Update Neutrophil Attractant/Activation Protein-l (NAP-l [Interleukin-8]) Edward J. Leonard and Teizo Yoshimura Immunopathology Section, Laboratory of Immunobiology, National Cancer Institute, Frederick, Maryland

Neutrophil attractant/activation protein-I (NAP-I [interleukin-8]) is an 8,400 0 protein that is a chemoattract ant and granule release stimulus for neutrophils. NAP-I was first purified from culture fluids of lipopolysaccharide-stimulated human blood mononuclear leukocytes. It was subsequently isolated from lipopolysaccharide-stimulated lung macrophages, mitogen-stimulated lymphocytes, and virus-infected fibroblasts. Interleukin-I or tumor necrosis factor induces NAP-I mRNA in many cells, including monocytes, fibroblasts, and endothelial cells. NAP-I belongs in a family of host defense small proteins, which have a degree of sequence and structural similarity. Noteworthy are the four half-cystine residues in each protein, which are in register when the protein sequences are suitably aligned. Based on cloning data and N-terminal sequence analyses, NAP-I is secreted as a 79 residue protein after cleavage of a 20 residue signal peptide. The commonly isolated 77 and 72 residue forms are probably extracellular cleavage products. NAP-I has considerable charge heterogeneity. Charge and length variants all have chemotactic activity. In contrast to many chemoattractants, NAP-I does not attract monocytes. Intradermal injection of NAP-I causes neutrophil infiltration. The wide spectrum of cell sources and production stimuli suggests that NAP-I mediates neutrophil recruitment in host defense and disease.

The macrophage is the predominant cell occupying the normal lung acinus, and it is widely held to be the first line of defense against noxious agents that elude removal mechanisms in the more proximal regions of the respiratory tree. In addition to its phagocytic and microbicidal activities, the lung macrophage -like the tissue macrophage - releases many different mediators that contribute to host defense through both local and systemic actions (1). Local action includes release of chemoattractants that recruit blood leukocytes to the challenge site. This was shown in several studies (reviewed in reference 2) in which bronchoalveolar lavage (BAL) macrophages obtained from animals after intratracheal insufflation of noxious stimuli secreted chemoattrac-

(Received in original form March 30, 1990 and in revised form April 11, 1990) Address correspondence to: Edward 1. Leonard, M.D., Chief, Immunopathology Section, Laboratory of Immunobiology, NCI, FCRF, Building 560, Room 12-71, Frederick, MD 21701. Abbreviations: o-l-proteinase inhibitor, aI-PI; adult respiratory distress syndrome, ARDS; bronchoalveolar lavage, BAL; connective tissue activating peptide-III, CTAP-III; interleukin, IL; lipopolysaccharide, LPS; leukotriene B4 , LTB4 ; monocyte chemoattractant protein-I, MCP-I; melanoma growth-stimulating activity, MGSA; neutrophil attractant/activation protein-I, NAP-I (also called interleukin-8); nuclear magnetic resonance, NMR; platelet factor 4, PF-4; phytohemagglutinin, PHA; tumor necrosis factor, TNF.

Am. J. Respir. Cell Mol. Bioi. Vol. 2. pp. 479-486, 1990

tants in tissue culture and caused neutrophil infiltration when transferred to lungs of normal animals. During the I980s, a major effort was made to identify chemically the macrophage-derived chemoattractants. In addition to leukotriene B4 (LTB4 ) , macromolecules ranging in estimated mass from 1,500 to 10,000 D were partially purified. Recently, we and others isolated, sequenced, and cloned an 8,400 D protein chemoattractant for neutrophils (neutrophil attractant/activation protein-I [NAP-I], also known as interleukin-8 [IL-8]). NAP-I was first purified from culture fluids of lipopolysaccharide (LPS)-stimulated human blood monocytes. Because it is also produced by LPS-stimulated human lung BAL macrophages (2), we believe that NAP-I is the 10 kD alveolar macrophage-derived protein described by Merrill and colleagues in 1980 (3). This review will focus primarily on NAP-I, which has generated at least 30 reports since our original description in 1987. We will also consider briefly other attractants that cause neutrophil recruitment. Isolation and Biochemical Characterization In at least two of the three laboratories that independently isolated NAP-I, the work was initiated as a search for a mediator of neutrophil recruitment. Schroeder and Christophers (4), looking for the proximate cause of neutrophil-rich lesions of psoriasis, isolated an NAP-I-like protein from extracts of 2 to 10 g of desquamated skin vacuumed from the bed sheets of hospitalized psoriatic patients (Schroeder, personal communication). This certainly demonstrates the

