Introduction

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Major basic protein (MBP) is a 13.9-kD protein localized to the crystalloid core of eosinophil secondary granules and implicated in many of the proinflammatory actions of eosinophils (1). Through its many actions, MBP contributes to generation of the inflammatory milieu and to the airway hyperreactivity characteristic of chronic asthma. Of interest among the activities reported for MBP is its capacity to activate neutrophils, as shown by our findings that MBP in concentrations similar to those detected in sputum of symptomatic asthmatic patients stimulates neutrophil superoxide anion production (2) and increases neutrophil expression of CD11b/CD18 (3). Two other basic proteins present in eosinophil granules, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) (1), were without effect at a concentration optimal for MBP (2, 3).

Together, the results of several studies have suggested the involvement of neutrophils in the pathophysiology of acute asthma. Acute exacerbations of asthma and status asthmaticus are associated with increased levels of neutrophils in sputum and in bronchoalveolar lavage fluid (4), and neutrophils were found to predominate within the airway submucosa of patients who died of a "sudden-onset form of fatal asthma" (7). Importantly, neutrophil presence in the airway in acute asthma is superimposed on the eosinophilia characteristic of asthma (4). That eosinophil products, and in particular MBP, may contribute to neutrophil activation is supported by two observations. First, both eosinophils and neutrophils are activated during acute exacerbations of asthma (5, 6), and second, the activated neutrophils are localized to the same sites as the activated eosinophils and discharged granule basic proteins (7).

Interleukin (IL)-8 is a potent chemoattractant and activator of neutrophils (8). In the lungs, IL-8 appears to be the primary chemoattractant for neutrophils (11), and accordingly is directly implicated in the neutrophil accumulation that follows acute respiratory infection (12). Although IL-8 is produced by a wide variety of cell types (13), it is of particular interest, given its biologic activity, that IL-8 is also synthesized and released by neutrophils in response to a number of inflammatory mediators (14). Thus, IL-8 production by neutrophils can contribute to additional neutrophil recruitment and, importantly, can also enhance or prolong neutrophil activation in an autocrinelike manner. Earlier results had shown that MBP can stimulate IL-8 production by eosinophils in a transcription- dependent manner (19). We therefore postulated that MBP may likewise stimulate IL-8 production by neutrophils. The results presented here confirm this hypothesis and show that the MBP acts through both transcriptional and posttranscriptional events.

	Materials and Methods

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Materials

Protamine sulfate, lipopolysaccharide (LPS) (Escherichia coli serotype 055:B5), dithiothreitol (DTT), leupeptin, aprotinin, pepstatin A, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), H₂O₂, actinomycin D, and IGEPAL CA-630 were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI 1640, penicillin/streptomycin, L-glutamine, and 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethane sulfonic acid (Hepes) buffer solution were purchased from Gibco Life Technologies (Grand Island, NY). Sources of additional reagents were as follows: 14-kD polymer of L-arginine (Miles Scientific, Naperville, IL); lymphocyte separation medium (Mediatech, Hernedon, VA); human serum albumin (Calbiochem, La Jolla, CA); [-³²P]deoxyuridine triphosphate ([-³²P]UTP) (Amersham, Arlington Heights, IL); and [-³²P]adenosine triphosphate ([-³²P]ATP) (Andotek, Irvine, CA). A specific enzyme-linked immunosorbent assay (ELISA) for IL-8 was purchased from Biosource International (Camarillo, CA), and specific ELISAs for macrophage inflammatory protein (MIP)-1 and MIP-1 were purchased from R&D Systems (Minneapolis, MN). Oligonucleotides corresponding to consensus binding sites for nuclear factor (NF)-B, activator protein (AP)-1, AP-2, and Oct-1 were purchased from Promega (Madison, WI). An oligonucleotide corresponding to the consensus binding site for NF-IL-6 was generously provided by Dr. Steven Ackerman (University of Illinois at Chicago).

Eosinophil Granule Proteins

MBP, ECP, EDN, and eosinophil peroxidase (EPO) were isolated as described previously from eosinophils of patients with the hypereosinophlic syndrome (20, 21). The proteins were pure as judged by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and staining with Coomassie brilliant blue R250. The EPO was enzymatically active as determined with a previously described method (22). Endotoxin content in the granule proteins was measured with the Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA).

