Alfred Walz

Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils.

Introduction Neutrophil accumulation in a tissue is characteristic of inflammation and is observed in a variety of pathological conditions as disparate as infection, trauma, ischemia, and cancer. The process of tissue infiltration is best understood in bacterial infection, where neutrophils are selectively attracted in large numbers to phagocytose and kill the invaders. In other conditions neutrophils are presumably recruited as scavengers of damaged tissue or unwanted extracellular deposits like immune complexes or fibrin. Phagocytosis is accompanied by the release of granule enzymes, superoxide, H202, and a variety of bioactive lipids. Several of these products are required for the killing and digestion of microorganisms. They also induce inflammation and tissue damage, however, which is normally observed after neutrophil accumulation. Several neutrophil chemoattractants have been characterized in recent years; the best known are the anaphylatoxin C5a (1), formylmethionyl peptides of bacterial origin (2), plateletactivating factor (PAF; 3),1 and leukotriene B4 (LTB4; 4). These stimuli have different origins and modes of formation, and their occurrence in disease must thus be expected to vary in accord with the underlying pathophysiological process. C5a is formed upon complement activation via the classical pathway after interaction of microorganisms with antibodies or the formation of immune complexes, or via the alternative pathway after the nonimmune recognition of foreign materials. In bacterial infections, on the other hand, formylmethionyl peptides (which are released by the microorganisms) are likely to be the major attractants. PAFand LTB4 are of special interest because they can be generated by the neutrophils themselves and may thus function as autoor paracrine amplifiers of the responses elicited by other stimuli (5). It has been shown that CSa, formylmethionyl peptides, PAF, and LTB4 act via unrelated receptors, suggesting that neutrophil recruitment can result from the concerted action of multiple stimuli.

The Functional Role of the ELR Motif in CXC Chemokine-mediated Angiogenesis

In this study, we demonstrate that the CXC family of chemokines displays disparate angiogenic activity depending upon the presence or absence of the ELR motif. CXC chemokines containing the ELR motif (ELR-CXC chemokines) were found to be potent angiogenic factors, inducing both in vitro endothelial chemotaxis and in vivo corneal neovascularization. In contrast, the CXC chemokines lacking the ELR motif, platelet factor 4, interferon -inducible protein 10, and monokine induced by -interferon, not only failed to induce significant in vitro endothelial cell chemotaxis or in vivo corneal neovacularization but were found to be potent angiostatic factors in the presence of either ELR-CXC chemokines or the unrelated angiogenic factor, basic fibroblast growth factor. Additionally, mutant interleukin-8 proteins lacking the ELR motif demonstrated potent angiostatic effects in the presence of either ELR-CXC chemokines or basic fibroblast growth factor. In contrast, a mutant of monokine induced by -interferon containing the ELR motif was found to induce in vivo angiogenic activity. These findings suggest a functional role of the ELR motif in determining the angiogenic or angiostatic potential of CXC chemokines, supporting the hypothesis that the net biological balance between angiogenic and angiostatic CXC chemokines may play an important role in regulating overall angiogenesis.

Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups

Neutrophils have been implicated in the pathogenesis of the adult respiratory distress syndrome (ARDS). We have measured concentrations of the neutrophil attractant interleukin-8 in blood and bronchoalveolar lavage fluid (BAL) from patients at risk of ARDS. We studied 29 patients from three groups at risk of developing ARDS: multiple trauma (n = 16), perforated bowel (n = 6), and pancreatitis (n = 7). ARDS developed in 7 of these patients. Interleukin-8 in BAL and blood samples taken on initial hospital presentation was measured by a sandwich enzyme-linked immunosorbent assay. The mean BAL interleukin-8 concentration was significantly higher for the patients who subsequently progressed to ARDS than for the non-ARDS group (3.06 [SE 2.64] vs 0.053 [0.010] ng/mL, p = 0.0006). There was no difference between the groups in plasma interleukin-8 (6.23 [2.60] vs 5.12 [2.22] ng/mL, p = 0.31). Immunocytochemistry suggested that the alveolar macrophage is an important source of interleukin-8 at this early stage in ARDS development. This study provides evidence of a relation between the presence of interleukin-8 in early BAL samples and the development of ARDS. The early appearance of interleukin-8 in BAL of patients at risk of ARDS may be an important prognostic indicator for the development of the disorder and reinforces the likely importance of neutrophils and the effects of their accumulation and activation in the pathogenesis of many cases of ARDS.

The CXC Chemokine Receptor 2, CXCR2, Is the Putative Receptor for ELR+ CXC Chemokine-Induced Angiogenic Activity

We have previously shown that members of the ELR+ CXC chemokine family, including IL-8; growth-related oncogenes α, β, and γ; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR+ CXC chemokines has not been determined. Because all ELR+ CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR+ CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR+ CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR+ CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR+ CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR+ CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2−/− mice. We thus conclude that CXCR2 is the receptor responsible for ELR+ CXC chemokine-mediated angiogenesis.

Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes

Human blood mononuclear cells were cultured for 24 h in the presence of LPS (100 ng per 5 x 10(6) cells), and a monocyte-derived neutrophil-activating factor (NAF) was purified to apparent homogeneity from the conditioned media. The purification consisted of ammonium sulphate precipitation, gel filtration, chromatography on phosphocellulose followed by hydroxylapatite, and reversed-phase HPLC on C4 and CN-propyl columns. Amino acid sequence analysis (32 of 50 presumed residues) shows that NAF is a novel peptide with little homology to known ones. Crude and pure NAF stimulated human neutrophils to release granule enzymes and to produce superoxide and H2O2.

Mechanism of neutrophil activation by NAF, a novel monocyte-derived peptide agonist.

The rise in cytosolic free Ca2+, shape change, superoxide formation, and granule exocytosis induced in human neutrophils by N‐formyl‐Met‐Leu‐Phe (fMLP) and by a newly discovered activating peptide, neutrophil‐activating factor, termed NAF, were compared. NAF was effective in the concentration range of 0.1‐10 nM and was 10‐to 100‐fold more potent than fMLP. In qualitative terms, the single responses to either stimulus were remarkably similar: they showed virtually identical onset and initial kinetics, and were all inhibited by pretreatment of the neutrophils with Bordetella pertussis toxin. In addition, the respiratory burst elicited by either stimulus was inhibited by 17‐hydroxywortmannin and staurosporine. Two conclusions are drawn from these results: 1) neutrophil activation by NAF (as by fMLP) is dependent on a GTP‐binding protein and on protein kinase C; 2) a similar, or even identical, mechanism of signal transduction must be assumed on stimulation of human neutrophils with NAF, fMLP, and other chemotactic agonists. Human monocytes, lymphocytes, and platelets did not show cytosolic free Ca2+ changes when exposed to NAF, which suggests that NAF is selective for the neutrophils.— Thelen, M.; Peveri, P.; Kernen, P.; von Tscharner, V; Walz, A.; Baggiolini, M. Mechanisms of neutrophil activation by NAF, a novel monocyte‐derived peptide agonist. FASEB J. 2: 2702‐2706; 1988.

Alteration of Monocyte Function Following Major Injury

The macrophage exerts its stimulatory and regulatory functions within the specific immune response via the interleukin 1 (IL-1) and prostaglandin E2 (PGE2), respectively. In a screening study of macrophage-related variables following injury, a total of 58 patients (mean age, 32 years; mean injury Severity Score, 38), macrophagic phenotyping with the monoclonal antibody Leu M3 and serial measuring of the antagonistic monokines IL-1 and PGE2 and of the macrophage-activating lymphokine interferon gamma were carried out on posttrauma days 0, 1, 3, 5, 7, 10, 14, and 21. The posttraumatic course was characterized by significant monocytosis, showing a peak value of 32% of Leu M3-positive cells compared with 15% of these cells in normal control subjects. During the posttrauma course, the macrophagic PGE2 output was significantly elevated up to eightfold on days 5 and 7 compared with that of control subjects (0.441 +/- 0.14 ng/mL vs 0.052 +/- 0.01 ng/mL). Conversely, macrophagic IL-1 synthesis was significantly suppressed until day 10. Levels of interferon gamma were suppressed to a significant degree during the two-day observation period, with a trend to slow recovery at the end of week 3. These data suggest that a negative regulatory macrophagic function may be the event initiating posttraumatic immunosuppression. To restore impaired macrophagic T-helper cell interaction, cyclo-oxygenase inhibition and substitution of interferon gamma may be useful to potentiate facilitatory macrophagic function and to block inhibitory macrophagic activity.

Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. The role of epithelial neutrophil activating protein.

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. One of the striking consequences of liver injury is the associated pulmonary dysfunction that may be related to the release of hepatic-derived cytokines. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and demonstrated that this injury causes the production and release of hepatic-derived TNF, which mediates a neutrophil-dependent pulmonary microvascular injury. In this study, we have extended these previous observations to assess whether an interrelationship between TNF and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein-78 (ENA-78), exists that may be accountable for the pathology of lung injury found in this model. In the context of hepatic ischemia/reperfusion injury, we demonstrated the following alterations in lung pathophysiology: (a) an increase in pulmonary microvascular permeability, lung neutrophil sequestration, and production of pulmonary-derived ENA-78; (b) passive immunization with neutralizing TNF antiserum resulted in a significant suppression of pulmonary-derived ENA-78; and (c) passive immunization with neutralizing ENA-78 antiserum resulted in a significant attenuation of pulmonary neutrophil sequestration and microvascular permeability similar to our previous studies with anti-TNF. These findings support the notion that pulmonary ENA-78 produced in response to hepatic-derived TNF is an important mediator of lung injury.

Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.

Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase-3.

Activated neutrophils secrete two forms of IL‐8 with 77 and 72 amino acids, IL‐8(77) and IL‐8(72), along with proteinases that could process these cytokines. Significant conversion of IL‐8(77) to more potent, N‐terminally truncated forms was observed upon incubation with neutrophil granule lysates and purified proteinase‐3. IL‐8(72) was considerably more resistant to proteolytic processing than IL‐8(77). The present observations indicate that neutrophil proteinases released in inflamed tissues convert IL‐8 to more active forms and therefore tend to conserve or enhance, rather than decrease IL‐8 activity.