480

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990

remarkable stability of the molecule. Our interest was in the capacity of the tissue macrophage to generate an acute inflammatory reaction by secretion of multifunctional cytokines like IL-l. According to two reports, IL-I was a neutrophil chemoattractant in vitro, and it was also claimed that intradermal injection of IL-I caused local infiltration of neutrophils (reviewed in reference 5). However, we found that highly purified or recombinant IL-I had no chemotactic activity in vitro. This suggested that the reported chemotactic activity of IL-I in vitro was due to a contaminating factor. We therefore added LPS, a potent stimulus of IL-I secretion, to human monocytes, and purified neutrophil chemotactic activity from the culture fluid. To anticipate the final result, NAP-I was separated from IL-I (5). Furthermore, IL-I, a product of LPS-stimulated monocytes, also caused monocytes to express mRNA for NAP-I (6). Thus, at least one basis for Il.d-induced neutrophil infiltration was stimulation of NAP-l secretion by tissue macrophages. Purification of NAP-l In our original purification procedure (7), 100 p,g of pure NAP-I was obtained from 3 liters of culture fluid collected 24 h after stimulation of human blood mononuclear leukocytes with LPS. After removal of culture fluid FCS albumin on an anion exchanger (DEAE-Sepharose), NAP-I was purified to homogeneity in three high-resolution steps that included gel filtration, CM-HPLC, and reverse-phase HPLC. We now obtain the same amount of pure NAP-I from onetenth the starting material by using an immunoaffinity column of Sepharose IgG monoclonal anti-NAP-I (8). Conditioned medium from stimulated monocytes is applied to the column, and after an extensive wash with 2 M NaCl to remove unbound andnonspecifically bound protein, NAP-I is eluted with pH 2.5 glycine buffer. Protein in the acid eluate is almost all NAP-I. Mass and Charge Heterogeneity of NAP-l After N-terminal sequence analysis of the first 40 residues of NAP-l (7), an oligonucleotide probe was made to screen a eDNA library generated from LPS-stimulated human blood mononuclear cells. DNA sequencing of a positive clone revealed an open reading frame coding for 99 residues (6), the C-terminal 72 of which corresponded to the NAP-I that was isolated by our original multistep purification method. However, when irnmunoaffinity-purified NAP-I was analyzed by CM-HPLC, four closely spaced but well-resolved protein peaks were seen. The chemotactic activities (VI p,g) of the peaks were comparable. SDS-PAGE showed two closely spaced bands in the 6.5 kD region, the relative intensities of which differed for the four peaks. Sequence analysis revealed three NAP-I proteins-one with the 72 residue sequence, and two more with an additional 5 and 7 residues at the N-terminus. The longer forms were converted to the 72 NAP-I when the stimulated mononuclear cell culture fluid was separated from cells and incubated overnight at 37° C (8). Thus, it appears that NAP-I is expressed as a 99 residue protein. The hydrophobicity of the first 20 residues is consistent with a signal peptide, the secreted form being 79 residue NAP-l (NAP-l a) after cleavage of the C-E bond at position 20. Proteolytic cleavage of NAP-Ia in the

tissue culture medium accounts for 77 and 72 residue NAP-I (NAP-I{j and NAP-I 'Y). Despite arginyl-lysyl bonds in the remainder of the molecule, no cleavage of NAP-l y in culture fluid occurred. This suggests that NAP-I'Y is resistant to proteolysis and may persist at sites of inflammation. Van Damme and associates found that three fourths of the NAP-I isolated from fibroblast-conditioned medium was NAP-I{j (9); this form has also been purified from culture fluids of stimulated T lymphocytes (10). The relative amounts of the different NAP-Is recovered may reflect conditions of isolation procedures and differences in proteolytic enzyme content of the culture fluids. The resolution by CM-HPLC of the NAP-I proteins into four separate peaks suggests charge heterogeneity. Remarkably, each of the NAP-I proteins is distributed about equally between two adjacent peaks. NAP-Ia is distributed between peaks I and II; NAP-l'Y between peaks II and III; and NAP-I{j between peaks III and IV (8). Thus, each isolated NAP-I appears to have two apparent charge forms, by the criterion of CM elution. Schroeder and coworkers (II) reported four different isoelectric points (4.7, 4.9, 6.4, and 6.9) by thinlayer isoelectric focusing for a preparation of NAP-I that was a single-chain species by sequence analysis. On the other hand, we reported an NAP-I pI by electrofocusing of about 8 (5); because all of our NAP-I species were bound to the CM-HPLC column at a pH of 6.5, they should have had an apparent net positive charge at that pH, and therefore a pI above 6.5. The point of this paragraph is not to confuse the reader with conflicting data but to emphasize that a protein with a given primary structure may exhibit widely different net charges, depending on assay conditions (12), tertiary structure (13), and well-known post-translational modifications such as deamidation, or addition of phosphate or sugar residues. For example, serum albumin has a pI of about 5 in the native state, whereas in 8 M urea it is 6.0, closer to the value estimated from the sum of its acidic and basic side chains (13). It is possible that NAP-lor closely related molecules may have basic or acidic pIs, depending on their posttranslational state or on methods of measurement. Intrachain Disulfides and Tertiary Structure NAP-I belongs to a family of several small protein cytokines that are thought to participate in the response to injury (7). These proteins comprise single chains with molecular masses in the 8,000 to 10,000 D range. A distinguishing feature is the occurrence of four half-cystine residues in the chain. When the sequences of the different proteins are appropriately aligned, the proteins fall into two subgroups according to the positions of the first two half-cystines (C-X-C or C-C), and all four half-cystine residues are in register. Proteins in the human C-X-C subgroup include platelet factor 4 (PF-4) (14), melanoma growth-stimulating activity (MGSA) (15), connective tissue activating peptide-III (CTAP-III) (16), NAP-2 (a truncated form of CTAP-III with chemotatic activity) (17, 18) and NAP-I. The genes for PF-4, MGSA, and NAP-I all map to chromosome 4q12-q21 (19). Monocyte chemoattractant protein-I (MCP-I) is the one human cytokine in the C-C subgroup with a known functional activity (20). Begg and colleagues showed that the half-cystines in {j-thromboglobulin (CTAP-III(des 1-4)) existed as disulfide