Neutrophil Isolation

Neutrophils were isolated under sterile conditions from the venous blood of healthy adult volunteers through density gradient centrifugation as previously described (23). The neutrophils were resuspended in RPMI 1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (RPMI).

Incubation Conditions for Measurement of Chemokine Release

Neutrophils (10⁶ cells) were incubated with the stimuli in RPMI containing 10% heat-inactivated (56°C; 45 min) autologous serum (added separately to the incubation contents) in 24-well tissue culture plates for the indicated times at 37°C in a 5% CO₂ atmosphere. Spontaneous IL-8 production was determined with cells incubated under the same conditions in the absence of stimulus, and the effect of the MBP vehicle buffer (25 mM sodium acetate, 150 mM sodium chloride, pH 4.3) was examined by addition of the buffer in the same volumes as required for MBP addition. Total incubation volume was 0.5 ml. The reactions were terminated by centrifugation at 400 × g for 10 min at 4°C, and the cell-free supernatants were stored at 20°C until the levels of IL-8, MIP-1, and MIP-1 were measured with a specific ELISA.

Ribonuclease Protection Assay

Neutrophils (10⁷ cells/ml) were incubated with MBP or LPS in RPMI supplemented with 10 mM Hepes, pH 7.4, and containing 10% heat-inactivated autologous serum in 15-ml round-bottom polypropylene tubes for the indicated times at 37°C in an oscillating water bath. Total incubation volume was 2 ml. For some samples, actinomycin D (5 µg/ ml) was added 1 h before or after incubation with the stimuli, as indicated in the text. The reactions were terminated by centrifugation at 300 × g for 10 min at 4°C. The cell pellets were washed once with phosphate-buffered saline (PBS) at 4°C, and total RNA was isolated from 2 × 10⁷ neutrophils using Trizol reagent (Life Technologies, Inc., Gaithersburg, MD). For the ribonuclease (RNase) protection assay, we used the RiboQuant multiprobe RNase protection assay (hCK-5; PharMingen, San Diego, CA). The complementary DNA (cDNA) probes were radiolabeled with [-³²P]UTP, and the assay was performed exactly as described elsewhere (24). Positive messenger RNA (mRNA) bands were quantified densitometrically using a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA), and mRNA levels of the chemokines were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Electrophoretic Mobility Shift Assay

Neutrophils (10⁷ cells/ml) were incubated with MBP, EDN, or LPS in RPMI 1640 supplemented with 10 mM Hepes, pH 7.4, in 15-ml round-bottom polypropylene tubes for 20 min or 1 h at 37°C. Total incubation volume was 1 ml. The reactions were stopped by transfer of the mixtures to microcentrifuge tubes and centrifugation at 300 × g for 10 min at 4°C. Nuclear extracts were prepared from the cell pellets as described by Osborn and colleagues (25) using a modification of the protease inhibitor cocktail described by McDonald and coworkers (26). Briefly, 10⁷ cells were washed twice in ice-cold Buffer A (10 mM Hepes, pH 7.9; 1.5 mM MgCl₂; 10 mM KCl; and 0.5 mM DTT) containing a protease inhibitor cocktail (4 mM DFP, 2 mM each of PMSF and AEBSF, and 10 µg/ml each of leupeptin, aprotinin, and pepstatin). Cells were collected between washes by centrifugation at 300 × g for 5 min at 4°C. The cell pellets were resuspended in ice-cold Buffer A (and protease inhibitor cocktail) containing 0.1% of the nonionic detergent IGEPAL CA-630, and were incubated on ice for 10 min. The nuclei were pelleted by centrifugation at 12,000 × g for 10 min at 4°C, and were resuspended in 15 µl of ice-cold Buffer C (20 mM Hepes, pH 7.9; 25% glycerol; 420 mM NaCl; 1.5 mM MgCl₂; 0.5 mM ethylenediaminetetraacetic acid [EDTA]; and 0.5 mM DTT) containing the protease inhibitor cocktail. The nuclei were incubated on ice for 15 min and, after centrifugation at 12,000 × g for 10 min at 4°C, the supernatants were transferred to a new microcentrifuge tube containing 30 µl of ice-cold Buffer D (20 mM Hepes, pH 7.9; 20% glycerol; 50 mM KCl; 0.5 mM EDTA; and 0.5 mM DTT) with the protease inhibitor cocktail. The samples were stored in aliquots at 70°C until analysis. Protein concentration was measured with the BioRad Protein Assay (BioRad Laboratories, Hercules, CA). Nuclear proteins (8 µg) were incubated with [-³²P]ATP-labeled double-stranded DNA (dsDNA) oligonucleotide probes (50,000 cpm) corresponding to the consensus binding sites for NF-B, AP-1, and NF-IL-6 for 20 min at room temperature. Inhibition of binding through use of a 50-fold molar excess of unlabeled specific probe or an irrelevant probe (AP-2 for NF-B, and Oct-1 for AP-1 and NF-IL-6) was evaluated to determine the specificity of the detection. Samples were run on a 5% polyacrylamide gel and exposed to X-OMAT-AR film (Eastman Kodak, Rochester, NY) at 70°C.

