Cell Motility Factors. ed. by I. D. Goldberg

© 1991 Birkhauser Verlag Basel/Switzerland

Neutrophil chemotactic factors Liana Harvath Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA Summary. Polymorphonuclear leukocytes (neutrophils) are recruited to inflammatory sites by a variety of soluble mediators (chemoattractants) that stimulate neutrophil directed migration (chemotaxis). Many neutrophil chemoattractants such as neutrophil activating proteins, leukotriene B4 (LTB4), platelet activating factor, and complement-derived C5a, are generated endogeneously by host cells or enzymatic cleavage of host proteins. Other chemoattractants such as N-formyl peptides are generated exogenously by bacteria that invade the host. Oxidative modification of methionine residues or changes in the amino acid sequence of peptide chemoattractants dramatically alter their chemoattractive properties. Many of the well-defined neutrophil chemotactic factors and studies of their structure-function relationships will be reviewed.

Introduction Leukocyte migration is a primary host defense mechanism for the mobilization of phagocytic leukocytes to inflammatory sites and the development of cell-mediated immune responses. Initiation of migratory responses occurs when signal molecules interact with leukocyte plasma membrane receptors triggering cytoskeletal reorganization and cell shape change. Motile responses may be random, chemokinetic, chemotactic, or haptotactic, and are classified by in vitro assays that quantify the directional responses of cells. Random migration (unstimulated motility) and chemokinetic migration (stimulated random movement of cells in the absence of a stimulus gradient) are motile responses that do not have consistent directionality. In contrast, chemotactic and haptotactic responses are directional; they occur when cells are exposed to a signal gradient and the cells migrate toward an increasing concentration of the stimulus. Chemotactic responses occur when the signal is in a fluid phase gradient whereas haptotactic responses occur when the stimulus gradient is attached to a substratum. The relative importance of random, chemokinetic, chemotactic, and haptotactic motility in vivo has not been determined. All of these motile responses are probably involved in the mobilization of leukocytes to inflammatory sites. Neutrophils are the most rapidly motile leukocytes and are generally the first leukocytes to appear at an inflammatory site. A variety of neutrophil chemoattractants has been identified (Table 1). Some chemoattractants such as N-formyl peptides are generated by a source

36 Table I. Neutrophil chemoattractants Attractant N-fonnyl peptides

Exogeneous Endogenous

C5a

References Schiffmann et al. (1974) Jensen et al. (1969); Ward and Newman (1969) Ford-Hutchinson et al. (1980); Malmsten et al. (1980) Goetz! and Pickett (1980) Palmer et al. (1980)

PAF

Goetz! et al. (1980)

Platelet factor 4

Deuel et al. (1981)

Neutrophil activating proteins (NAPs) NAP-l NAP-2 NAP-3 NAP-4

Yoshimura et al. (1987) Walz and Baggiolini (1989) Moser et al. (1990) Schroder et al. (1990)

Neuropeptides Substance P (J -endorphin met-enkephalin

Marasco et al. (1981) Van Epps and Saland (1984) Van Epps and Saland (1984)

Laminin

Terranova et al. (1986)

other than the host (exogenous), e.g., bacteria. Many chemoattractants are generated by the host (endogenous) and include: a cleavage product of the fifth component of complement termed C5a, arachidonic acid lipoxygenation metabolites such as LTB4 , platelet activating factor, neuropeptides, and cytokines such as neutrophil activating proteins. Neutrophils have distinct plasma membrane receptors for most chemoattractants. Structural modifications of chemoattractants that alter the primary amino acid sequence, stereochemistry, or oxidative state of methionine residues frequently alter their chemotactic activity. Many of the most well-defined neutrophil chemoattractants, studies of their structure-function relationships, and examples of the effects of structural modifications on chemotactic activity will be reviewed.