Update

bridges, the first linked to the third, and the second linked to the fourth (21). The same order of half-cystine pairing was recently shown for NAP-l by nuclear magnetic resonance (NMR) (22). This pairing is apparently required for biologic activity, which is abolished if the NAP-I is reduced and alkylated (23). The NMR analysis of NAP-I gives a remarkably detailed picture of its three-dimensional structure. The conclusions are that the bulk of the NAP-I molecule up to residue 54 is in the form of a {3-sheet, whereas the C-terminus comprises a long a-helix that sits on the surface of the {3-sheet. The residues in the a-helix are arranged so that the hydrophobic amino acid side chains face the {3-sheet and the hydrophilic amino acids are on the solvent side of the helix. NAP-I is a dimer in solution, due to interaction of {3-sheet complementary residues; the paired a-helices, separated by about 14 A (center to center), are thought to have the potential for presenting their hydrophilic residues to the NAP-I receptor. The investigators speculate that other proteins in this family may have similar structures in solution, including an amphiphilic a-helix. In this regard, it is of interest that the Cterminal dodecapeptide of PF-4 is a chemoattractant for human neutrophils (24), and though the potency is 1,000 times less than NAP-I, efficacy (percentage of input neutrophils migrating) is comparable (Leonard et al., unpublished observations). Cellular Aspects of NAP-l Neutrophil receptors for NAP-I. Studies with other pure chemoattractants, like fMLP and C5a, have established that neutrophils have saturable plasma membrane binding sites for these agonists. Binding at temperatures above 0° C is associated with internalization of the ligand-receptor complex, and appearance of new receptors on the cell surface that are thought to originate in part from an intracellular receptor pool (25). The new receptors provide for continuing responsiveness as the cell migrates up a concentration gradient of chemoattractant. Human neutrophils also have saturable binding sites for radioiodinated NAP-I. From analysis of the binding curves, Samanta and collaborators concluded that there- are about 20,000 receptors per cell, with a ligand Kd of about 10-9 M (26). Internalization of the NAP-I receptor complex is associated with NAP-l degradation and reappearance of receptors on the cell surface. Neutrophillysosomes participate in this process, as shown by inhibition with agents that block lysosomal function by altering pH (27). Using flow cytometry, we found saturable binding of NAP-l-FITC at 0° C to human neutrophils. The symmetry of the histograms shows that all neutrophils bind; there is no evidence for a subpopulation without NAP-l receptors (28). We also measured the capacity of structurally related attractants to inhibit NAP-l-FITC. Binding inhibition curves were generated by equilibrating neutrophils with mixtures of 2 x 10-7 M NAP-l-FITC and increasing concentrations of unlabeled ligand. As expected, NAP-I inhibited binding of NAP-I FITC. Binding was also inhibited by NAP-2 (Leonard et at. , unpublished observations), a truncated form of the structurally related CTAP-III molecule (17), which was recently shown to be a chemoattractant for neutrophils (18). Thus, it appears that NAP-2 interacts with the NAP-I receptor. How-

481

ever, the chemotactic C-terminal dodecapeptide ofPF-4 (PF4(59-70», another member of the C-X-C protein family, had no effect on NAP-I-FITC binding. It is interesting to relate these results to the NMR study of NAP-I described above, which suggests that all members of the C-X-C protein family may have similar (3-sheet-ahelix structures in solution and that the ligand-binding surface is the hydrophilic face of the a-helix. The chemotactic activity ofPF-4(59-70) supports the idea that the C-terminus is the receptor-binding region; the absence of NAP-I-FITC binding inhibition shows that despite the structurally similar a-helix motif, PF-4 and NAP-I receptors appear to be distinct. The NAP-2 data raise questions about how the N-terminal part of the molecule affects the receptor interactive site. The parent CTAP-III molecule is not chemotactic for neutrophils and has only borderline capacity to inhibit NAPI-FITC binding. After cleavage of the N-terminal 15 residues, the resulting NAP-2 had both chemotactic activity and NAP-I receptor-binding capacity. What is the structural basis for the appearance of chemotactic activity after N-terminal cleavage? NMR studies ofPF-4, CTAP-III, and NAP-2 may provide answers to some of these structure-function questions. Experiments on specific desensitization have also provided information about ligand-receptor relationships in neutrophils. When cells receive two successive doses of an agonist at relatively short time intervals, loss of cellular responsiveness to the second dose of the same agonist is referred to as specific desensitization. For example, with neutrophil specific granule enzyme release as the endpoint, specific desensitization is observed with the second of two successive doses of C5a, whereas enzyme release occurs when C5a is followed by an unrelated agonist like NAP-I. In earlier studies, this was cited as evidence that NAP-I and C5a were different attractants, interacting with functionally different receptors (29, 30). Conversely, lack of response by NAP-I-treated neutrophils to a purified attractant from LPSstimulated endothelial cells suggests that the endothelial cell product and NAP-I are structurally related, since they appear to interact with the same receptor (31). Stimuli and cellular sources for NAP-l production. To define the role of NAP-I as a host-defense cytokine, it is essential to know the stimuli for its synthesis, the cells that produce it, and the time course and amounts of its release. Responses have been determined by isolation and sequencing of the product, densitometry of Northern blots for NAP-I mRNA, detection of NAP-l mRNA by in situ hybridization, or immunoassay of NAP-l in solution. Table 1 presents a list of cells that have been reported to make NAP-I mRNA or secrete NAP-I. The product has been isolated and identified by partial N-terminal sequencing from culture fluids of LPSstimulated monocytes (7, 32, 33) and BAL macrophages (2), phytohemagglutinin (PHA) or Con A-treated lymphocytes (10, 35), and fibroblasts treated with poly I:C or infected with measles virus (9). By cross-desensitization criteria, LPS-stimulated endothelial cells also secrete an NAP-lrelated chemoattractant (31). The biologic significance of NAP-l message formation or secretion by such a wide variety of cells is a matter for speculation. Perhaps this is a metazoan call for neutrophil help in response to injurious

482

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 21990

TABLE I

NAP-J cell sources and stimuli for production Identification by Cell Mononuclear cells! Lung macrophages Lymphocytes Endothelial cells Fibroblasts Keratinocytes Epithelial cells, hepatocytes

Stimulus LPS LPS, IL-I, TNF LPS TNF PHA, Con A LPS IL-I, TNF, LPS IL-I, TNF Rubella, poly I:C IL-I, not TNF IL-I, TNF, not LPS

mRNA*

N-terminal sequence!