Statistical Analysis

Statistical significance was determined by the paired t test or by analysis of variance (ANOVA) and Fisher's protected least significant differences test for repeated measures. A value of P < 0.05 was accepted as significant.

	Results

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Concentration-Dependence and Time Course for MBP-Stimulated IL-8 Production

The capacity of MBP to stimulate neutrophil IL-8 production was evaluated over the concentration range of 0.1-10 µM MBP. As shown in Figure 1, incubating neutrophils with 0.1 µM or 0.3 µM MBP for 18 h at 37°C produced negligible IL-8 release. MBP in concentrations of 1-10 µM, however, stimulated the release of up to approximately 8 ng/ml IL-8 in a concentration-dependent manner. To avoid possible cytotoxic effects that may be associated with high concentrations of MBP (1), the time course for MBP-stimulated IL-8 production was determined with 2 µM MBP. The results, in Figure 2, show that a statistically significant level of IL-8 release occurred after 2 h of incubation with MBP, and that the level of MBP-induced IL-8 release then increased with time for up to 10 h of incubation. An additional 10 h of incubation with MBP resulted in only a minimal further increase in the amount of IL-8 released.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Concentration requirements for MBP-stimulated IL-8 release. Neutrophils (2 × 106/ml) were incubated with MBP for 18 h at 37°C. The net amount of IL-8 release is given as the mean ± SEM for three to five experiments. Spontaneous IL-8 production was 85 ± 63 pg/ml. The MBP vehicle buffer in volumes required for the MBP addition did not stimulate any IL-8 release. The MBP did not interfere with the measurement of IL-8. (*P < 0.05 compared with spontaneous value by ANOVA.)

View larger version (13K): [in this window] [in a new window]   Figure 2.   Time course for MBP-stimulated IL-8 release. Neutrophils (2 × 106/ml) were incubated with 2 µM MBP for 2, 5, 10, and 20 h at 37°C. The net IL-8 release at each time point is given as the mean ± SEM for four experiments. Spontaneous IL-8 release was undetectable at 1 and 2 h, and was 71 ± 37 pg/ml, 124 ± 59 pg/ml, and 152 ± 91 pg/ml at 5, 10, and 20 h, respectively. (*P < 0.05 compared with the spontaneous value at the same time point by the paired t test.)

Stimulation of Neutrophil IL-8 Production by Eosinophil Granule Basic Proteins

The ability of the three other basic eosinophil granule proteins to likewise stimulate neutrophil IL-8 production at a concentration effective for MBP was evaluated by incubating neutrophils with 2 µM MBP, ECP, EDN, or EPO for 18 h at 37°C. MBP stimulated the release of approximately 1,800 pg/ml IL-8 (Figure 3); however, neither ECP nor EDN stimulated IL-8 release, and EPO stimulated the release of 130 pg/ml IL-8, a level that was not statistically significant. The addition of 40-1,000 µM H₂O₂ did not enhance the ability of EPO to stimulate IL-8 release, and EPO was similarly ineffective at lower concentrations (0.03-0.3 µM) reported to induce shape changes in neutrophils (27) (results not shown). The abilities of a 14-kD polymer of L-arginine (Poly-R) and of protamine, a 4.8-kD protein containing 66% arginine (28), to stimulate IL-8 production were also evaluated in the same experiments in order to further assess the contribution of the basic charge of the MBP molecule to its activity. Incubation of neutrophils with 0.3 µM Poly-R, which corresponds on a weight basis to the arginine content of 2 µM MBP (1), or with 2 µM protamine, resulted in the release of less than 70 pg/ml IL-8 (Figure 3). In the same experiments, 100 ng/ml LPS stimulated the release of 1,280 pg/ml IL-8.