Exogenous chemoattractants N -formyl peptides

In 1975 Schiffmann and colleagues reported that a series of synthetic N-formylmethionyl peptides stimulated neutrophil chemotaxis. The

37 most potent of the synthetic peptides in their study was N-formylmethionyl-Ieucyl-phenylalanine (fMet-Leu-Phe). Schiffmann et aI. postulated that N-formyl peptides were released by bacteria during protein synthesis and that the peptides stimulated neutrophil inflammatory responses to bacterial invasion. Nearly ten years later, Marasco et aI. (1984) identified fMet- Leu-Phe in a reverse-phase high performance liquid chromatography column fraction from Escherichia coli culture fluid. Other N-formyl peptides have been identified from Streptococcus sanguis cultures (Miyake et aI., 1983) and Staphylococcus aureus culture supernatant (Rot et aI., 1986). The identification and isolation of N-formyl pep tides from bacterial culture supernatants and the demonstration of the peptides' chemotactic activities indicate that N-formyl peptides are likely signals for recruiting neutrophils to sites of bacterial invasion. Synthetic N-formyl peptides have been extensively studied as probes of neutrophil activation because the peptides stimulate a variety of biochemical and physiological responses in addition to chemotaxis. When N-formyl peptides bind to neutrophil receptors, rapid changes occur in cytosolic calcium levels, membrane potentials, and cyclic nucleotide levels (White et aI., 1983; Hatch et aI., 1977; Simchowitz et aI., 1980; Smolen et aI., 1980). These events are accompanied by activation of oxygen radical production, lysosomal enzyme release, and cell membrane ruffling (Boxer et aI., 1979; Becker et aI., 1974; Zigmond, 1981; Zigmond and Sullivan, 1979). Several studies have investigated the structural requirements for N-formyl peptides as agonists of neutrophil function and indicate that N-formyl peptides containing 4 amino acids appear to have the greatest chemotactic potency and efficacy. Optimal potency occurs when the N-terminus is formylated, methionine appears in position I, phenylalanine appears in position 3, and a nonpolar amino acid appears in position 4 (Showell et aI., 1976; Freer et aI., 1980; Freer et aI., 1982; Toniolo, 1984; Rot et aI., 1987). Oxidation of the methionine residue of fMet-Leu-Phe to form either the methionine sulfoxide or methionine sulfone derivative eliminates the chemotactic activity of the molecule for human neutrophils (Fig. 1). The oxidized derivatives of fMet-Leu-Phe bind to neutrophil receptors and stimulate oxidative metabolism but fail to stimulate neutrophil chemotaxis (Harvath and Aksamit, 1984). Activated neutrophils oxidize the methionine sulfur atom of fMet-Leu-Phe to form the sulfoxide derivative (Tsan and Chen, 1980; Tsan and Denison, 1981; Clark and Szot, 1982). These observations have led to the proposal that neutrophil oxidative metabolic reactions may be a mechanism by which neutrophils modulate the inflammatory response in vivo. Neutrophils are also one of the best sources of an enzyme, methionine sulfoxide peptide reductase, which catalyzes the reduction of peptide methionine sulfoxide residues to methionine residues (Fliss et aI., 1982). Neutrophils have the potential to oxidize methionine residues of inflammatory mediators such as

38

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2500

2000

fMet-Leu-Phe

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fMet-Leu-Phe sulfoxide

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fMet-Leu-Phe sulfone

11)

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-

1500

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1000

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CI

:E

500

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0 -11

-7

-5

log [N-formyl

peptidel (Molar)

-9

-3

Figure I. Effects of methionine oxidation on the chemotactic activity of fMet-Leu-Phe. The oxidized derivatives of fMet-Leu-Phe, fMet-Leu-Phe sulfoxide (0) and fMet-Leu-Phe sulfone (/'::,), were prepared as previously described (Harvath and Aksamit, 1984) and compared with nonoxidized fMet-Leu-Phe (e) in an in vitro chemotaxis assay. Data are presented as the number of migrating human neutrophils/mm 2 filter surface in the polycarbonate membrane assay (Harvath and Aksamit, 1984). The arrow indicates the random migration response of neutrophils to medium alone.

