD44 with src-related kinases.

The complex formation of murine CD44 with the src-like protein tyrosine kinases, lck and lyn, was investigated. In accordance with previous observations, stable CD44-lck and CD44-lyn complexes were detected in nonstimulated lymphoid T- and B-cells, respectively. In addition, a direct modulation of lck and lyn by CD44 was observed as revealed by the CD44-dependent translocation of these enzymes to the Triton X-100 resistant cell fraction. To clarify which receptor domain is responsible for the association, peptide binding assays were performed. Interestingly, the synthetic peptide pCD44 (ILAVCIAVNSRRR), which corresponds to the plasma membrane-cytoplasmic interface region of murine CD44, exhibited a high capacity to bind lck and lyn. A single amino acid modification in the position of the cysteine residue completely abolished this interaction, while the truncation of the three tandem arginines significantly decreased it. Remarkably, similar sequences were found in a number of other molecules including subunits of receptors recognizing antigens, immunoglobulins, extracellular matrix components, accessory molecules, cytokines and also in certain viral gene products. Synthetic peptides corresponding to the homologous regions found in CD28 and FcepsilonRIbeta were also studied and comparable lck-lyn-binding potentials were detected. These data suggest a novel interaction between src-family kinases and CD44, CD28, FcepsilonRIbeta, and provide a simple model for the association of src-like kinases with transmembrane proteins.

Membrane-bound ezrin is involved in B-cell receptor-mediated signaling: potential role of an ITAM-like ezrin motif.

Ezrin is a cytoskeleton-plasma membrane linker molecule which is implicated in the T-cell antigen receptor signaling as one of the major tyrosine phosphorylated components. Its function in B-lymphocyte activation has not yet been clarified. Here we studied the potential involvement of ezrin in the B-cell receptor (BCR) signaling in BL41 Burkitt lymphoma cells. Our data demonstrate that ezrin, which shows predominantly cytosolic distribution in unstimulated cells, undergoes only a moderate tyrosine phosphorylation in response to BCR triggering, with no concomitant translocation of the protein from the cytosol to the plasma membrane. Instead, BCR-independent stimulants like oxidant stress induced by phenylarsine oxide, resulted in rapid redistribution of ezrin to the plasma membrane. When BCR triggering was preceded by membrane recruitment of ezrin, it became one of the main and earliest substrates of tyrosine kinases activated by BCR. No detectable influence on distribution or phosphorylation of ezrin was triggered by the tyrosine phosphatase inhibition by orthovanadate, suggesting that these effects of phenylarsine oxide are not attributable to its tyrosine phosphatase inhibitory capacity. The notion that BCR-mediated phosphorylation of ezrin negatively correlates with activation events such as phosphorylation of tyrosine kinase, syk and induction of calcium mobilization response, suggests that ezrin might be implicated in the regulation of transmembrane signaling and cellular responsiveness. As will be discussed, the regulatory function of ezrin may be due to an immunoreceptor tyrosine-based activation motif (ITAM)-like sequence.

Rapid desensitization of B-cell receptor by a dithiol-reactive protein tyrosine phosphatase inhibitor: uncoupling of membrane IgM from syk inhibits signals leading to Ca2+ mobilization.

B-cell antigen receptor (BCR)-mediated calcium response can be blocked by phenylarsine oxide (PAO), a dithiol group-reactive protein tyrosine phosphatase inhibitor. We have examined the mechanism of this inhibition in BL41 Burkitt lymphoma cells. PAO-dependent inhibition is not restricted to the BCR-mediated functions, as evidenced by the failure of the same cells to mobilize Ca2+ in response to CD19 cross-linking. In contrast, calcium response induced by a putative syk activator, H2O2, exhibited only a moderate sensitivity to PAO, demonstrating that PAO did not cause general suppression of all the functions leading to Ca2+ mobilization. BCR cross-linking or H2O2 treatment leads to the induction of almost complete non-responsiveness for the reciprocal stimulation. Since BCR cross-linking did not generate non-responsiveness to H2O2 in the presence of PAO, and PAO-treated cells remained responsive to syk activation by H2O2, we suppose that PAO may inhibit BCR-mediated signal transduction events upstream of syk activation. This assumption was supported by additional data, indicating that PAO was able to modulate functions of at least 2 different protein tyrosine kinase enzymes involved in BCR-mediated signaling. PAO induced rapid and dose-dependent tyrosine phosphorylation of lyn and selectively inhibited BCR-mediated tyrosine phosphorylation of syk. The results presented in this paper demonstrate that PAO may provoke cellular desensitization process by alteration of the signal transducer functions of lyn and syk tyrosine kinase enzymes.

The alternative splicing of human Fc gamma RII mRNA is regulated by activation of B cells with mIgM cross-linking, interleukin-4, or phorbolester.