View larger version (14K): [in this window] [in a new window]   Figure 3.   Stimulation of IL-8 release by eosinophil granule basic proteins. Neutrophils (2 × 106/ml) were incubated with the indicated concentrations of eosinophil granule proteins, protamine (Prot), Poly-R, or 100 ng/ml LPS for 18 h at 37°C. IL-8 release is expressed as the mean ± SEM for four to eight experiments, after correction for spontaneous release. The level of spontaneous IL-8 release was 192 ± 113 pg/ml, and IL-8 release in the presence of the MBP vehicle buffer was 93 ± 54 pg/ml. (*P < 0.05 compared with the spontaneous value by ANOVA.)

MBP-Stimulated IL-8 Production by Neutrophils Is Independent of Endotoxin

As reported previously by others (14), and as shown earlier (Figure 3), LPS is an effective stimulus for neutrophil IL-8 production. Measurement of endotoxin content in the granule protein preparations indicated that a 2 µM concentration of the granule proteins contained endotoxin levels of 0.1 ng/ml (MBP), 0.8 ng/ml (ECP), 0.7 ng/ml (EDN), and 1.8 ng/ml (EPO), which did not correlate with the abilities of the proteins to stimulate neutrophil IL-8 production (Figure 3). Additional results showed that heating MBP for 5 min at 100°C abrogated by more than 90% the ability of 2 µM MBP to stimulate IL-8 production (n = 3; data not shown). In contrast, heating did not reduce the activity of LPS in the same experiments.

Induction of IL-8 mRNA Synthesis by MBP

To assess the effect of MBP stimulation on IL-8 mRNA synthesis, as well as to screen for the synthesis of additional chemokines, an RNase protection assay was performed after incubating neutrophils with 5 µM MBP or, as a positive control, 1 µg/ml LPS for 1 h and 3 h at 37°C. IL-8 mRNA was detected at 1 h and to a lesser extent at 3  h in unstimulated neutrophils, but incubation with MBP produced additional 2-fold increases (relative to the unstimulated cells) in the level of IL-8 mRNA at both 1 h and 3 h (Figures 4A and 4B). MBP also stimulated increases in mRNA for MIP-1 and MIP-1 (5-fold and 3-fold, respectively) at 1 h; however, the increases were absent at the 3-h time point. Incubation with LPS stimulated increases in the level of IL-8 mRNA at the 3-h time point and in the levels of MIP-1 and MIP-1 mRNA at both time points (Figures 4A and 4B). Of note was that preincubating the neutrophils with 5 µg/ml actinomycin D for 1  h at 37°C before addition of the stimuli blocked all the observed increases in IL-8 mRNA, as well as in MIP-1 and MIP-1 mRNA (Figure 4A). Preincubation with 5 µg/ml actinomycin D also blocked MBP-stimulated release of IL-8 protein (n = 3; data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 4.   Induction of chemokine mRNA synthesis by MBP and LPS. (A) Neutrophils (107/ml) were preincubated in the presence or absence of 5 µg/ml actinomycin D (Act. D) for 1 h at 37°C. The cells were then incubated with 5 µM MBP or 1 µg/ml LPS, or in RPMI alone (Spont), for 1 or 3 h. Expression of IL-8, MIP-1, MIP-1, released on activation, normal T-cell-expressed and secreted (RANTES), MCP-1, IP-10, I-309, L-32, and GAPDH mRNA was determined by RNase protection assay. Only positive mRNA bands are labeled; note that IL-8 mRNA and MIP-1 mRNA each run as a doublet. (B) The positive mRNA bands of the three chemokines (A) were quantified densitometrically and were normalized to the density of GAPDH mRNA. The results are presented as the fold increase in mRNA level for each of the three chemokines relative to the mRNA level in unstimulated cells (Spont).

