Neutrophils are the first to be recruited to a site of infection or a diseased site. Among various inflammatory mediators, CXC chemokines including IL-8 (CXCL8), MIP-2 (CXCL2), and KC (CXCL1) are the most critical for such recruitment. Neutrophils have been considered as effector cells that kill bacteria or destroy affected tissues mainly through the production of reactive oxygen species. Recent studies, however, revealed that neutrophils are involved in the production of chemokines in response to a variety of stimulants including LPS, TNF-alpha, and IFN-gamma, thereby contributing to immunomodulation. These functions are also regulated by selectins during infiltration into various sites. In this review, I summarize the current knowledge on this area and propose that neutrophils are a fascinating target for basic as well as clinical scientists.

Activated monocytes produce a variety of cytokines that are involved in inflammation, such as IL-1, TNF, chemotactic factors, transforming growth factor R, platelet-derived growth factor, and IFN-a and -Q (1). Chemotactic factors released at foci of injury or bacterial invasion are thought to mediate directed migration of leukocytes into inflammatory sites. Since the leukocyte composition of the inflammatory infiltrate depends on the temporal stage of the lesion (2) and the nature of the stimulus (3), it follows that some chemoattractants should be specific for a given type of leukocyte. We recently showed that LPS-stimulated monocytes produce a chemotactic factor that attracts neutrophils, but not monocytes (4). We purified this factor to homogeneity and described the N1-12-terminal sequence of the first 42 amino acids (5). We now report molecular cloning and sequencing ofthe full-length cDNA for this monocyte-derived neutrophil chemotactic factor (MDNCF)' and the deduced amino acid sequence of the entire molecule. Specific cDNA probes also enabled us to test the capacity of a number of cytokines to induce MDNCF mRNA expression in human PBMC. The stimulation of MDNCF mRNA expression by IL-1 and TNF suggests that the local pro-inflammatory action of these cytokines may be mediated by induction of chemotactic factor secretion. Volume 167 June 1988 1883-1893 Materials and Methods cDNA Cloning of MDNCF and Nucleotide Sequence . Normal human PBMC were first fractionated by Ficoll-Hypaque and plastic adherent cells (> 90% nonspecific esterase-positive monocytes), were cultured in RPMI-1640 medium supplemented with 1% FCS and 10 ug/ml LPS (Serotype 055:1155; Difco Laboratories Inc., Detroit, MI) for 6 h at 37°C . Total RNA

We describe here the involvement of calciumactivated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin la (IL-la) into mature IL-la. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-la and the release of mature IL-la either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-la and -P. The proteolytic processing of pre-IL-la was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-la was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 IM) and that treatment of pre-IL-la with purified CANP yielded the 17-kDa mature form of IL-la, which has an amino terminus identical with that reported for mature human IL-la. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-la is selectively mediated by CANP.Interleukin 1 (IL-1), produced by a variety of cells, is widely known to manifest multiple biological activities on various types of target cells (1). Two biochemically distinct forms of IL-1, IL-la and -/3, have been genetically cloned (2-4). Although both types of IL-1 are lacking in a signal peptide, they have been reported to be present not only in the cytosol (5) but also in the cell membrane (6) and extracellularly. Since mature IL-1 has a molecular mass of 17 kDa, precursor IL-1 (pre-IL-1; molecular mass of 33-35 kDa) presumably is processed by an as-yet-unidentified proteolytic enzyme(s) to generate mature IL-1.A calcium ionophore has been reported to augment production of extracellular IL-1 by lipopolysaccharide (LPS)-stimulated human adherent mononuclear cells through the entry of exogenous calcium into cells (7). LPS also has been shown to increase intracellular calcium in macrophages (8).On the other hand, there are some cell types, such as HTB9 5637, which constitutively produce pre-IL-1 but do not generate and release mature IL-1 effectively (ref. 9; unpublished results). We, therefore, decided to test the possibility that calcium plays some roles in release and/or processing of IL-1 by comparative studies of this cell line and human adherent mononuclear cells. We examined the effects of calcium ionophore on the release and processing of IL-1 in detail, and we identified a major calcium-dependent proteolytic processing enzyme of pre-IL-la as calcium-activated neutral protease (CANP ...

