Proteoglycans are ubiquitous components of cell surface membranes, basement membranes, and extracellular matrices in various tissues. They belong to a family of macromolecules that consist of core proteins to which glycosaminoglycans (GAGs), sulfated polysaccharides, are attached. Because of the high sulfate and carboxyl group content of their GAG moieties, proteoglycans have strong negative charges. This property allows them to interact with a wide range of proteins, including growth factors, enzymes, cytokines, chemokines, lipoproteins, and adhesion molecules. In this article, I will focus on the interaction of chondroitin sulfate proteoglycans with L-and P-selectin and CD 44, and chemokines, all of which have been implicated in leukocyte trafficking, and will provide evidence that a nonspecific electrostatic interaction is not the sole factor determining the interaction. I will also discuss biological implication of the interaction of chondroitin sulfate proteoglycans with L-and P-selectin and CD 44, and chemokines.

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To clarify roles of eosinophils in allergic inflammation, biological activities of granule proteins in rat eosinophils were examined. Granule proteins from rat eosinophils (1×10⁶ cells) dissolved in 1 ml of medium showed no cytotoxic activity on ⁵¹Cr-labeled rat bronchial epithelial cells and rat skin fibroblasts (5×10⁵ cells) when incubated at 37°C for 6 h as determined by the release of radioactivity into the medium. In addition, the lysate of rat eosinophils showed no cytotoxic activity on a human bronchial epithelial cell line NCI-H 292 cells at concentrations enough to express RNase activity. These findings strongly suggest that the granule proteins of rat eosinophils have very little cytotoxic activity.
We cloned rat eosinohil major basic protein (MBP), eosinophil-associated ribonuclease (EAR) -1, EAR-2, and the α chain of interleukin-5 receptor (accession numbers to DDBJ, EMBL, and NCBI nucleotide sequence database for these proteins are D 50568, D 88586, AB 005291 and AB 056101, respectively) . Expression of rat pre-EAR-1 (mature EAR-1 having a signal peptide) or pre-EAR-2 (mature EAR-2 having a signal peptide) in E. coli (M 15) suppressed the growth of E. coli. We then prepared recombinant rat pre-EAR-1 and pre-EAR-2, and examined the effects of these proteins on the growth of S. aureus (Cowan I) and E. coli (LE 392) . It was demonstrated that they inhibited the growth at 0.1 to 1μM. The growth was almost completely suppressed at 1μM. These findings suggest that pre-EAR-1 and pre-EAR-2 have bactericidal activity. At such concentration, no cytotoxic activity was observed. Then, we prepared recombinant rat mature EAR-1 and EAR-2, and examined the effect of these proteins on the growth of E. coli (LE 392) to compare the activity of mature EAR-1 and EAR-2 with that of pre-EAR-1 and pre-EAR-2. It was shown that these proteins have almost the same inhibitory activity on the growth of E. coli (LE 392) ; at 1μM, the growth of E. coli (LE 392) was completely suppressed.
As to the RNase activity, mature EAR-2 showed stronger activity than mature EAR-1 at 0.1 to 1μM, and no RNase activity was detected in pre-EAR-1 and pre-EAR-2. These findings indicate that the linking of mature EAR to its corresponding signal peptide diminishes the RNase activity. No RNase activity in pre-EAR might be important for the synthesis of EAR in the cells.
Our findings suggest that eosinophils might not express cytotoxicity by releasing their granule proteins in the inflammatory locus. Eosinophils might be useful for the host defense against infection by single strand RNA viral pathogens such as respiratory syncytial virus (RSV) and human immunodeficiency virus (HIV) and several types of bacteria.

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In recent years, new concepts have been formulated for the pathogenesis and therapeutic management of ulcerative colitis (UC) and Crohn's disease (CD) . In the inflamed intestinal mucosa, analysis of the types of immune response ongoing has revealed that there is predominantly a T-helper cell type 1 response in CD, with exaggerated production of IL-12 and IFN-γ, whereas the lesion seems more of an antibody-mediated hypersensitivity reaction in UC. Despite these differences, downstream inflammatory events could be similar in both conditions. IL-1β, IL-6, IL-8 and TNF-α are produced in excess in both UC and CD. New medical therapies that inhibit the bioactivity of TNF-α represent a major breakthrough in the treatment of CD. The mouse chimeric monoclonal antibody infliximab against TNF-α is effective for treating active CD, closing fistulas, and maintaining remission. Side effects occurring in CD patients treated with infliximab include human anti-chimeric antibodies, worsening infections, or malignancies of unknown relationships. This review seeks to summarize analysis about manipulation of cytokines especially focusing into TNF-α in inflammatory bowel diseases and studies in which anti TNF-α antibody has been used in the treatment of CD.