fMet-Leu-Phe via their oxidative metabolic intermediates, and they have the enzymatic potential to reduce the methionine sulfoxide residues in peptide mediators to methionine. It remains unknown which reaction predominates at an inflammatory site in vivo. If the environment of an inflammatory site favors the oxidation of methionine, it appears that upon oxidation, molecules such as fMet-Leu-Phe would lose their chemoattractant activity for neutrophils. The human N-formyl peptide receptor is a distinct membrane protein containing 350 amino acids (Boulay et aI., 1990). The receptor associates with a 40-kDa guanine nucleotide binding protein (Polakis et aI., 1988). Two isoforms of the receptor exist which differ by a single amino acid. It remains to be determined whether the two isoforms represent the high and low affinity states of the receptor which have been described (Koo et aI., 1981; Mackin et aI., 1982; Seligmann et aI., 1982). Endogenous chemoattractants C5a

Activation of either the classical or alternative pathways of complement results in the production of C5a, a 74-amino acid cationic glycopeptide derived from the amino terminal end of the C5 alpha chain

39 (Hugli and Muller-Eberhard, 1978; Hugli, 1981; 1986). C5a is one of the most potent humoral neutrophil chemoattractants thus far identified (Jensen et ai., 1969; Ward and Newman, 1969). In addition, C5a is an anaphylatoxin inducing smooth muscle contraction (Marceau and Hugli, 1984) and increasing vascular permeability (Hugli, 1984). In human blood, carboxypeptidase N cleaves the carboxy terminal arginine from C5a to form the less potent derivative, C5a des Arg (Fernandez et ai., 1978). As illustrated in Fig. 2, removal of the carboxy terminal arginine residue results in the loss of chemotactic potency for human neutrophi1s (Webster et ai., 1980; Yancey et ai., 1989). The biological activities of C5a and C5a des Arg appear to be modulated in vivo by serum proteins. Gc-globu1in (vitamin D-binding protein) in human blood binds to C5a and C5a des Arg and acts as a cochemotaxin to enhance the chemotactic potency of C5a and C5a des Arg (Perez et ai., 1988; Kew and Webster, 1988). Chemotactic factor inactivator (CFI) is a serum protein that decreases the chemotactic activity of C5a and is the predominant inactivator of C5a contained in ammonium sulfate-fractionated serum (Ward and Ozols, 1973; Robbins et ai., 1987; Kruetzer et ai., 1979). CFI appears to prevent binding of Gc-g1obulin to C5a, thereby decreasing the chemotactic capacity of C5a (Robbins and Hamel, 1990). The precise regulation of C5a and C5a des Arg chemotactic activites by Gc-globulin and CFI remains undefined; both proteins appear to regulate the inflammatory activity of C5a and C5a des Arg in vivo. 5000 !!!.

4000

-

3000

"C

2000

..c:

~

C5a

---0--

C5a des Arg

Co

~

::J

Q)

Z

Q)

~

Cl

~

1000

a

10"

10

'0

10

9

10. 8

10

7

10

6

log [Ligand] (Molar)

Figure 2. In vitro chemotactic activities of human serum-derived C5a and C5a des Arg for human neutrophils. Human serum derived C5a (.) and C5a des Arg (0) were prepared as previously described (Yancey et ai., 1989) and evaluated for their effects on human neutrophil chemotaxis in the polycarbonate membrane assay. Data are presented as the number of migrating neutrophils/mm 2 filter surface. The arrow indicates the random migration response of neutrophils to medium alone. Bars represent 1 SD of three replicate experiments for each condition.

40 Native C5a has a molecular weight of approximately 11,000 Da (Fernandez and Hugli, 1976). Nearly 25% of the molecular mass is due to carbohydrate which is deglycosylated with endoglycosidase H (Gerard and Hugli, 1981). Studies with deglycosylated native C5a and recombinant C5a from Escherichia coli that lack the carbohydrate at asparagine64 demonstrate normal biological activity (Fernandez and Hugli, 1976; Franke et aI., 1988). The chemotactic activity ofC5a is due to the protein sequence and not the carbohydrate moiety of the molecule. Neutrophils express C5a-specific receptors on their plasma membranes (Chenoweth and Hugli, 1978). The C5a binding site appears to be formed by a single polypeptide with a molecular mass of 40 to 50 kDa (Johnson and Chenoweth, 1985; Rollins and Springer, 1985) and associates with an inhibitory guanine nucleotide binding protein (Siciliano et aI., 1990).