7, 32, 33 6 2 34 10, 35* 3111 34 36

37' 9

36 cited in 38

Definition ofabbreviations: NAP-I = neutrophil attractant/activation protein-l (also known as interleukin-8); LPS = lipopolysaccharide; IL-I = interleukin-l; TNF = tumor necrosis factor; PHA = phytohemagglutinin; Con A = concanavalin A. * References in this column are to detection of NAP-I mRNA, but not secreted product. t Citations in this column are for N-terminal sequencing. Papers on purification of NAP-I to homogeneity without sequencing are cited in reference 38. t After separation of monocytes and lymphocytes by elutriation, the cell responding to LPS by secretion of NAP-I was the monocyte (5). In reference 10, the cells were purified T lymphocytes; in reference 35, they were unseparated blood mononuclear cells. II Although this highly purified material was not sequenced, it was biochemically similar to NAP-I and cross-desensitized with NAP-I. , Fibroblasts also secreted melanoma growth-stimulating activity (MGSA). MGSA was also purified and sequenced from extracts of psoriatic scales (cited in reference 37).

*

agents (LPS, viruses) or to cytokines released (IL-l, tumor necrosis factor [TNF]) in response to injurious agents. Secretion of NAP-l by a virus-infected cell without intervention of immune system leukocytes suggests that infectionrelated neutrophil recruitment may occur via a simple NAPl-mediated pathway. For example, secretion of NAP-l by cells of the nasal mucosa might account for the practically exclusive presence of neutrophils in secretions associated with the common cold. Wf: recently developed a sandwich ELISA for measurement of NAP-l in biologic fluids. The ELISA utilizes a mouse monoclonal and a rabbit polyclonal anti-NAP-1. Its sensitivity limit is about 1 ng/ml, and it does not detect the structurally related proteins PF-4 or CTAP-III. The concentration of NAP-l in culture fluid ofBAL human macrophages 24 h after the addition of LPS was 860 ± 40 ng/ml (SEM for six normal subjects), compared to 40 ± 15 ng/ml for unstimulated macrophages. Based on ELISAs of culture fluids 24 h after LPS stimulation, the amount of NAP-l produced by either blood monocytes or lung macrophages was comparable (0.5 to 1.0 picogram/cell). NAP-I was not the only neutrophil chemoattractant in the 24 h lung macrophage culture fluids, since after absorption of 95 % of the NAP-Ion an immunoaffinity column (as determined by ELISA of the pass-through effluent) there was much more chemotactic activity than could be accounted for by residual NAP-l (2). In studies on the time course of attractant release by LPSstimulated lung macrophages, Rankin et al. (submitted for

publication) showed that LTB4 was released early, with a peak at 3 h and a decline to zero at 24 h. NAP-l was detectable by ELISA at 5 h, but reached considerably higher concentrations at 24 h. Zymosan, tested as a phagocytic stimulus, also caused secretion of NAP-I. The chemotactic activity other than NAP-I in 24 h culture fluids is not LTB4 but is probably another protein. Since LPS is one of the causes of the adult respiratory distress syndrome (ARDS), the relevance of these studies to the pathogenesis of ARDS needs no elaboration. Quantitative NAP-l ELISAs of ARDS lung fluids will be of great interest. Sticherling and associates (39) have also reported a sandwich ELISA for NAP-I, using mouse monoclonals for both capture and detection antibodies. Their work and ours show that more than one epitope on the NAP-l molecule is immunogenic, since two reactive sites on the antigen are required in a sandwhich ELISA. Actions of NAP-l in vitro. We will first summarize the in vitro actions of NAP-I and then relate them to the results of in vivo studies. In contrast to many other well-studied attractants, such as C5a, fMLP, and LTB4 , which attract both neutrophils and monocytes, NAP-l is not a chemoattractant for monocytes at concentrations as high as 3 X 10-8 M (7). In a survey of different human blood leukocytes (28), we found that NAP-I chemotactic efficacy (percentage of input cells migrating at the optimal attractant concentration) was 30% for neutrophils, 16% for basophils, 9% for T lymphocytes, and 0 % for eosinophils (except for a small increment above a high random migration if eosinophils were stimulated by fMLP during the isolation). The NAP-l concentration eliciting optimal responses by each cell type was approximately 10-8 M. NAP-I-stimulatedneutrophils released a marker of specific granule contents (vitamin B12 binding protein) (30), and when treated with cytochalasin B they released /3-glucuronidase from azurophil granules (29, 30). NAP-l also stimulated a metabolic burst of 1 to 2 min in duration, as measured by release of about 10 nmol of superoxide per 3 X 106 neutrophils. The response was comparable to that observed with C5a (30). The concentration of NAP-I eliciting these reactions is not certain, since the study was done with partially purified material (30). However, the investigators state that similar results were obtained with pure NAP-I. NAP-l also caused histamine release from human basophils but only at a concentration (10-6 M) that was 1 to 2 orders of magnitude higher than the chemotaxis optimum (40). Dahinden and colleagues reported histamine release at NAP-l concentrations of 10-8 M to 10-7 M, provided that basophils were pretreated with IL-3 for as little as 10 min prior to the addition of NAP-l (41). Of possible relevance to lung disease is a report that NAP-l caused contraction of guinea pig lung parenchyma, a response that was completely abolished by pretreatment with indomethacin (42). The cell secreting the spasmogen (presumably a product of the cyclooxygenase pathway) in response to NAP-l has not yet been identified. . Gimbrone and coworkers (43) reported that neutrophils have enhanced adherence to cultured endothelial cells stimulated by IL-l, TNF, or LPS. Neutrophil adherence was measured by adding labeled neutrophils to endothelial cells in 96-well plates and removing nonadherent neutrophils by cen-