Absence of MBP-Stimulated MIP-1 and MIP-1 Production

With MBP stimulating transient increases in MIP-1 and MIP-1 mRNA (Figure 4A), the ability of MBP to stimulate release of MIP-1 or MIP-1 protein was evaluated. Results obtained in two separate experiments showed that incubating neutrophils with 2 µM MBP for 18 h at 37°C stimulated the release of up to 3,400 pg/ml IL-8, but caused no release of MIP-1 and negligible release of MIP-1 (Table 1). In the same experiments, 100 ng/ml LPS stimulated the release of up to approximately 1,400 pg/ml IL-8, 700 pg/ml MIP-1, and 2,400 pg/ml MIP-1.

Effect of MBP Stimulation on the Binding Activities of NF-B, AP-1, and NF-IL-6

The finding (Figure 4A) that actinomycin D blocked the MBP-stimulated increase in IL-8 mRNA indicated the involvement of a transcriptional event in MBP-induced IL-8 release. With activation of NF-B alone or in combination with AP-1 or NF-IL-6 responsible for the transcriptional regulation of IL-8 synthesis in many cells (29), the ability of MBP to activate NF-B, AP-1, and NF-IL-6 was evaluated in an electrophoretic mobility shift assay. As shown in Figure 5, incubating neutrophils with 5 µM MBP for 20 or 60 min at 37°C did not stimulate an increase in the binding activities of NF-B or AP-1. In the same experiments, 1 µg/ml LPS produced an increase in NF-B binding activity after 20 min of incubation, and increases in the binding activities of NF-B and AP-1 after 60 min. A 50-fold molar excess of the specific unlabeled oligonucleotide blocked detectable binding activity of both NF-B and AP-1 (Figure 5). The NF-B binding activity was also partly inhibited by an unlabeled irrelevant oligonucleotide (AP-2), indicating that a portion of the observed NF-B binding activity was nonspecific. No NF-IL-6 binding activity was detected in any of the experiments (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 5.   Effect of MBP and LPS stimulation on binding activities of NF-B and AP-1. Neutrophils (107 cells/ml) were incubated with 5 µM MBP, 5 µM EDN, 1 µg/ml LPS, or in RPMI buffer alone for 20 or 60 min at 37°C. The binding activities were determined by electrophoretic mobility shift assays as described in MATERIALS AND METHODS. A 50-fold molar excess of unlabeled probe (Specific) or unlabeled nonspecific probe (AP-2 for NF-B and Oct-1 for AP-1) were included in the assay as indicated. The results of a single experiment that is representative of two additional experiments are shown.

Effect of MBP on the Stability of IL-8, MIP-1, and MIP-1 mRNA

In the absence of an MBP effect on the binding activity of NF-B or AP-1, the possibility that MBP might act posttranscriptionally to stabilize IL-8 mRNA was evaluated. Neutrophils were incubated with 5 µM MBP or 1 µg/ml LPS for 2 h at 37°C, and 5 µg/ml actinomycin D was then added (time zero). The level of IL-8 mRNA and, for comparison, the levels of MIP-1 and MIP-1 mRNA, were measured with the RNase protection assay at time zero and after an additional 1, 2, and 4 h of incubation at 37°C in the presence of the actinomycin D. The results show that more than 80% of the IL-8 mRNA in the MBP-stimulated neutrophils was still present after 4 h of incubation with the actinomycin D, whereas 50% of the IL-8 mRNA in unstimulated neutrophils was degraded by approximately 2.5 h (Figure 6). Incubation with MBP also increased the stability of MIP-1 mRNA (T_1/2 ~ 2 h in MBP-stimulated cells, versus T_1/2 < 1 h in unstimulated cells). In contrast, incubation with LPS enhanced the mRNA stability of all three chemokines (Figure 6).

View larger version (13K): [in this window] [in a new window]   Figure 6.   Effect of MBP and LPS stimulation on the stability of IL-8 (upper panel), MIP-1 (middle panel), and MIP-1 (lower panel) mRNA. Neutrophils (107/ml) were incubated with 5 µM MBP, 1 µg/ml LPS, or in RPMI alone for 2 h at 37°C. Actinomycin D (5 µg/ml) was added (time zero), and total RNA was isolated at time zero and after an additional 1, 2, and 4 h of incubation at 37°C. Expression of IL-8, MIP-1, and MIP-1 mRNA was assessed with an RNase protection assay. Levels of IL-8, MIP-1, and MIP-1 mRNA were normalized to that of GAPDH mRNA, and the average of two experiments is presented as the percent of mRNA at time zero.