Arachidonic acid released from membrane phospholipids undergoes oxygenation by two distinct pathways: the 5-lipoxygenase and the cyclooxygenase pathways. Oxygenation by the 5-lipoxygenase pathway generates a series of unstable hydroperoxy-eicosatetraenoic acids (OOHETES or HPETES), of which 5-00HETE is predominantly generated in neutrophils (Borgeat and Samuelsson, 1979). The OOHETES are critical intermediates in the generation of a family of complex HETES, termed leukotrienes, that contain additional polar groups and three conjugated double bonds (Samuelsson, 1982). Of the leukotrienes, LTB4 exhibits the greatest activity as an inflammatory mediator. LTB4 is a potent neutrophil chemotactic and chemokinetic factor (FordHutchinson et aI., 1980; Malmsten et aI., 1980; Goetzl and Pickett, 1980; Palmer et aI., 1980). Structural modifications of LTB4 dramatically affect the molecule's chemotactic activity. Acetylation of the hydroxyl groups or alterations in the double bonds of the triene portion of the molecule reduce the chemotactic potency (Goetzl and Pickett, 1980) suggesting that the free hydroxyl groups and the sequence of conjugated double bonds are critical determinants. As illustrated in Fig. 3, stereochemical changes in the LTB4 molecule dramatically affect its chemotactic activity. The differences in chemotactic activities of stereoisomers are directly related to differences in their binding affinities (Goldman and Goetzl, 1984). Neutrophils usually oxidize LTB4 to form the omega-oxidation products, 20-0H- and 20-COOH-LTB4 (Samuelsson, 1982). Omega-oxidation products have significantly reduced chemotactic potency, suggesting that further oxidation of LTB4 to these products represents a mechanism for the inactivation of LTB4 in vivo (Samuelsson, 1982).

41 OH

OH COOH

OH

~:~COOH

~-OH

12(R,S)-6-trans-LTB. ~

~

e

2500,-------------------------------, ______

2000 --0--

LTB4

12(R,S)-6-trans-L TB4~

~ 1500

z

"0 (1)

n;

1000

0, 500

::iE

0+---~--'---~-'--~---'--~---4

-13

-11

-9 log [L TB4] Molar

-7

-5

Figure 3. In vitro chemotactic activities of LTB. and trans-6-LTB. for human neutrophils. The stereoisomers, LTB4 (.) and trans-6-LTB 4 (0) were compared in the polycarbonate membrane chemotaxis assay. Data are presented as the number of migrating neutrophilsjmm 2 filter surface. The arrow indicates the random migration response of neutrophils to medium alone. Specific details of the migration assay for LTB4 have been previously described (Harvath et aI., 1987).

LTB4 has been established as an important inflammatory mediator in vivo (Bray, 1986; Thorsen, 1986). Elevated concentrations of LTB4 are present in psoriatic lesions and rheumatoid arthritis synovial fluid (Brain et aI., 1984; Klickstein et aI., 1980). Intradermal injection of LTB4 stimulates neutrophil accumulation at the injection site (Soter et aI., 1983; Movat et aI., 1984), whereas intravenous injection of LTB4 induces a rapid and reversible neutropenia (Bray et aI., 1981). These responses to LTB4 administration are characteristic of an inflammatory mediator. Pharmacologic agents that inhibit the 5-lipoxygenase pathway of arachidonic acid metabolism significantly decrease inflammatory reactions (Allen and Littlewood, 1982; Kassis, 1985), suggesting that LTB4 and the 5-lipoxygenase pathway of arachidonic acid metabolism are important components of the inflammatory response. Stereospecific binding sites for LTB4 have been identified on human neutrophils (Goldman and Goetzl, 1984; Kreisle and Parker, 1983). A 60-kDa plasma membrane protein appears to contain the binding site for LTB4 (Goldman et aI., 1985). Like N-formyl peptide and C5a

42 receptors, neutrophil LTB4 receptors appear to associate with a 40-kDa guanine nucleotide-binding protein (Goldman et aI., 1987). Platelet activating factor (P AF)