Update

483

Figure 1. Hematoxylin and eosin stained 2-JLm sections of human skin biopsies taken 3 h after intradermal injection of 40 JLI of 2 x 10-7 M NAP-I. The upper panel shows that neutrophil infiltration was confined to vascular regions, with little migration into surrounding dermis. Most neutrophils were morphologically intact, as shown in the lower panel.

trifugation of the inverted plate after a lO-min incubation at 37° C. If NAP-l was added with the neutrophils, adherence was diminished. The molecular basis for the altered adherence has not been established. As noted above, NAP-l causes many neutrophil alterations related to the plasma membrane, including ligand-receptor internalization and granule exocytosis. The effect of NAP-Ion the plasma membrane interaction of neutrophils with endothelial cells is of interest because of the role of adhesion in the movement of leukocytes across vessel walls. Actions of NAP-l in vivo. Colditz and associates (44) in-

jected pure recombinant NAP-l (human protein sequence) intradermally into rabbits. Indium-labeled homologous neutrophils and iodinated BSA were given intravenously at the same time. Skin sites were biopsied 4 h later for histology and counting of radioactivity as indices of neutrophil infiltration and increased vascular permeability to albumin. Microscopy showed dermal edema and neutrophil infiltration, especially in proximity to venules, but no other types of leukocytes. At the highest concentration of NAP-l injected (5 x 10-6 M), counts of indium-labeled neutrophils and iodinated BSA were about half that obtained with intradermal

484

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990

endotoxin. Actinomycin D, an inhibitor of protein synthesis, eliminated the endotoxin response but had no effect on the response to NAP-I. Using the same radioactive markers for neutrophil infiltration and increased vascular permeability, Rampart and colleagues (45) found that the minimal response to a 10-8 M concentration of intradermally injected NAP-I was greatly increased when PGE 2 was mixed with the challenge agonist. Plasma protein extravasation was dependent on neutrophils, since it did not occur in neutropenic rabbits. Taken together, these two studies show that human NAP-l acts across species barriers to cause dermal neutrophil infiltration in rabbits. As shown previously for the attractant C5a (46), the magnitude of both neutrophil infiltra.tion and plasma extravasation is greatly increased by the vasodilator, PG&, to the NAP-I challenge, and the increased vasopermeability is neutrophil dependent. The greater response to endotoxin than to NAP-I is thought to be due to the fact that endotoxin induces endothelial cell expression of adhesion molecules for neutrophils, as well as synthesis and release of NAP-I. Intradermal injection of synthetic or recombinant NAP-I into human skin at concentrations of 2 X 10-7 M or 10-6 M caused perivascular neutrophil infiltration that was detectable at 30 min and that increased progressively at 1 and 3 h (Figure 1). Neutrophil counts for biopsies taken 3 h after injection of 2 X 10-7 M or 1 X 10-6 M NAP-I were 161 ± 41/mm 2 dermis (n = 15) compared to 4 ± 3 (n = 6) for saline or inactive protein controls. The number of cells detected by anti-CD45 antibody, a lymphocyte marker, was not increased over the saline control number. NAP-I caused no wheal-and-flare, induration, or tenderness in any subject, and acid toluidine blue staining of biopsy sections showed intact mast cells without degranulation (47). It is of considerable interest to compare the differences in responses of normal human subjects to intradermal NAP-I and C5a. In contrast to the restricted action of NAP-I, intradermal injection of C5a into human skin had global effects: wheal-andflare due to mast cell degranulation, and neutrophil infiltration accompanied by degranulation and leukocytoclasis (48). The time courses of the responses were also different. Whereas neutrophil infiltration after intradermal injection of NAP-I increased progressively over the 3-h observation period, the response to intradermal C5a was maximal at 20 min and declined thereafter, based on visual estimates of reaction intensity. These results suggest that there may be significant differences in vivo between complement-mediated neutrophil infiltration (typified by the Arthus reaction) and the reactions initiated by NAP-I release. In contrast to the experiments on rabbits and humans, intradermal injection into rat ears of 20 Jotl of 10-10 M recombinant NAP-I caused lymphocyte as well as neutrophil infiltration (35). However, in another study of the response in rats to intradermal NAP-I, no accumulation of lymphocytes was seen over a concentration range of 10-11 M to 10-5 M (R. Zwahlen and M. Baggiolini, personal communication). This evidence, along with the absence oflymphocyte infiltration in humans and in rabbits, suggests that lymphocytes do not participate in acute NAP-I-induced inflammation. NAP-I induction of basophil histamine release in vitro is not reflected in the human in vivo study, but interactions with IL3-stimulated basophils or mast cells remain to be explored.

