Publisher Summary This chapter focuses on interleukin-8 (IL-8) and related chemotactic cytokines—namely, CXC and CC chemokines. IL-8 is the best known member of a new class of cytokines that are widely studied because of their ability to attract and activate leukocytes, and their potential role as mediators of inflammation. IL-8 was originally isolated from the culture supernatants of stimulated human blood monocytes and was identified as a protein of 72 amino acids with a molecular weight of 8383. The three-dimensional structure of IL-8 has been studied by nuclear magnetic resonance spectroscopy and X-ray crystallography. In concentrated solution, and on crystallization, IL-8 is present as a dimer. The first CC chemokine was identified after cloning by differential hybridization from human tonsillar lymphocytes and was termed LD78. The CC and CXC chemokines are similar in size and have an overall structure that is characterized by the two intrachain disulfide bonds, short N-terminal and long C-terminal sequences. It discusses the role of chemokines in pathology with skin inflammation because psoriasis was the first disease to be linked to overproduction of IL-8. Several independent studies document the occurrence of high levels of IL-8 in the synovial fluid of inflamed joints of patients with different forms of rheumatic diseases, osteoarthritis, and gout.

Interleukin-8 and related chemotactic cytokines--CXC and CC chemokines.

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.

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

Granules of the Human Neutrophilic Polymorphonuclear Leukocyte

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.

https://rupress.org/jcb/article-pdf/97/1/52/1389524/52.pdf

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.

INCIDENCE AND PROBLEMS OF MULTIPLE-ORGAN-FAILURE SYNDROMES The panelists for the discussion were C. James Carrico, MD, from Seattle; Jonathan Meakins, MD, DSc, FRCSC, FACS, from Montreal; Donald Fry, MD, from Cleveland; and Ronald V. Maier, MD, from Seattle. Dr Carrico: Multiple-organ-failure (MOF) syndrome is a process that occurs following 7% to 22% of emergency operations and between 30% and 50% of operations for intra-abdominal sepsis; MOF syndrome carries a mortality that varies from 30% to 100% depending on the number of organs involved. Treatment usually involves support of the organ (system) function and control of sepsis. 1,2 In developing a clinical description, we can use a classic description of respiratory failure from the late 1960s as a model. Rather than a single organ we need to describe the function of several systems and follow the changes through a series of stages, beginning with the patient who has recently experienced one

Multiple-Organ-Failure Syndrome

Postnuclear supernates from homogenates of purified neutrophil polymorphonuclear leukocytes (PMNs) from human blood were fractionated by zonal sedimentation and isopycnic equilibration in sucrose gradients. The fractions were characterized biochemically by measuring protein content and the activities of eight enzymes. Selected fractions were further analyzed by electron microscopy. In both centrifugation systems, azurophil and specific granules could be resolved almost completely. Azurophil granules sediment three to four times faster than the specifics and have an average density of 1.23. They contain all the peroxidase of the cells, large portions of four lysosomal hydrolases, and about half of the total lysozyme, and therefore appear to be, in biochemical terms, very similar to the azurophil granules of rabbit PMNs. The specific granules, which have an average density of 1.19, contain the remaining half of the lysozyme but appear to be free of the other components of the azurophil granules, and of alkaline phosphatase. Isopycnic equilibration disclosed a minor lysosomal population, which strongly overlaps the specific granules, and made possible the identification of a membrane-fraction which is characterized by the presence of the thiol-sensitive acid 4-nitrophenyl phosphatase and of alkaline phosphatase.

https://rupress.org/jcb/article-pdf/63/1/251/1071299/251.pdf

Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes

Gelatinase is a metallo-proteinase that acts specifically on denatured collagen. In human neutrophils, this enzyme is localized in small, morphologically still unidentified storage organelles that are resolved from the specific and the azurophil granules upon subcellular fractionation by differential sedimentation. When neutrophils isolated from freshly drawn blood are exposed to soluble stimuli such as N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, phorbol myristate acetate, or the calcium ionophore A 23187, or are induced to phagocytose opsonized zymosan, they rapidly release gelatinase in large amounts (30-70% of the cellular content in 10 min). When neutrophils from donor blood, which had been stored for 24 h at 4 degrees C are used, extensive release even occurs without added stimuli by simply warming to 37 degrees C. Gelatinase release appears to occur by secretion because it is not dependent on phagocytosis. It is paralelled by the release of specific granule contents (vitamin B(12)-binding protein), but is more rapid and much more extensive. It is, however, dissociated from the discharge of azurophil granules (as assessed by beta-glucuronidase). In addition, it was found that gelatinase release does not depend on the activation of the respiratory burst, although the two responses are often observed in parallel. Release is not due to cell damage as the cytoplasmic enzyme lactate dehydrogenase is fully retained. The distinct subcellular distribution and kinetics of release of gelatinase reported in this paper uncover a novel, truly secretory compartment of human neutrophils, which is highly responsive to stimulation. Gelatinase and possibly other enzymes stored in this secretory organelle may be involved in the early events of neutrophil mobilization, the response to chemotactic signals and diapedesis.

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Release of Gelatinase from a Novel Secretory Compartment of Human Neutrophils

The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.

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Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.

Polymorphonuclear leukocytes (PMNs) are one of the main sources of enzymes responsible for tissue damage in inflammatory processes. These enzymes are stored in two types of cytoplasmic granules. Azurophil granules contain lysosomal hydrolases, neutral serine proteinases, and bactericidal elements (myeloperoxidase and lysozyme). Specific granules contain collagenase, lysozyme and lactoferrin but lack lysosomal hydrolases. PMNs store all four classes of tissue proteinases, carboxyl, thiol and serine proteinases in the azurophil granules, and metallo proteinases in the specific granules. Three serine proteinases have been identified, elastase, cathepsin G and a third enzyme, which together account for a large proportion of the protein of the azurophil granules. In the course of phagocytic events, all these enzymes are released extracellularly. The neutral proteinases degrade proteoglycans and collagen. In vitro, they stimulate B-lymphocytes, which suggests that they may have immuno-potentiating activity when they are released at sites of chronic inflammation.

The polymorphonuclear leukocyte.

Neutrophils and macrophages produce, store and release large amounts of various acid and neutral proteinases. The two main proteinases of neutrophils are elastase and cathepsin G. They are localized in the azurophil granules, together with proteinase 3 and the acid cathepsins B and D. In addition neutrophils contain collagenase in the specific granules, acid proteinases in the C-particles and plasminogen activator in organelles with the characteristics of secretory vesicles. The granule-bound proteinases are released during phagocytosis while plasminogen activator is apparently secreted. In macrophages, the acid hydrolases are bound to lysosomes while the neutral proteinases are confined to secretory vesicles. The main mechanism of enzyme release in macrophages is secretion. Lysosomal hydrolases are also released by phagocytosis. Enzyme secretion is a characteristic property of activated or inflammatory macrophages. Macrophages become activated after phagocytosis of certain particles and the metabolic burst appears to be an initial event in the activation process. The action of neutrophils and of purified elastase or plasmin on cartilage was tested. These experiments indicate that neutrophil-mediated degradation of cartilage proteoglycans is largely dependent on elastase.

Ursula Bretz

Papers

Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes

Release of Gelatinase from a Novel Secretory Compartment of Human Neutrophils

Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes.

The polymorphonuclear leukocyte.

Cellular mechanisms of proteinase release from inflammatory cells and the degradation of extracellular proteins.