The lipid, 1-0-Alkyl-2-acetyl-sn-glyceryl-3-phosphory1choline, referred to as PAF (Pinckard et aI., 1979; Demopoulos et aI., 1979), is a neutrophil chemoattractant (Goetzl et aI., 1980) that is generated by a variety of cells including endothelial cells and leukocytes. Studies with 2-acyl-analogues, such as the 2-acetyl-, 2-maleyl-, 2-succinyl-, and 2-phthalyl-analogues, have indicated that the 2-acyl-substituent is a functionally critical determinant for chemoattractant activity (Goetzl et aI., 1980). All four of these analogues retain chemotactic activity for neutrophils. At suboptimal chemotactic concentrations, the maleyl-, succinyl-, and phthalyl-analogues enhance neutrophil chemotactic responses to C5a (Goetzl et aI., 1980). PAF, like N-formyl peptides, C5a, and LTB4, stimulates neutrophil enzyme release and activates the respiratory burst (O'Flaherty et aI., 1981; Smith et aI., 1984; Shaw et aI., 1981). Like C5a, PAF also functions as an anaphylatoxin, inducing smooth muscle contraction (Stimler and 0' Flaherty, 1983) and increasing vascular permeability (Humphrey et aI., 1984). PAF receptor activity has been identified on neutrophil plasma membranes (Stewart and Dusting, 1988), however, the primary structure and subunit composition remain unknown. Platelet factor 4 (PF4)

PF4 is a 70-amino acid protein that is synthesized by bone marrow megakaryocytes, stored in platelet IX-granules, and released after platelet activation (Broekman et aI., 1975; Kaplan et aI., 1979; Holt and Niewiarowski, 1985; Deuel et aI., 1977; Hermodson et aI., 1977). PF4, at concentrations found in normal human serum, is chemotactic for neutrophils in vitro (Deuel et aI., 1981). The chemotactic potency of PF4 for neutrophils is approximately 60-70% of the activity of C5a in vitro (Bebawy et aI., 1986). Recombinant PF4, expressed in Escherichia coli, and native PF4 have similar chemotactic properties (Park et aI., 1990). The chemoattractant activity appears to be related to the carboxy-terminal domain of PF4 (Osterman et aI., 1982; Goldman et aI., 1985). The carboxy-terminal dodecapeptide of PF4 is a more effective neutrophil chemoattractant than the intact PF4 molecule (Leonard et aI., 1990). PF4 is a member of a multigene family, the small inducible genes (SIG), which appear to be important in coagulation, inflammation, and cell growth (reviewed by Park et aI., 1990). Like most chemoattractants,

43 PF4 stimulates neutrophil lysosomal enzyme release; unlike most chemoattractants, it does not appear to stimulate neutrophil oxidative metabolism (Bebawy et aI., 1986). PF4 has sequence similarity to the neutrophil activating protein family of chemoattractants (described below). Neutrophil activating proteins (NAPs)

A cytokine family of structurally similar proteins, referred to as neutrophil activating proteins (NAPs) has emerged as a class of neutrophil chemoattractants. NAPs are single chain proteins of 8-10 kDa and most contain 4 highly conserved cysteine residues that form two disulfide linkages. NAP-I, also referred to as interleukin-8 (IL-8), was first identified as a product of lipopolysaccharide (LPS)-stimulated monocytes (Yoshimura et aI., 1987). Subsequent studies have shown that NAP-l is also generated by stimulated lymphocytes, lung macrophages, endothelial cells, fibroblasts, keratinocytes, epithelial cells, and hepatocytes (reviewed by Leonard and Yoshimura, 1990). NAP-l is secreted as a 79-residue protein after cleavage of a 20-residue signal peptide, referred to as NAP-IO( (Leonard and Yoshimura, 1990). Proteolytic cleavage of NAP-IO( in tissue culture medium results in the formation of 77- and 72-residue NAP-1P and NAP-ly, respectively. The 0(, p, and y forms of NAP-l all possess neutrophil chemoattractant activity. Recombinant NAP-l expressed in Escherichia coli is biologically equivalent to native NAP-l and is a potent neutrophil chemoattractant (Lindley et aI., 1988). Intradermal injection of human NAP-l into rabbits, rats, and humans induces perivascular neutrophil infiltration, indicating that human NAP-l is an active inflammatory stimulus in other species as well as humans (Colditz et aI., 1989; Rampart et aI., 1989; Larsen et aI., 1989; Leonard and Yoshimura, 1990). Human neutrophils express plasma membrane receptors for NAP-l (Samanta et aI., 1989). Structurally related PF4 does not bind to neutrophil NAP-l receptors, however, NAP-2 interacts with the NAP-l receptor (Leonard et ai., 1990). NAP-2 is a 70-amino acid protein with structural homology to NAP-l and PF4 (Walz and Baggiolini, 1989). NAP-2 is formed when a IS-residue fragment is cleaved from the N-terminus of connective tissueactivating peptide III (CTAP-III). CTAP-III is released from platelet-O(granules during platelet stimulation. In vitro, monocyte proteases can cleave the IS-residue fragment from CTAP-III and generate NAP-2 (Walz and Baggiolini, 1990). NAP-2, like NAP-I, is a neutrophil chemoattractant that stimulates neutrophil free calcium changes and enzyme release (Walz et ai., 1989). CTAP-III is not a neutrophil