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Angiogenesis is a very important process not only in embryogenesis but also in a variety of severe diseases such as cancer and diabetes. Recently, molecular mechanism of angiogenesis has been extensively studied, and several signaling systems were found to be involved in this process. Among these, VEGF (vascular endothelial growth factor) family and its receptors (VEGFR-1, -2 and-3) regulate the earliest step of blood vessel formation in embryogenesis. VEGFR-2 (KDR/Flk-1) minus mice lack both blood vessel formation and hematopoiesis and die at E 8.5. VEGFR-1 (Fit-1) minus mice are also lethal at a similar stage due to vascular dysorganization. VEGFR-3 functions at the middle stage of angiogenesis and lymphangiogenesis. On the other hand, Angiopoietin-Tie system and Ephrin-Eph system are involved in the interrelationship between endothelial cell and smooth muscle cell and the differentiation of arterial and venous endothelial cell, respectively. Thus, highly organized signaling systems are required for physiological angiogenesis. In the pathological processes such as tumor angiogenesis, VEGF appears to play a central role in new blood vessel formation. To regenerate the blood vessels, stimulation of VEGF-VEGF receptor system seems most effective, and VEGF-A and VEGF-E may be useful tools since they stimulate most potent signal transducer, VEGFR-2.

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Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (α-/β-defensins and cathelicidins), which contribute to the innate host defense by killing invaded microorganisms. Recently, we have evaluated the lipopolysaccharide (LPS) -neutralizing activities of cathelicidin peptides human LL-37 and guinea pig CAP 11, using CD 14⁺ murine macrophage cell line RAW 264.7 and murine endotoxin shock model. LL-37 and CAP 11 inhibited the binding of LPS to RAW 264.7 cells, and suppressed the LPS-induced TNF-α mRNA and protein expression by RAW 264.7 cells. Interestingly, LL-37 and CAP 11 possessed the LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS-binding protein (LBP) that mediates the transport of LPS to CD 14. Moreover, LL 37 and CAP 11 bound to the cell surface CD 14 and inhibited LPS-binding to the cells. Furthermore, in murine endotoxin shock model, LL-37-or CAP 11 -administration inhibited the binding of LPS to CD 14⁺ cells (peritoneal macrophages) and suppressed the LPS-induced TNF-α expression by these cells. Together these observations indicate that cathelicidin peptides LL 37 and CAP 11 likely exert the protective actions in endotoxin shock by blocking the binding of LPS to CD 14⁺ cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD 14.

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In order to investigate the mechanism for secretion of macrophage migration inhibitory factor (MIF) in cultured human fibroblasts, we compared with the secretion of interleukin-6 (IL-6) as stimulated with lipopolysaccharides (LPS) and H₂O₂. MIF content of the medium of 2.0×10⁶ cells/20 ml after 20 h culture of non-stimulated fibroblasts was 0.30±0.06 ng/ml, whereas LPS-stimulation (10μg/ml) led to increase only 1.5 fold as compared with the nonstimulated cells. In contrast, a significant increase of IL-6 was induced by LPS-stimulation (6048±488 pg/ml in LPS-stimulated cells vs 58±36 pg/ml in control cells) . On the other hand, the higher concentrations of H₂O₂ (0.6 mM-1.2 mM) caused an increase of MIF secretion into the culture medium irrespective of LPS-stimulation, showing that 1.2 mM H₂O₂-stimulation for 20 h was increased to 40 fold as compared with the nonstimulated cells. However, the lower concentrations (0.1 mM-0.4 mM) did not cause this. Interestingly, H₂O₂-stimulation not only failed to increase IL-6 production from fibroblasts, but also repressed induction of IL-6 by LPS-stimulation in a dose dependent manner. Genistein, a tyrosine kinase inhibitor and H-7, a protein kinase C inhibitor also inhibited IL-6 secretion but not MIF secretion in both LPS-and H₂O₂-stimulated fibroblasts. From analyses of trypan blue exclusion, formazan formation, morphological changes, and intracellular MIF content by Western blot, we found that MIF secretion by H₂O₂ seems to be mainly due to cell-death and subsequently leakage of intracellular MIF. Taken together, these results suggest that MIF secretion differs from IL-6 via LPS-mediated signaling pathways.

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Recently, various kinds of tissue specific stem cells, such as hematopoietic stem cells or neural stem cells, have been identified. It has been well known the embryoid body formed by aggregated embryonic stem (ES) cells differentiate into cardiac muscle cells, but cardiac progenitor/stem cells, which are supposed to exist during cardiac differentiation, have not been identified. Here, we intended to characterize cardiac progenitor/stem cells from ES cells using FACS sorting system. We considered that flk 1 might be a good marker for isolating cardiac progenitor/stem cells. Because flk 1 is one of the early markers of the anterior lateral mesoderm where vasculogenesis and also cardiomyogenesis originate from paired regions of the cardiac crescent. Differentiation of ES cells was induced in suspension culture in medium without leukemia inhibitory factor (LIF) . At 3.75 day from induction, the cells were stained with anti-flk 1 antibody, and cells with flk 1+ or fikl- were sorted respectively. Each subpopulation was co-cultured on OP 9 stromal cell. The differentiation of cardiac muscle cells was examined at day 10. Simultaneously, the gene expression analysis for Nkx-2.5 was carried out using RT-PCR.
No colonies of contracting cardiac muscle cells were identified on tissue culture plate or type 4-collagen corted plates. Contracting muscle cells were identified only after plating on OP 9 cells, although no colonies on NIH 3 T 3 cells. These results suggest that OP 9 cells supply a signal indispensable for cardiac cell differentiation. However, there were no significant difference in numbers of contracting colonies and expression of Nkx 2.5 between flk 1 + and flk 1- cells. In this sense, the significance of flk 1 for cardiac cell differentiation still requires further discussion. Further examination remains to be done about signaling molecules for cardiac muscle cell differentiation.

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