The human type two IgG binding receptors (Fc gamma RII) are encoded by three genes (Fc gamma RIIA, -B and C) resulting in at least six protein isoforms generated by alternative mRNA splicing. Surface expression of Fc gamma RII has been shown to be modulated during B cell activation, although data characterizing the isoform(s) expressed are not available. The extracellular as well as the transmembrane domains of various Fc gamma RII are highly homologous. Only the intracellular domains vary between the different Fc gamma RII isoforms, suggesting differences in signal transduction. Using reverse transcriptase and polymerase chain reaction of mRNA obtained from resting tonsil B cells, we show that the majority of Fc gamma RII mRNA species to be of b2 type, although b1 type and a low level of Fc gamma RIIa type are also present. Culturing the cells for 18 h in the presence of 2.5 U/ml interleukin-4 or 10 micrograms/ml affinity-purified anti-IgM F(ab')2 fragments induced a switch in alternative splicing, resulting in a significant increase of Fc gamma RIIb1 mRNA expression, while the synthesis of Fc gamma RIIb2 mRNA was down-regulated. Stimulation of B cells with 100 ng/ml phorbol 12-myristate 13-acetate induced similar alteration, although only after 48-h treatment. The accumulation of Fc gamma RIIb1 and the reduction of both Fc gamma RIIb2 and Fc gamma RIIa mRNA in activated cells is accompanied by the enhanced expression of Fc gamma RII on the cell surface, representing most probably the Fc gamma RIIb1 isoform. Heat-aggregated IgG inhibited the anti-IgM-induced proliferation of resting but not that of activated B cells, suggesting that aggregation of Fc gamma RIIb2 constitutively expressed on resting B cells might be responsible for the prevention of inadequate activation of resting B cells.

Interaction of signaling molecules with human Fc gamma RIIb1 and the role of various Fc gamma RIIb isoforms in B-cell regulation.

The low-affinity type-IIb IgG Fc-binding receptors (Fc gamma RIIb) are expressed on B cells. When cross-linked with mIgM Fc gamma RIIb are known to down-regulate B-cell activation by interrupting signal transduction upstream from G-protein-activated events. We have studied Fc gamma RII isoforms expressed on resting and activated B cells and the interaction of Fc gamma RIIb1 with molecules transducing the antigen receptor-mediated signals. Expression of Fc gamma RII isoforms was studied by reverse transcription and polymerase chain reaction. Resting B cells express both Fc gamma RIIb2 and Fc gamma RIIb1 isoforms. Activation with anti-IgM or IL-4 induces the splicing of Fc gamma RIIb1 mRNA, while the alternative splicing of Fc gamma RIIb2 mRNA is down-regulated, resulting in the surface expression of Fc gamma RIIb1. Functional differences were found between the two isoforms in inhibiting B-cell activation, suggesting that Fc gamma RIIb2 might influence the threshold of signals necessary for activation of resting B cells, while Fc gamma RIIb1 may regulate in later phases of antibody response. To explore the mechanism by which Fc gamma RII may uncouple antigen receptor-mediated signal transduction, we have investigated the association of signaling molecules with Fc gamma RII. Beside the protein tyrosine kinase (PTK) fyn, protein kinase C (PKC) was found to be co-isolated with Fc gamma RIIb1, suggesting a tight connection between these kinases and Fc gamma RII. We suggest that PKC might be responsible for the activation-induced phosphorylation of Fc gamma RII on serine residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenylarsine oxide (PAO) blocks antigen receptor-induced calcium response and tyrosine phosphorylation of a distinct group of proteins.

Antigen receptor (AgR) crosslinking by antigens or AgR-specific antibodies induces a cascade of enzymatic events in lymphocytes which involves activation of several non-receptor tyrosine- and serine/threonine kinases, phosphatases, phospholipases, etc. Here we show data demonstrating that a thiol group-reactive protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide (PAO), uncouples a crucial part of the signaling events induced by anti-IgM or anti-Leu-4 (CD3) in human tonsil B lymphocytes, BL41 and Daudi B cell lines and Jurkat T lymphoma cells. PAO treatment (10 microM) resulting in distinct modification of AgR-induced tyrosine phosphorylation pattern inhibited the AgR-mediated calcium response (Ca++ release and influx) of all of these cells completely. Since this treatment did not alter the cell viability and the binding capacity of the AgR crosslinking antibodies, alteration of the tyrosine phosphorylation pattern and blockage of the calcium response indicate prompt inactivation of essential signal transduction element(s).

A trypsin-like serine protease activity on activated human B cells and various B cell lines.