	Discussion

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These results described show that MBP is an effective stimulus for IL-8 production by neutrophils. As such, MBP joins a prominent list of inflammatory reactants known to stimulate IL-8 release by neutrophils (14, 32). The level of IL-8 production stimulated by MBP is comparable to that stimulated by optimal concentrations of LPS, tumor necrosis factor (TNF)-, IL-1, and formylmethionyl leucyl phenylalanine (14), indicating a similar efficacy for MBP and these stimuli. MBP, however, is less potent than many of the other inflammatory reactants, with MBP stimulating IL-8 release at concentrations above 1 µM. Although the concentrations of MBP achieved locally within tissues cannot be quantified, MBP levels of up to approximately 90 µg/ml have been measured in the sputum of symptomatic asthma patients (33). These values, together with an MBP content of approximately 9 µg/10⁶ eosinophils (34), suggest that the 2 µM concentration of MBP, which was consistently found to be effective in the present study, can at least be approached within the tissues. The results presented here clearly exclude any possible LPS contamination as a basis for the MBP activity. Indeed, whereas LPS stimulates a prolonged (at least 20 h) release of IL-8 by neutrophils (14, 16), release of IL-8 stimulated by MBP tended to reach a plateau with respect to time after approximately 10 h of incubation. Given this, the time course for MBP-stimulated IL-8 release is similar to the time course for IL-8 release stimulated by TNF- or IL-1 (14).

These results also show that among the basic proteins present in eosinophil granules, MBP is unique in its ability to stimulate neutrophil IL-8 production, at least at a concentration consistently effective for MBP. In this regard, the present findings are in agreement with earlier observations that ECP, EDN, and EPO do not share the ability of MBP to stimulate neutrophil superoxide anion production at a concentration optimal for MBP (2; S. Page and L. Thomas, unpublished observation). Although it is possible that higher concentrations of ECP, EDN, or EPO may stimulate IL-8 release by neutrophils, such concentrations would begin to exceed concentrations plausibly expected to be achieved clinically. Further, it is worth noting that when expressed in µg/ml protein, the 2 µM concentrations of the 21-kD ECP and 66-kD EPO (1) are 50% and almost 5-fold greater, respectively, than that of the same concentration of 13.9-kD MBP. EPO also did not stimulate significant levels of IL-8 production at the concentrations (0.1-0.3 µM) previously reported to stimulate neutrophil aggregation and adherence (27). In addition, although the enzymatic activity of EPO is responsible for some EPO actions (35, 36), EPO did not stimulate IL-8 release in the presence of its substrate, H₂O₂. This finding may partly reflect the use of chloride, which is less optimal than iodide or bromide (35, 37), as a halide for EPO enzymatic activity, but it has also been reported that the enzymatic activity of EPO is inhibited after binding of EPO to neutrophils (27).

A striking feature of the MBP molecule is its strongly basic charge (1), and, indeed, many of the activities described for MBP have been attributed to its charge. The failure, however, of both ECP, which has a charge similar to that of MBP (pI values of 10.8 and 10.9, respectively) and a similar arginine content (14% each), and of EPO, which is likewise strongly basic (pI of 10.8) (1), to stimulate neutrophil IL-8 release suggests that the basic charge alone does not account for the activity of MBP. In support of this conclusion, it has been found that protamine, a 4.8- kD polypeptide of which 66% consists of arginine (28), and a 14-kD polymer of L-arginine (Poly-R), at a concentration equivalent to the arginine content of MBP, also do not stimulate IL-8 release. Interestingly, it has recently been reported that MBP also stimulates TNF- production by murine bone marrow-derived mast cells after priming by stem-cell factor (38). In contrast, poly-L-arginine was found to stimulate TNF- release independently of priming by stem-cell factor (38).