In summary, the major effect of intradermal NAP-I is neutrophil infiltration, without signs of histamine release from tissue mast cells or infiltration of other types of leukocytes. Role of NAP-l and other attractants in the development of acute inflammation. It may be useful to divide the seemingly endless list of chemoattractants into rapid-deployment and follow-up groups. The rapid-deployment group comprises products that can be quickly generated by enzymatic actions on cellular or extracellular substrates. Rapid synthesis and release of LTB4 by LPS-stimulated lung macrophages is an example. A two-step example would be activation ofNADPH oxidase to generate superoxide anion, which acts on a blood plasma precursor to form a neutrophil attractant (49). Enzymatic cleavage of many different proteins generates chemoattractants. The attractant moiety may be a free peptide (collagen fragments, or the peptides cleaved from fibrinogen) or a small protein (C5a, NAP-2). Chemoattract ant activity may be generated by enzymatic cleavage of a single peptide bond of a protein, without fragment release. The resultant two parts of the molecule remain associated because of a disulfide linkage (C5 activated by limited trypsin proteolysis [50]) or because of noncovalent interactions (proteolytically cleaved o-l-proteinase inhibitor [aI-PI] [51]). In contrast to the rapid-deployment group, the follow-up group of attractants comprises proteins that are synthesized de novo in response to an inflammatory signal, and are released after a time interval of 3 to 6 h after challenge. NAP-I is the bestdocumented example, though other cytokines such as TNF and granulocyte/macrophage colony-stimulating factor (GMCSF) have been reported to be neutrophil attractants. If we consider LPS as the prototypical insult to lung macrophages, the target cells respond by releasing LTB4 early and NAP-I later (within 6 h, [Rankin et al., submitted for publication]). The potential for an explosive inflammatory response exists. Possibilities include but are not limited to the following. (1) LPS-induced macrophage secretion of IL-I and TNF, which can stimulate macrophages, endothelial cells, and fibroblasts to secrete NAP-I. (2) Release of neutrophil elastase, which after cleavage of the serine protease bait site of aI-PI, forms a 1:1 complex that is chemotactic for neutrophils (51). aI-PI also becomes an attractant (52) after catalytic cleavage by a metalloprotease from rat macrophages (not yet isolated from human macrophages). (3) Activation of C5 by non-complement pathway enzymes. This has been documented for platelet granule release products (53); a similar activation by macrophage or neutrophil enzymes is possible. (4) Release by activated C5 of lung mast cell granule contents, which include an enzyme with chymotrypsin activity that is reported to generate a neutrophil attractant from IgG (54). (5) Release of CTAP-III from platelets, and conversion of NAP-2 (CTAP-III(des 1-15» by elastase (shown for porcine elastase [17] but not yet for neutrophil elastase). Future Directions It will not be easy to determine what, among the above possibilities, operate in vivo. Is there as much redundancy in the system as the long list of attractants might suggest? One approach to this question will be to establish ligand-receptor relationships. For example, NAP-I and NAP-2 interact with the same neutrophil receptor. This redundancy may be more

Update

apparent than real, since in tissue injury with platelet activation, NAP-2 would be generated early and NAP-I secretion would follow. Another approach is to continue our search for deficiencies. We sometimes find no cause for many episodes of pneumonia per year in babies evaluated for immunodeficiency. Would we find new causes (and thus establish in vivo relevance) by testing, for example, blood monocyte NAP-I production in response to LPS, or by measuring monocyte release of enzymes capable of activating C5? There is, of course, also great interest in how the inflammatory response is regulated, and turned off at the appropriate time. With respect to a locally produced cytokine like NAP-I, the obvious possibilities include regulation of production and a decrease in concentration of secreted molecules by oxidative or enzymatic inactivation, or by diffusion out of the site. We need to learn more about NAP-I susceptibility to proteolysis. As described earlier, the shortest NAP-I molecule found after proteolytic cleavage in monocyte culture fluids was the 72 residue form, despite additional potential proteolytic cleavage sites. Diffusion of this relatively small protein into the venous circulation almost certainly occurs. Alpha-macroglobulins, circulating molecular traps, are associated with a number of cytokines (55); this could contribute to vascular clearance. Preliminary reports of other cytokine-binding proteins include plasma IgG anti-ILIa (56) and soluble cell receptors for specific cytokines isolated from human urine (57). Thus, there are new areas to explore in the regulation of these potent mediators. References I. Sibille. Y, and H. Y. Reynolds. 1990. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am. Rev. Respir. Dis. 141: 471-501.2. Sylvester, 1.,1. A. Rankin, T. Yoshimura, S. Tanaka, and E. 1. Leonard. 1990. Secretion of neutrophil attractant/activation protein by lipopolysaccharide-stimulated lung macrophages determined by both enzyme-linked immunosorbent assay and N-terminal sequence analysis. Am. Rev. Respir. Dis. 141:683-688. 3. Merrill, W. W., G. P. Naegel, R. A. Matthay, and H. Y. Reynolds. 1980. Alveolar macrophage-derived chemotactic factor. Kinetics of in vitro production and partial characterization. J. Clin. Invest. 65:268-276. 4. Schroeder, 1.-M., and E. Christophers. 1986. Identification of C5l1d,,,,. and an anionic neutrophil-activating peptide (ANAP) in psoriatic scales. J. Invest. Dermatol. 87:53-58. 5. Yoshimura, T., K. Matsushima, 1. 1. Oppenheim, and E. 1. Leonard. 1987. Neutrophil chemotactic factor produced by lipopolysaccharide (LPS) stimulated human blood mononuclear leukocytes. 1. Partial characterization and separation from interleukin-l (IL-I). J. Immunol. 139:788-793. 6. Matsushima, K., K. Morishita, T. Yoshimura et al. 1988. Molecular cloning of cDNA for a human monocyte derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin-l and tumor necrosis factor. J. Exp. Med. 167:1883-1893. 7. Yoshimura, T., K. Matsushima, S. Tanaka et al. 1987. Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc. Natl. Acad. Sci. USA 84:9233-9237. 8. Yoshimura, T., E. A. Robinson, E. Appella et al. 1989. Three forms of monocyte-derived neutrophil chemotactic factor (MDNCF) distinguished by different lengths of the amino-terminal sequence. Mol. Immunol. 26: 87-93. 9. Van Damme, 1., B. Decock, R. Conings, 1.-P Lenaerts, G. Opdenakker, and A. Billiau. 1989. The chemotactic activity for granulocytes produced by virally infected fibroblasts is identical to monocyte-derived interleukin 8. Ear. J. Immunol. 19:1189-1194. 10. Gregory, H., 1. Young, 1.-M. Schroeder, U. Mrowietz, and E. Christophers. 1988. Structure determination of a human lymphocyte derived neutrophil activating peptide (LYNAP). Biochem. Biophys. Res. Commun. 151: 883-890. II. Schroeder, J.-M., U. Mrowietz, and E. Christophers. 1988. Identification of different charged species of a human monocyte derived neutrophil activating peptide (MONAP). Biochem. Biophys. Res. Commun. 152: 277-284.