We have studied the trypsin-like serine protease activity of human tonsillar B lymphocytes. The lysate of the low-density, in vivo activated B cells as well as the lysate of cells stimulated with anti-human IgM F(ab')2 show elevated trypsin-like serine protease activity compared to the resting subset as monitored by the cleavage of Tos-Gly-Pro-Arg-pNA. The cleavage is sensitive to N-tosyl-L-lysyl-chloromethyl ketone and benzamidine but not to iodoacetamide. Experiments with intact cells give similar results. The finding that the intact cells hydrolyze the substrate, while their supernatant does not, suggests that the protease activity is cell membrane associated. It is possible that C3 is a substrate of the enzyme since the activated B cells cleave C3, whereas the resting B cells do not, and also C3 inhibits the enzyme-substrate reaction. In addition to the ex vivo B cells, we studied the serine protease activity of certain well-characterized B cell lines. The results show a correlation between the phenotype and the enzyme expression of the cell lines. BL41, an Epstein-Barr virus (EBV)-negative Burkitt lymphoma line, with a resting phenotype, has low activity, while its EBV genome-carrying convertants E95-A-BL41, E95-C-BL41, EHR-A-BL41 and BL41/95 that have the phenotype of activated B cells, have high proteolytic activity. The lymphoblastoid cell line WW-1-LCL which has the phenotype of an immunoblast, has the highest serine protease activity. On the basis of the above data, we suggest that a rather tight correlation exists between the degree of activation and the appearance of serine protease(s) on the surface of human B cells.

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.

Modulation of type II Fc gamma receptor expression on activated human B lymphocytes.

We have monitored Fc gamma RII expression during the activation of human B lymphocytes by simultaneous analysis of monoclonal antibody (mAb) binding and EA rosetting. The expression of Fc gamma RII showed a biphasic time course. Initially, a transient increase of Fc gamma RII with no ligand-binding capacity was observed with mAb staining as early as 10 min after stimulation by the F(ab')2 fragment of anti-human IgM or phorbol 12-myristate 13-acetate and then after 3 to 24 h a decrease in the number of Fc gamma RII+ cells was seen. Trypsin-like serine protease activity also appeared in the lysate of activated B cells at this time. On the 2nd day of activation a significant enhancement of Fc gamma RII expression was observed, mainly on enlarged blast cells as monitored by both mAb and by ligand binding (EA rosette). At the same time, soluble fragments of Fc gamma RII with the ability to bind human Fc were detected in the supernatant of activated B cells, probably as a result of proteolytic cleavage. These findings suggest that activated B cells are identical with the population of mononuclear cells which shed Fc gamma R when incubated at 37 degrees C. The ability of activated but not resting B cells to release Fc gamma RII correlates with the expression of early activation markers and with the appearance of a trypsin-like serine protease activity of the same cells; thus, the release of Fc gamma RII occurs in the early G1 phase of cell cycle as a result of proteolysis. Later the release of Fc gamma RII is accompanied by the enhancement of Fc gamma RII expression before the cells reach the S phase. The fragments of cleaved Fc gamma RII had an apparent molecular mass of 33 and 14-18 kDa under nonreducing conditions, and upon reduction fragments of smaller size were observed.

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules.

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.

Distinctive role of IgG1 and IgG3 isotypes in Fc gamma R-mediated functions.

Polyclonal and monoclonal anti-Rh (D) antibodies of IgG1 and IgG3 subclass were evaluated for their capacity to sensitize erythrocytes and (i) to trigger monocyte and K-cell mediated antibody-dependent cellular cytotoxicity (ADCC); (ii) to mediate binding to monocyte and lymphocyte Fc gamma R; (iii) to stimulate phagocytosis by monocytes. All antibodies were equally effective in mediating monocyte or activated U937 cell ADCC but IgG1 was more active than IgG3 in K-cell mediated ADCC. IgG3-sensitized erythrocytes inhibited IgG1-induced lysis, suggesting that each subclass engages the same Fc gamma R receptor but that lysis requires a further 'signal' that the IgG3 molecule can not deliver. Two monoclonal IgG3 anti-D antibodies were shown to have higher binding (two times) and phagocytic (three times) indices than IgG1 antibody for monocytes; similar differences were observed for polyclonal IgG1 and IgG3 antibodies. The same pattern was observed in an EA rosette assay when a total lymphocyte population was used; however, this difference was not seen with a B-cell depleted (T+ null cell) lymphocyte population.

PHA- and Con A-induced chemiluminescence of human blood mononuclear cells and granulocytes in luminol or lucigenin.

Phytohemagglutinin (PHA) and concanavalin A (Con A) can evoke a chemiluminescence (CL) response both in granulocytes and blood mononuclear cells. We have used two parallel systems to compare the quantity and quality of oxygen radicals produced during activation. While the luminol-enhanced CL response is linked to the myeloperoxidase-H2O2-chloride system, the lucigenin-dependent light production measures only the superoxide radical. PHA produced higher CL response in the presence of luminol than with lucigenin. Con A showed high CL response only in the lucigenin-enhanced system. The results suggest that while Con A induces mostly superoxide production, the membrane stimulus evoked by PHA produces light through the myeloperoxidase-H2O2-halide system. Granulocytes are less sensitive to PHA than the blood mononuclear cells. The sensitivity of the responses to several scavengers and enzymes support the differential production of oxygen radicals following activation via these two lectins.