Earlier results demonstrated the capacity of MBP to stimulate IL-8 production by eosinophils via a transcription-dependent mechanism, as shown by inhibition of MBP-induced increases in IL-8 mRNA by actinomycin D (19). The results presented here indicate that MBP-stimulated IL-8 release by neutrophils is likewise the result of new protein synthesis, since actinomycin D blocked the MBP-stimulated increases in both IL-8 mRNA synthesis and IL-8 protein release. MBP-induced IL-8 synthesis appears, however, to occur independently of an increase in the binding activity of NF-B, which alone or in combination with AP-1 or NF-IL-6 regulates IL-8 gene transcription in many cell types (29). MBP stimulation was likewise not accompanied by an increase in the binding activity of AP-1, and no NF-IL-6 binding activity was detected in the neutrophil extracts in our experiments. In accordance with previous reports (26, 39), LPS-induced activation was accompanied by increases in the binding activities of both NF-B and AP-1, although a portion of the observed increase in NF-B binding activity was nonspecific. Recently, it has been suggested that activation of the transcription factor nuclear factor of activated T-cells (NF-AT) by a sustained increase in the cytosolic free calcium concentration may contribute to the regulation of IL-8 synthesis in neutrophils (40). MBP, however, stimulates an increase in cytosolic free calcium concentration only in cytochalasin B-treated neutrophils (23), which were not used in the present study. Alternatively, the possibility that the transcriptional regulation of MBP-induced IL-8 synthesis in neutrophils requires a novel transcription factor(s), or is due to the inhibition or removal of a repressor protein such as Oct-1, which negatively regulates IL-8 synthesis (41), cannot be excluded.

The present results indicate that the regulation of MBP-stimulated IL-8 production in neutrophils may also occur posttranscriptionally through stabilization of IL-8 mRNA, as observed in other cells in response to various stimuli (42). The 3' untranslated region of IL-8 mRNA contains tandem repeats of adenine/uracil (AU)-rich regions that are often associated with unstable mRNA (46). Specific AU-binding proteins have been identified (47, 48), and the finding that cycloheximide induces large increases in IL-8 mRNA (43, 49) has implicated the binding of specific proteins to AU-rich regions in the targeting of IL-8 mRNA for degradation. Specific AU-binding proteins have not yet been identified in neutrophils, but a similar mechanism probably occurs in neutrophils. Cycloheximide induces large increases in neutrophil IL-8 mRNA levels (14, 50), and the inhibition of LPS-stimulated IL-8 production by IL-10 (51, 52) is correlated with a decreased stability of IL-8 mRNA that depends on protein synthesis (51). IL-10 likewise inhibits LPS-stimulated production of MIP-1 and MIP-1 by a similar mechanism (51). Interestingly, MBP also stabilized MIP-1 mRNA, but not for the same duration as IL-8 mRNA. Thus, the findings that MBP stimulated larger but more transient increases in MIP-1 and MIP-1 mRNA, but did not stimulate MIP-1 or MIP-1 protein production, suggest that prolonged stability of IL-8 mRNA plays a major role in the action of MBP. It is worth noting in this context that the IL-8 mRNA level in unstimulated neutrophils was increased at 1 h in the present experiments. Adherence to plastic has been shown to induce increases in neutrophil IL-8 mRNA levels (14), and the possibility that adherence contributes to the increase observed here cannot be excluded. In any event, the increased IL-8 mRNA level found in unstimulated neutrophils would provide a basis for the posttranscriptional effect of MBP.

Although MBP was first recognized for its cytotoxicity (1), it is now clear that this protein also exhibits many noncytotoxic activities, including the stimulation of neutrophil IL-8 production. Besides being a potent chemoattractant and stimulus for neutrophils (8, 10), IL-8 is also a chemoattractant for T-lymphocytes (53) and for IL-5-primed eosinophils (54), as well as a stimulus for basophils primed by IL-3 (55). In addition, recent findings obtained with the human mast cell line HMC-1 indicate that IL-8 may also be chemotatic for mast cells (56). Thus, MBP-induced IL-8 production by neutrophils could contribute to inflammatory events characteristic of chronic asthma. However, we propose that the effect of MBP on neutrophil IL-8 release may be even more relevant to the pathophysiology of acute asthma. In addition to promoting additional neutrophil influx, IL-8 release could enhance or prolong neutrophil activation in an autocrinelike manner. The resulting increased release of elastase and other neutrophil mediators (57) could exacerbate changes such as formation of the mucus plugs observed in fatal asthma (7). An association between eosinophil infiltrates and exacerbations in chronic bronchitis (58) suggests that MBP-stimulated IL-8 production may also have implications for other inflammatory lung diseases.