485

12. Auron, P. E., S. 1. C. Warner, A. C. Webb etal. 1987. Studies on the molecular nature of interleukin I. J. Immunol. 138:1447-1456. 13. Salaman, M. R., and A. R. Williamson. 1971. Isoelectric focusing of proteins in the native and denatured state. Biochem. J. 122:93-99. 14. Deuel, T. E, R. M. Senior, D. Chang, G. L. Griffin, R. L. Heinrikson, and E. T. Kaiser. 1981. Platelet factor 4 is chemotactic for neutrophils and monocytes. Proc. Natl. Acad. Sci. USA 78:4584-4587. 15. Richmond, A., E. Balentien, H. G. Thomas et al. 1988. Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to J3-thromboglobulin. EMBO 1. 7:2025-2033. 16. Castor, C. W. 1988. Regulation of connective tissue metabolism. In Arthritis and Allied Conditions. 11th ed. D. 1. McCarty, editor. Lea & Febiger, Philadelphia. 242-256. 17. Castor, C. w., D. A. Walz, C. G. Ragsdale et al. 1989. Connective tissue activation. XXXIII. Biologically active cleavage products of CTAP-III from human platelets. Biochem. Biophys. Res. Commun. 163:1071-1078. 18. Walz, A., B. Dewald, V. von Tschamer, and M. Baggiolini. 1989. Effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue-activating peptide III, and platelet factor 4 on human neutrophils. J. Exp. Med. 170:1745-1750. 19. Modi, W. S., M. Dean, H. N. Seuanez, N. Mukaida, K. Matsushima, and S. 1. O'Brien. 1990. Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily. Hum Genet. 84:185-187. 20. Yoshimura, T., N. Yuhki, S. K. Moore, E. Appella, M. 1. Lerman, andE. 1. Leonard. 1989. Human monocyte chemoattractant protein-l (MCP-I). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE. FEBS Lett. 244:487-493. 21. Begg, G. S., D. S. Pepper, C. N. Chesterman, and E 1. Morgan. 1978. Complete covalent structure of human J3-thromboglobulin. Biochemistry 17: 1739-1744. 22. Clore, G. M., E. Appella, M. Yamada, K. Matsushima, andA. M. Gronenborn. 1990. The three-dimensional structure of interleukin-8 in solution. Biochemistry 29: 1689-1696. 23. Tanaka, S., E. A. Robinson, T. Yoshimura, K. Matsushima, E. J. Leonard, and E. Appella. 1988. Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor. FEBS Lett. 236: 467-470. 24. Goldman, D. W., A. L. Hannah, and E. 1. Goetzl. 1985. Inhibition of human receptor-mediated uptake of N-formyl-met-Ieu-phe by platelet factor 4(59-70). Immunology 54:163-172. 25. Fletcher, M. P., and 1. 1. Gallin. 1980. Degranulating stimuli increase the availability of receptors on human neutrophils for the chemoattractant frnet-leu-phe. J. Immunol. 124:1585-1588. 26. Samanta, A. K., 1. 1. Oppenheim, and K. Matsushima. 1989. Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils. J. Exp. Med. 169: 1185-1189. 27. Samanta, A. K., J. J. Oppenheim, and K. Matsushima. 1990. Interleukin 8 (monocyte-derived neutrophil chemotactic factor) dynamically regulates its own receptor expression on human neutrophils. J. Bioi. Chem. 265: 183-189. 28. Leonard, E. J., A. Skeel, T. Yoshimura, K. Noer, S. Kutvirt, and D. van Epps. 1990. Leukocyte specificity and binding of human neutrophil attractant/activation protein-I (NAP-I). J. Immunol. 144:1323-1330. 29. Schroeder, J.-M., U. Mrowietz, E. Morita, and E. Christophers. 1987. Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin I activity. J. Immunol. 139:3474-3483. 30. Peveri, P., A. Walz, B. Dewald, and M. Baggiolini. 1988. A novel neutrophil-activating factor produced by human mononuclear phagocytes. J. Exp. Med. 167:1547-1559. 31. Schroeder, 1.-M., and E. Christophers. 1989. Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells. J. Immunol. 142:244-251. 32. Walz, A., P. Peveri, H. Aschauer, andM. Baggiolini. 1987. Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Commun. 149:755-761. 33. Van Damme, J., 1. van Beeumen, G. Opdenakker, and A. Billiau. 1988. A novel, NH,-terminal sequence characterized human monokine possessing neutrophil chemotactic, skin-reactive, and granulocytosis-promoting activity. J. Exp. Med. 167:1364-1376. 34. Strieter, R. M., S. L. Kunkel, H. 1. Showell et al. 1989. Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-IJ3. Science 243:1467-1469. 35. Larsen, C. G., A. O. Anderson, E. Appella, J. 1. Oppenheim, and K. Matsushima. 1989. The neutrophil-activating protein (NAP-I) is also chemotactic for T lymphocytes. Science 243:1464-1466. 36. Larsen, C. G., A. 0. Anderson, 1. 1. Oppenheim, and K. Matsushima. 1989. Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-l or tumor necrosis factor. Immunology 68: 31-36.

