Human umbilical cord blood-derived mesenchymal stromal cells display a novel interaction between P-selectin and galectin-1.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been shown to exert immunomodulatory properties that have great potential in therapies for various inflammatory and autoimmune disorders. However, intravenous delivery of these cells is followed by massive cell entrapment in the lungs and insufficient homing to target tissues or organs. In targeting to tissues, MSCs and other therapeutic cells employ similar mechanisms as leucocytes, including a cascade of rolling and adhesion steps mediated by selectins, integrins and their ligands. However, the mechanisms of MSCs homing are not well understood. We discovered that P-selectin (CD62P) binds to umbilical cord blood (UCB)-derived MSCs independently of the previously known sialyl Lewis x (sLex)-containing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1, CD162). By biochemical assays, we identified galectin-1 as a novel ligand for P-selectin. Galectin-1 has previously been shown to be a key mediator of the immunosuppressive effects of human MSCs. We conclude that this novel interaction is likely to play a major role in the immunomodulatory targeting of human UCB-derived MSCs.

Nexin-1 inhibits the activity of human brain trypsin.

Trypsin and other trypsin-like serine proteases have been shown to play important roles in neural development, plasticity and neurodegeneration. Their activity is modulated by serine protease inhibitors, serpins. However, for human brain trypsin, trypsin-4, no brain-derived inhibitors have been described. Here, we report that nexin-1 inhibits trypsin-4, and forms stable complexes only with this trypsin-isoenzyme. This result suggests that nexin-1 could modulate trypsin activity in brain where both nexin-1 and trypsin-4 are expressed.

Differential susceptibility of transferrin glycoforms to chymotrypsin: a proteomics approach to the detection of carbohydrate-deficient transferrin.

Transferrin exhibits heterogeneity in glycosylation characteristic of pathological changes in alcohol abuse and congenital disorders in glycosylation. This study investigated an alternative approach in the detection of carbohydrate-deficient transferrin based on the premise that glycosylation may afford some degree of protection to proteolytic action. Differential susceptibility to proteolysis by chymotrypsin was demonstrated for normal glycosylated and nonglycosylated recombinant human transferrin, using reverse-phase (RP) HPLC, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and LC-tandem mass spectrometry (MS/MS). Peptide fragmentation profiles were consistent with a predominantly high-specificity cleavage pattern of chymotrypsin. The observed peptide fragmentation profile showed that the C-lobe of recombinant full-length nonglycosylated transferrin (rhTf-NG) appeared to be preferentially cleaved, while cleavage of the N-lobe was restricted to the N-terminal and link sequence regions. Although chymotryptic cleavage sites abound in the N-lobe, their resistance to cleavage was independent of glycosylation. Compared to previous studies of lactoferrin, our data suggest disparity in the role by which glycosylation exerts a protective effect in the siderophilin family. It was clear from the transferrin digestions analyzed by HPLC that N-linked glycosylation did confer protection from proteolysis by chymotrypsin. After fragmentation, a range of peptides representing previously cryptic epitopes were identified as potential candidates for an immunological approach to differentiate between the different transferrin glycoforms. Based on its proximity to the Asn413 glycosylation site, a 15-mer peptide, m/z 1690.472 (NKSDNCEDTPEAGYF), was identified as a suitable candidate for raising anti-peptide antibodies for subsequent immunological detection. This novel approach could form the basis for an alternative assay or reference method for the detection of carbohydrate-deficient transferrin.

Site-specific protease activity of the carboxyl-terminal domain of Semliki Forest virus replicase protein nsP2.