44 chemoattractant and does not stimulate neutrophil function. NAP-2 appears to be a less potent neutrophil chemoattractant than NAP-I (Leonard et aI., 1990). NAP-3 (Schroder et aI., 1990) is an LPS-stimulated monocyte-derived cytokine that has an identical amino terminal sequence to the 73-amino acid protein, known as melanoma growth-stimulatory activity (MSGA/ gro). MSGA/gro was identified on the basis of its mitogenic activity for Hs294T human melanoma cells and was found to be structurally related to fJ-thromboglobulin (Richmond et aI., 1988). MSGA/gro also has marked sequence similarity to NAP-I. Chemically synthesized MSGA/ gro is a potent neutrophil chemoattractant that stimulates neutrophil free calcium changes and enzyme release (Moser et aI., 1990). NAP-4 is an 8 kD protein isolated from human platelet lysates which demonstrates 71 % homology with PF4, but only weak homology with NAP-I, NAP-2, and NAP-3 (Schroder et aI., 1990). Unlike the other members of the PF4-like proteins, which contain four cysteine-residues at identical relative position, NAP-4 is lacking two ,of the four cysteineresidues. NAP-4 is a chemoattractant for human neutrophils and stimulates enzyme release, however, it has lower chemotactic potency than NAP-I. NAPs appear to play an important role in inflammation and regulation of the inflammatory response. NAP-2 and NAP-4 originate from platelets, whereas NAP-l and NAP-3 are synthesized by LPS- or cytokine-stimulated cells. The striking sequence homology of NAPs with PF4 suggest that these molecules were derived from a common ancestral gene. The genes for PF-4, MSGA/gro, and NAP-l all map to chromosome 4q12-q21 (Modi et aI., 1990). The structure and biochemical properties of NAP receptors remain unknown. PF4 does not appear to interact with the neutrophil NAP-l receptor, however, NAP-2 binds to the NAP-l receptor (Leonard et aI., 1990), indicating that PF4 and NAP-l have distinct receptors and that NAPs may share a common receptor on neutrophils.

Neuropeptides Substance P is an undecapeptide present in the peripheral and central nervous system (Jessell et aI., 1983) and is a hypotensive, vasodilatory, and smooth muscle contracting agent (Bury and Mashford, 1976). The four carboxy-terminal amino acids of substance P (Phe-Gly-Leu-MetNH 2 ) resemble, in reverse, the amino acid sequence of fMet-Leu-Phe (Marasco et aI., 1981). Substance P stimulates neutrophil chemotaxis and lysosomal enzyme release (Marasco et aI., 1981). At chemotactic concentrations, substance P binds to neutrophil N-formyl peptide receptors. The concentration required for half-maximal inhibition of specific