Upon inflammation, neutrophils and subsequently monocytes infiltrate into the involved site. Neutrophils perform functions such as bacterial killing or tissue destruction and then undergo apoptosis, whereas monocytes differentiate into macrophages at the site. Macrophages and other phagocytes finally clear apoptotic neutrophils, leading to resolution of the inflammation. One of the key steps during inflammation is leukocyte infiltration, which is controlled chiefly by chemokines for neutrophils and monocytes. The production of these chemokines is regulated positively or negatively by iNOS-derived NO. Although the mechanisms underlying such dual effects of NO remain unknown, the level of NO and duration of NO exposure appear to be determining factors. The clearance of apoptotic neutrophils without causing further proinflammatory responses, on the other hand, is another key event during inflammation. The production of proinflammatory cytokines appears to be actively suppressed by TGF-β and NO, which are produced by phagocytes upon interaction with apoptotic cells. Overall, NO plays a critical role during inflammation and therefore, remains a potential target for developing therapeutics for inflammatory diseases.

Objective: To investigate whether concentrations of plasma adiponectin constitute a significant coronary risk factor, with particular focus on the relation between plasma concentrations of adiponectin and the development of acute coronary syndrome (ACS). Subjects and methods: Plasma concentrations of adiponectin were measured in 123 patients with coronary artery disease (CAD) and in 17 control participants. Patients were divided into three groups according to condition type: acute myocardial infarction (AMI) group (n = 59), unstable angina pectoris (UAP) group (n = 28), and stable angina pectoris (SAP) group (n = 36). Results: Plasma concentrations of adiponectin correlated negatively with body mass index (r = 20.18, p , 0.05), serum triglyceride (r = 20.25, p , 0.01), and fasting glucose concentrations (r = 20.21, p , 0.05), but correlated positively with age (r = 0.26, p , 0.01), high density lipoprotein cholesterol concentrations (r = 0.35, p , 0.01), and low density lipoprotein particle size (r = 0.37, p , 0.01). Plasma concentrations of adiponectin in patients with ACS, in both the AMI and UAP groups, were significantly lower than those in patients with SAP and in the control group (ACS, 6.5 (3.0) mg/ml; SAP, 11.3 (5.9) mg/ml; control 12.8 (4.3) mg/ml; p , 0.01). Additionally, plasma concentrations of adiponectin in patients with CAD (7.9 (4.6) mg/ml, p , 0.01) were significantly lower than in the control group. There were, however, no significant differences between patients with SAP and the control group (p = 0.36). Multiple logistic regression analysis showed that smoking, fasting glucose concentration, and low log adiponectin concentration correlated independently with the development of an ACS. Conclusions: The findings suggest that measurement of plasma concentrations of adiponectin may be of use for assessing the risk of CAD and may be related to the development of ACS.

Apoptotic cells are phagocytosed as soon as they appear in vivo. In this study, we first determined precisely at what stage apoptotic cells are phagocytosed by macrophages, and then examined the subsequent cytokine production. Phagocytosis was confirmed by flow cytometry and confocal laser microscopy, whereas the subsequent response was examined by ELISA and RT-PCR for quantitative and semiquantitative measurement of the protein and mRNA levels of cytokines, respectively. Even the cell populations containing very early apoptotic cells, such as IL-2-dependent CTLL-2 cells cultured in the absence of IL-2 for 4 h and a murine leukemic cell line, P388 cells, treated with etoposide for 5 h, were phagocytosed by macrophages. Although the cell populations containing the very early apoptotic cells used in this study were FITC-Annexin V-negative and did not show a decrease in cell size as compared with untreated cells, they showed a very small increase in phosphatidylserine on the cell surface, as detected with Cy3-Annexin V, and a decrease in mitochondrial membrane potential, indicating that the cell populations had already started the apoptotic process. Phagocytosis of such populations containing very early apoptotic cells was inhibited by phospho-l-serine much more significantly than Arg-Gly-Asp-Ser. In addition, macrophages hardly produced either proinflammatory or anti-inflammatory cytokines after phagocytosis, thus being an almost null response. These results are contrary to the generally accepted concept that the phagocytosis of apoptotic cells leads to the production of anti-inflammatory cytokines, suggesting instead that cells starting to undergo apoptosis are quickly phagocytosed by macrophages without any inflammation in vivo.

Neutrophil infiltration is mainly mediated by ELR+ CXC chemokines and is regulated through interactions between various stimulants and cells, which are perhaps more complex than previously thought. In this review, I describe the current view of the roles of neutrophil-selective chemokines such as IL-8, MIP-2, and KC by choosing several in vivo models. I also illustrate how these three chemokines are induced and how they are involved in neutrophil infiltration. Finally, I introduce synthetic compounds with high efficacy in the prevention of ischemia reperfusion injury.

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ESlike cells (E-1 and E-2) appear to maintain a normal diploid karyotype inde¢nitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-speci¢c embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells di¡erentiated to neural precursor cells in the presence of basic ¢broblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the di¡er-entiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.