The virus-specific components (nsP1-nsP4) of Semliki Forest virus RNA polymerase are synthesized as a large polyprotein (P1234), which is cleaved by a virus-encoded protease. Based on mutagenesis studies, nsP2 has been implicated as the protease moiety of P1234. Here, we show that purified nsP2 (799 amino acids) and its C-terminal domain Pro39 (amino acids 459-799) specifically process P1234 and its cleavage intermediates. Analysis of cleavage products of in vitro synthesized P12, P23, and P34 revealed cleavages at sites 1/2, 2/3, and 3/4. The cleavage regions of P1/2, P2/3, and P3/4 were expressed as thioredoxin fusion proteins (Trx12, Trx23, and Trx34), containing approximately 20 amino acids on each side of the cleavage sites. After exposure of these purified fusion proteins to nsP2 or Pro39, the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis, mass spectrometry, and amino-terminal sequencing. The expected amino termini of nsP2, nsP3, and nsP4 were detected. The cleavage at 3/4 site was most efficient, whereas cleavage at 1/2 site required 5000-fold more of Pro39, and 2/3 site was almost resistant to cleavage. The activity of Pro39 was inhibited by N-ethylmaleimide, Zn(2+), and Cu(2+), but not by EDTA, phenylmethylsulfonyl fluoride, or pepstatin, in accordance with the thiol proteinase nature of nsP2.

Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3-dependent myeloid progenitor cells.

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3-dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC-alpha and PKC-delta specifically caused rapid inhibition of IL-3-induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3-induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-delta and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-delta. Furthermore, PKC-delta inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-delta. In 32D cells, the inhibition of Jak2 either by PKC-delta, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-delta-mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation. (Blood. 2000;95:1626-1632)

Leukocyte adhesion--an integrated molecular process at the leukocyte plasma membrane.

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell-cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific beta2-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.

Leukocyte adhesion--a fundamental process in leukocyte physiology.

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte beta 2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.

The cytoskeletal association of CD11/CD18 leukocyte integrins in phorbol ester-activated cells correlates with CD18 phosphorylation.

Leukocyte adhesion is a regulated process, which involves CD11/CD18 leukocyte integrins. CD11/CD18 acidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated on serine and threonine after phorbol ester activation of T cells. Increased adhesiveness is believed to be mediated by regulating the overall avidity of cellular contact. CD11/CD18 integrins have earlier been reported to interact with several cytoskeletal proteins. We have now studied the involvement of the CD18 phosphorylation in cytoskeletal associations. We have investigated the distribution of phosphorylated CD18 between soluble, cytoskeletal and nuclear fractions of T cell detergent lysates. A significant amount of phosphorylated CD18 polypeptides was observed to fraction along with the cytoskeleton, while the majority of the cell surface CD18 molecules remained in the soluble fraction. Putative candidates for this altered cytoskeletal binding of CD11/CD18 were shown to be talin and filamin, which were observed to bind to CD18 cytoplasmic peptides and co-precipitate with CD18. The importance of the CD18 cytoplasmic domain in the regulation of the leukocyte adhesion was further strengthened by inhibition of phorbol ester-induced T cell adhesion with a phosphorylated lipopeptide corresponding to the cytoplasmic portion of the CD18. These results indicate that the induced CD18 phosphorylation and the altered cytoskeletal binding of the phosphorylated integrin complex may contribute to the increased avidity of CD11/CD18-mediated leukocyte adhesion.

Characterization of beta2 (CD18) integrin phosphorylation in phorbol ester-activated T lymphocytes.

Integrins are transmembrane proteins involved in cell-cell and cell-extracellular-matrix interactions. The affinity and avidity of integrins for their ligands change in response to cytoplasmic signals. This 'inside-out' activation has been reported to occur also with beta2 integrins (CD18). The beta2 integrin subunit has previously been shown to become phosphorylated in T lymphocytes on cytoplasmic serine and the functionally important threonine residues after treatment with phorbol esters or on triggering of T-cell receptors. We have now characterized the phosphorylation of beta2 integrins in T-cells in more detail. When T-cells were activated by phorbol esters the phosphorylation was mainly on Ser756. After inhibition of serine/threonine phosphatases, phosphorylation was also found in two of the threonine residues in the threonine triplet 758-760 of the beta2 cytoplasmic domain. Activation of T-cells by phorbol esters resulted in phosphorylation in only approx. 10% of the integrin molecules. Okadaic acid increased this phosphorylation to approx. 30% of the beta2 molecules, assuming three phosphorylation sites. This indicates that a strong dynamic phosphorylation exists in serine and threonine residues of the beta2 integrins.