486

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 2 1990

37. Schroeder, J.-M., M. Stricherling, H. H. Henneicke, W. C. Preissner, and E. Christophers. 1990. IL-Ia or tumor necrosis factor-a stimulate release of three NAP-lIIL-8-related neutrophil chemotactic proteins in human dermal fibroblasts. J. Immunol. 144:2223-2232. 38. Baggiolin, M., A. Walz, and S. L. Kunkel. 1989. Neutrophil-activating peptide-l/interleukin 8, a novel cytokine that activates neutrophils. J. Clin. Invest. 84:1045-1049. 39. Stichering, M., J.-M. Schroeder, and E. Christophers. 1989. Production and characterization of monoclonal antibodies against the novel neutrophil activating peptide NAP/IL-8. J. Immunol. 143:1628-1634. 40. White, M. V., T. Yoshimura, W. Hook, M. Kaliner, and E. J. Leonard. 1989. Neutrophil attractant/activation protein-l (NAP-I) causes human basophil histamine release. Immunol. Lett. 22:151-154. 41. Dahinden, C. A., Y. Kurimoto, A. L. de Week, I. Lindley, B. Dewald, and M. Baggiolini. 1989. The neutrophil-activating peptide NAF/NAP-I induces histamine and leukotriene release by interleukin 3-primed basophils. J. Exp. Med. 170:1787-1792. 42. Burrows, J. J., P. J. Piper, I. Lindley, M. Baggiolini, andJ. Westwick. 1989. Human recombinant neutrophil-activating factor/interleukin-B is a spasmogen of airway smooth muscle. Cytokine 1:100 (Abstr.), 43. Gimbrone, M. A., Jr., M. S. Obin, A. F. Brock et al. 1989. Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions. Science 246:1601-1603. 44. Colditz, 1., R. Zwahlen, B. Dewald, and M. Baggiolini. 1989. In vivo inflammatory activity of neutrophil activating factor, a novel chemotactic peptide derived from human monocytes. Am. J. Pathol. 134:755-760. 45. Rampart, M., J. Van Damme, L. Zonnekeyn, and A. G. Herman. 1989. Granulocyte chemotactic protein/interleukin-8 induces plasma leakage and neutrophil accumulation in rabbit skin. Am. J. Pathol. 135:21-25. 46. Williams, T. J., and P. J. Jose. 198 I. Mediation of increased vascular permeability after complement activation. Histamine-independent action of rabbit C5a. J. Exp. Med. 153:136-153. 47. Leonard, E. J., T. Yoshimura, S. Tanaka, and M. Raffeld. 1990. Neutrophil recruitment by intradermally injected neutrophil attractant/activating

48.

49.

50.

51. 52.

53. 54. 55. 56.

57.

protein-l (NAP-I) in human subjects. In Pathophysiologic and Therapeutic Roles of Cytokines. C. A. Dinarello, M. Kluger, M. Powanda, and J. J. Oppenheim, editors. Alan Liss, Inc., New York. (In press) Yancey, K. B., C. H. Hammer, L. Harvath, L. Renfer, M. M. Frank, and T. J. Lawley. 1985. Studies of human C5a as a mediator of inflammation in normal human skin. J. Clin. Invest. 75:486-495. Petrone, W. F., D. K. English, K. Wong, and J. M. McCord. 1980. Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma. Proc. Natl. Acad. Sci. USA 77: 1159-1163. Wetsel, R. A., and W. P. Kolb. 1982. Complement-independent activation of the fifth component (C5) of human complement: limited trypsin digestion resulting in the expression of biologic activity. J. Immunol. 128: 2209-2216. Banda, M. J., A. G. Rice, G. L. Griffin, andR. M. Senior. 1988. The inhibitory complex of human ol-proteinase inhibitor and human leukocyte elastase is a neutrophil chemoattractant. J. Exp. Med. 167:1608-1615. Banda, M. J., A. G. Rice, G. L. Griffin, and R. M. Senior. 1988. odproteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase. J. Bioi. Chem. 263:4481-4484. Weksler, B. B., and C. E. Coupal. 1973. Platelet-dependent generation of chemotactic activity in serum. J. Exp. Med. 137:1419-1430. Katunuma, N., N. Fukusen, and H. Kido. 1986. Biological functions of serine proteases in the granules of rat mast cells. Adv. Enzyme Regul. 25: 241-255. Sottrup-Jensen, L. 1989. o-Macroglobulins: structure, shape, and mechanism of proteinase complex formation. J. BioI. Chem. 264:11539-11542. Bendtzen, K., M. Svenson, A. Foomsgaard, L. K. Poulsen, and M. Hansen. 1989. Auto-antibodies to IL-la and TNFa in normal individuals and in infectious and immunoinflammatory disorders. Cytokine 1:89 (Abstr.). Novick, D., H. Engelmann, D. Wallach, and M. Rubinstein. 1989. Soluble cytokine receptors are present in normal human urine. Cytokine 1:149 (Abstr.).