45 binding of fMet-Leu-Phe is 1400-fold greater than half-maximal inhibition by fMet-Leu-Phe (Marasco et ai., 1981). Since the spasmogenic activity of substance P occurs at concentrations approximately 10,000fold lower than the neutrophil chemotactic concentration, it is unlikely that the effects of substance P on neutrophil chemotaxis are physiologically relevant in vivo. j3-endorphin, a 31-amino acid peptide hormone, and met-enkephalin, a penta peptide, are potent analgesics that have a broad spectrum of biologic activities which includes effects on lymphocyte proliferation and antibody production (Gilman et ai., 1982; Johnson et ai., 1982). j3-endorphin and met-enkephalin exhibit weak chemotactic activity for human neutrophils. Chemotactic responses are :-:;; 38% of the response to fMet-Leu-Phe with met-enkephalin and :-:;; 12% of the response to fMet-Leu-Phe with j3-endorphin (Van Epps and Saland, 1984). j3-endorphin and met-enkephalin do not bind to neutrophil N-formyl peptide receptors, but appear to bind to a distinct receptor. It remains unclear whether the j3-endorphin and met-enkephalin effects on human neutrophil chemotaxis in vitro have any physiologic relevance in vivo. The observations of neuropeptide effects on neutrophil chemotaxis and the demonstration that opiate receptors are present on human neutrophils (Lopker et ai., 1980) indicate that neuropharmacologic mediators may interact with phagocytic cells of the immune system and alter their function in vivo.

Lam in in Laminin, a large and abundant basement membrane-specific glycoprotein with a molecular mass of appriximately 1 x 106, binds to type IV collagen, to heparan sulfate proteoglycan, and to an integral membrane protein on the surface of some epithelial and neoplastic cells (reviewed by Martin et ai., 1988). Laminin can alter the shape of some cells and promote matrix deposition (McGarvey et ai., 1984; Martin et ai., 1984). Laminin is a chemoattractant for rabbit peritoneal neutrophils that have been harvested from an inflammatory exudate (Terranova et aI., 1986; Byrant et ai., 1987). The laminin chymotryptic fragment Cl, which retains cell binding activity, is also chemotactic for rabbit neutrophils (Bryant et ai., 1987) and suggests that proteolytic fragments of laminin may function as chemoattractants in vivo. Human and rabbit neutrophils have specific receptors for laminin on their plasma membrane surfaces (Bryant et ai., 1987). It appears that a cell-associated form of laminin may be required for neutrophil chemotaxis (Terranova et ai., 1986). The chemotactic activity of laminin for peripheral blood human neutrophils has not been well established. It is possible that neutrophils acquire chemotactic responsiveness to laminin

46 when they have been recruited to an inflammatory locus in vivo and that circulating peripheral blood neutrophils are relatively unresponsive to the laminin molecule.

Conclusions A variety of molecules has been discovered that exhibits a range of chemotactic potencies for neutrophils. Most of the chemoattractants are generated by the host as a result of leukocyte, platelet, or other host tissue stimulation (i.e., LTB 4 , PAF, PF4, NAPs) or by activation of the complement pathways (i.e., C5a). Many of the molecules demonstrate substantial chemotactic potency and efficacy in vitro and appear to be involved in recruiting neutrophils to inflammatory sites in vivo. Neuropeptides and laminin have less chemotactic potency, perhaps because they are continually present in the host and do not appear to be increased in concentration during inflammation. The bacterially-derived N-formyl peptides are extremely potent molecules for recruiting neutrophils to sites of bacterial invasion. The most active neutrophil chemoattractants share in common their ability to also stimulate neutrophil lysosomal enzyme release and the respiratory burst. Therefore, highly active chemoattractants efficiently recruit neutrophils to inflammatory sites and then stimulate other biochemical and metabolic events that are necessary for microbicidal and degradative function. Neutrophil chemoattractant receptors exhibit exquisite selectivity and specificity in their interaction with molecules. As illustrated in this review, oxidative modifications of methionine residues (Fig. 1), removal of one amino acid residue (Fig. 2), or changes in the stereochemistry (Fig. 3) of chemoattractants significantly affect their chemotactic activities. As progress is made in the characterization of neutrophil chemoattractant receptors, it will become clearer how these chemical modifications affect chemoattractant interactions with the receptor binding domains. This information should provide a better understanding of the basic mechanisms of neutrophil chemotaxis.

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