Leukocyte integrins and inflammation.

Leukocyte adhesion is of pivotal functional importance. Without adequate adhesion, T lymphocytes and natural killer cells are not cytotoxic, B cells cannot develop into antibody secreting plasma cells, leukocytes do not home into inflamed tissues and myeloid cells are not able to phagocytize or exhibit chemotactic responses. During evolution several leukocyte adhesion molecules have developed belonging to a few molecular families. Among these, the leukocyte-specific integrins (beta 2 integrins, CD11/CD18 molecules) are among the most important. Much progress has taken place during the past few years, and at present we have a considerable knowledge of their structure and function. Inflammation is critically dependent on integrin activity, and its regulation forms the topic of this short review.

A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50.

We have made a monoclonal anti-CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)-1 (CD54) and ICAM-2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM-1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM-1 and ICAM-3. The binding to ICAM-2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand-specific manner by engagement of CD44.

Treatment with okadaic acid reveals strong threonine phosphorylation of CD18 after activation of CD11/CD18 leukocyte integrins with phorbol esters or CD3 antibodies.

The CD11/CD18 leukocyte integrins comprise three heterodimers involved in leukocyte adhesion. CD11/CD18 avidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated in leukocytes after phorbol ester stimulation. The importance of phosphorylation in the regulation of CD11/CD18 avidity has, however, remained unclear. We have now activated T cells using phorbol esters, CD3, and CD44 Abs. Both phorbol ester and CD3 treatment activated protein kinase C. CD18 was shown to become more stably phosphorylated after phorbol ester treatment and more transiently so after CD3 stimulation. The phosphorylation was strongly augmented by okadaic acid, a serine/threonine phosphatase inhibitor. While phorbol ester treatment caused phosphorylation mainly on serine, in okadaic acid-pretreated cells, both phorbol ester treatment as well as CD3 stimulation revealed strong threonine phosphorylation. Since earlier mutational studies have demonstrated the functional importance of cytoplasmic threonine residues in CD18, the threonine phosphorylation reported here indicates the role of threonine phosphorylation in the regulation of CD11/CD18 avidity.

A peptide from ICAM-2 binds to the leukocyte integrin CD11a/CD18 and inhibits endothelial cell adhesion.

Numerous leukocyte functions depend on adhesive intercellular interactions. The leukocyte-specific integrins CD11a/CD18 (lymphocyte function-associated antigen-1 (LFA-1)) and CD11b/CD18 (complement type 3 receptor (Mac-1)), which bind to the intercellular adhesion molecules ICAM-1 and ICAM-2, play a key role in adhesion. Little is known about the binding in molecular detail. We have now defined a peptide region from the first immunoglobulin domain of ICAM-2 that is specifically involved in binding to CD11a/CD18. A synthetic peptide from this part of ICAM-2, covering residues 21-42, bound to purified CD11a/CD18 and inhibited the adhesion of endothelial cells to this integrin. It also inhibited the binding of B lymphoblastoid cells to endothelial cells. Leukocytes bound to the peptide coated on plastic. Several shorter peptides from the same region showed less or no activity.

Leukocyte cell adhesion proteins: from molecular dissection to clinical applications.

Leukocyte adhesion is needed for a number of leukocyte functions like immunoglobulin synthesis, T and NK-cell-mediated cytotoxicity, phagocytosis by granulocytes, and cellular accumulation in inflamed tissue. Several cell surface molecules involved in leukocyte-leukocyte and leukocyte-target cell interactions have recently been identified and characterized. Both the polypeptide and carbohydrate portions are important in leukocyte interactions. It is becoming increasingly apparent that it is possible to interfere with the normal functions of the leukocyte adhesion glycoproteins, and such applications may become important in medicine.

Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues.