INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Infiltration by polymorphonuclear neutrophils (PMNs) occurs in response to release of chemoattractant molecules by resident tissue cells. Infiltrating PMNs are important for clearance of invading microorganisms, but these cells can also cause significant tissue damage by releasing toxic molecules such as oxidants and proteinases (1). Interleukin 8 is one chemoattractant (2) for PMN that is important in inflammation associated with allergic rhinitis (3), bronchitis due to smoking (4), pulmonary fibrosis (5), psoriasis (6), rhinitis (7), inflammatory bowel disease (8), postischemic reperfusion cardiac injury (9), ozone-related bronchospasm (10), and other disorders. A previous investigation (11) demonstrated that nebulization of interleukin 8 into noses of human subjects causes a quantifiable degree of acute nasal inflammation.

Apoptosis (programmed cell death) is a process by which cells undergo a progression of events that culminate in cell death without release of injurious molecules, thus sparing surrounding tissue from damage (12). Apoptotic PMNs exhibit decreased cellular functions including chemotaxis and oxidant production (13). An agent that accelerates PMN apoptosis could have significant antiinflammatory effects through attenuation of acute inflammatory cell infiltration. Studies have shown that PMN apoptosis is accelerated or delayed by various environmental signals (12). However, all the exogenous factors that control PMN apoptosis have not been defined.

The integrin CR3 is important for neutrophil chemotaxis (14) and may be involved in apoptosis of these cells (15, 16). Expression of this integrin on neutrophil plasma membranes is increased with cellular activation, and the expression of this receptor is greatest at the leading front of the cell when it is migrating up a chemotactic gradient (14). This integrin also appears to be important in stimulated oxidant production by neutrophils (17) and may be a modulator of the cycling of chemoattractant receptors (14). In addition, two studies (15, 16) have suggested that CR3 ligation is associated with neutrophil apoptosis.

We have previously reported that bronchial secretions of normal human subjects contain a peptide that inhibits PMN chemotaxis and oxidant production (18, 19). The amino acid sequence of this peptide shows remarkable homology to a portion of influenza A nucleoprotein (19), a molecule that also inhibits PMN function (20). In the current study we examined whether a synthetic analog of this peptide can inhibit interleukin 8-induced nasal inflammation in normal humans, established some in vitro effects of this peptide, including specific binding to neutrophil CR3, and characterized the molecular source of this peptide, which is a 70-kDa secreted protein.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects

Normal nonsmoking human volunteers were recruited from the Birmingham, Alabama area. Volunteers had no history of medical illness and had no recent history of nasal symptoms. One group of volunteers (n = 7, three males, four females) took part in nasal challenge studies. Another group, distinct from those recruited for nasal exposures, supplied whole blood as a source of polymorphonuclear neutrophils (PMNs) for in vitro studies. All studies were performed under the guidelines of the Institutional Review Boards of the University of Alabama Medical School and the Birmingham Veterans Affairs Medical Center (VAMC).

Synthesis of Peptides

A peptide with the amino acid sequence NH₂-REGSYFFGDNA-COOH was synthesized with an acetylated amino terminal and amidated carboxy terminal (synthetic neutrophil inhibitor peptide, SNIP) at the University of Alabama at Birmingham, using a PE Biosystems (Foster City, CA) peptide synthesizer. A similar peptide was also biotinylated and fluoresceinated to allow detection. A peptide with the reverse sequence (NH₂-ANDGFFYSGER-COOH) and chemically blocked terminals was also synthesized for use as a control for in vitro studies. Accuracy of synthesis was determined by analysis of amino acid composition and amino acid sequence confirmation. Purification of peptide was performed by reversed phase high-performance liquid chromatography (HPLC) at the core facility. The peptide was suspended in sterile phosphate-buffered saline (PBS) prior to use.

Nasal Lavage and Exposures

The protocol for in vivo studies is summarized in Figure 1. Subjects (n = 7) first underwent nasal lavage with 10 ml of PBS. The subject tilted his/her head to a horizontal position while 5 ml of PBS was slowly instilled; the subject retained the fluid for 1-2 s and then forcibly expelled it into a 50-ml tube. This process was repeated a second time to reach a total of 10 ml of instilled PBS. As part of a baseline exposure the same nostril was then exposed to 1.32 mg of histamine in 330 µl of PBS (to increase vascular permeability prior to other exposures) followed by 330 µl of PBS (as control for administration of synthetic peptide in the subsequent exposure). Five minutes later 500 ng of interleukin 8 in 330 µl of PBS was nebulized. The exposure to PBS, followed by interleukin 8, was repeated four times so that a total of 2.5 µg of interleukin 8 was nebulized over 1 h. Nebulization of the solutions was performed with an atomizer (DeVilbis) and a Rosenthal dosimeter that accurately delivered the desired volume of solution.

View larger version (8K): [in this window] [in a new window]   Figure 1.   Protocol for in vivo studies. Subjects initially underwent nasal lavage (NL) and then were exposed to histamine followed by phosphate-buffered saline (PBS) or SNIP every 15 min just prior to exposure to interleukin 8. Nasal lavage was repeated 2, 3, and 4 h after the start of the procedure.

At least 2 wk after initial exposure subjects were rechallenged, using a similar protocol except that the volunteers received 30 µg of the peptide in 330 µl of PBS five times just prior to interleukin 8 nebulization, for a total of 150 µg of peptide (in place of the control PBS exposure for the baseline protocol). As part of each protocol nasal lavage was performed just prior to, as well as 2, 3, and 4 h after, histamine exposure.

Processing of Nasal Lavages

The volume of PBS returned after nasal lavage was measured. Total cell counts were performed on unconcentrated nasal lavage fluid by determining the number of cells per milliliter, using a hemocytometer and multiplying by the volume returned. Differential cell counts were performed on Wright-stained cytospin preparations as previously described (18) and total PMNs were calculated in a similar manner. Supernatant was separated from cellular material by centrifugation (400 × g for 8 min) and was frozen prior to assay of total protein and myeloperoxidase concentrations.

Measurement of Total Protein and Myeloperoxidase Concentrations in Nasal Lavages

Protein concentration in cell-free supernatant was measured with Coomassie blue as substrate (21) as previously described (20). Results were multiplied by volume returned of nasal lavage to obtain total micrograms of protein in each specimen. Myeloperoxidase (MPO) concentrations were determined with a commercially available enzyme-linked immunosorbent assay (ELISA; Oxis International, Portland, OR). Briefly, 100 µl of undiluted nasal lavage supernatant or MPO standard was added to the wells of a microtiter plate coated with a monoclonal antibody directed against MPO. The plate was incubated at 37° C for 2 h and then washed five times with wash buffer (1 M Tris-HCl [pH 7.8] with 3 M NaCl, 2% Tween 20, and 0.1% sodium azide), and then 100 µl of a biotin-linked polyclonal antibody generated against MPO in goats was added. The plate was incubated at 37° C for 1 h and washed five times in wash buffer, and then 100 µl of avidin-coupled alkaline phosphatase was added and the plate was incubated for 1 h at 37° C. The plate was then washed five times and 100 µl of a solution containing 4-nitrophenyl phosphate (pNPP), a substrate for alkaline phosphatase, was added; the color was allowed to develop for 15-25 min, and then the reaction was stopped with 1 M sodium hydroxide containing 0.1 M EDTA and the absorbance at 405 nm was determined on a microplate reader. The concentration of MPO in nasal lavage supernatants was determined by comparison with a standard curve. The concentration of MPO in supernatant (ng/ml) was multiplied by the volume of the sample to determine the total nanograms of MPO present.

Assessment of Inhibition of Chemotaxis by Inhibitor Peptide

PMNs were isolated from normal human volunteer whole blood by Ficoll-Hypaque gradient centrifugation followed by dextran sedimentation as previously described (18, 19). Inhibition of PMN chemotaxis in response to interleukin 8 was assessed with a modified Boyden chamber (18, 19). PMNs (2 × 10⁶ cells/ml in Hanks' balanced salt solution [HBSS] with 0.1% bovine serum albumin [BSA]) were exposed to SNIP at 200, 20, or 2 µg/ml (final concentration) or to control peptide (200 µg/ml) for 10 min at room temperature, and then chemotaxis of exposed cells in response to 3 nM recombinant interleukin 8 was examined. Interleukin 8 or PBS alone was placed in the bottom wells of the chemotaxis chamber and then a nitrocellulose membrane containing 8-µm pores was placed over the wells, followed by a rubber gasket and a top chamber, where the exposed cells were added. This pore size (8 µm) was used to eliminate alteration in deformability as the cause of SNIP effects, so as to examine effects on migration alone. The plates were incubated at 37° C for 1.5 h and then the membrane was removed, fixed, and stained. The maximal distance migrated was determined by the leading front method as previously described (19). Distance traveled by PMNs preexposed to buffer alone (negative control) was subtracted from the distance that peptide-exposed cells migrated in response to interleukin 8 prior to comparisons among the treatments.

Effects of SNIP on PMN chemotaxis in response to IL-8 were determined with nitrocellulose filters (3-µm pores) that were incubated for 1 h at room temperature in a 10-µg/ml concentration of fibrinogen (plasminogen free; Calbiochem, La Jolla, CA) BSA (GIBCO, Grand Island, NY) diluted in PBS. PMNs were exposed to SNIP or control peptide (200 µg/ml) and chemotaxis in response to IL-8 was immediately assessed and quantitated in a similar manner as described above.

Detection of PMN Apoptosis

Effects of peptide on PMN apoptosis were determined by two methods, one using a commercially available kit that detects in situ double-stranded, low molecular weight DNA fragments occurring with this process (in situ cell death detection kit, AP; Boehringer Mannheim, Indianapolis, IN), and the other using the polymerase chain reaction (PCR) to increase sensitivity for detection on agarose gels of DNA ladder formation caused by DNA fragmentation (ApoAlert LM-PCR ladder assay kit; Clontech, Palo Alto, CA). In the first method PMNs were diluted to 10 × 10⁶ cells/ml in HBSS with 0.1% BSA and then exposed to SNIP or control peptide (100 µg/ml) for 2 h at room temperature. At the end of exposure a monolayer preparation of cells was prepared on glass slides, using a Cytospin apparatus (Shandon, Pittsburgh, PA). The monolayer was fixed in 4% paraformaldehyde (in PBS, pH 7.4) and then cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4° C. The permeabilized cells were then exposed to a combination of terminal deoxynucleotidyltransferase from calf thymus and a fluorescein-labeled nucleotide mixture for 1 h at 37° C in a humidified chamber. This resulted in labeling of DNA strand breaks with material that contained fluorescein. The slides were rinsed in PBS three times and then an alkaline phosphatase-labeled antibody directed against fluorescein was added, and the slides were incubated for 30 min in a humidified chamber at 37° C. The slides were then washed three times in PBS and exposed to a substrate solution for alkaline phosphatase (Western Blue substrate; Promega, Madison, WI) for 10 min at room temperature. The slides were counterstained with hematoxylin and eosin, and then coverslips were mounted and 500 cells were viewed by a reader blinded to the specific exposure, to determine the percentage of positive cells, i.e., with darkly staining partial or whole nuclei.

For detection of DNA ladder formation DNA was extracted from cells exposed to SNIP or control peptide for 45 min, using a kit (Easy DNA kit; InVitrogen, Carlsbad, CA), and then suspended in PCR-grade water. DNA (350 ng) was then ligated to adapter primers and amplified by PCR (25 cycles) per manufacturer instructions. Samples were then fractionated on 2% agarose and stained with ethidium bromide to visualize DNA. Calf thymus DNA served as a positive control.

Bronchoalveolar Lavage and Processing of Fluid

Bronchoalveolar lavage was performed on lungs obtained from heart transplant donors as previously reported (19). At the time of death there was no evidence of lung infection or any history of previous disease in these lungs. Bronchoalveolar lavage was performed by cannulating a subsegmental orifice with a catheter, occluding the airway around the catheter with a suture, lavaging the distal airways with a total of 1 L of Hanks' balanced salt solution without calcium or magnesium (MHS), and retrieving eluant by gentle suction or gravity drainage. The lavage fluid was immediately placed on ice and cells were separated from supernatant by centrifugation (400 × g for 15 min). Cell-free supernatant was then frozen at 80° C until processing. Subsequently 100 ml of supernatant was extracted using C₁₈ Sep-Pak cartridges and eluted with methanol, and then resuspended in 1 ml of phosphate-buffered saline and used for Western blotting.

Western Blotting

Extracted bronchoalveolar lavage fluid was diluted 1:1 in sample buffer and then boiled and separated on a 12% polyacrylamide gel by electrophoresis under reducing conditions. Separated material was transferred to nitrocellulose for probing with antiserum. Complete transfer was established with prestained protein standards. Nitrocellulose filters were first blocked for 1 h in PBS containing 2% gelatin, and then antiserum generated in rabbits to an acetylated peptide containing the 11-amino acid NIP sequence (REGSYFFGDNA) (1:1,000) or antiserum generated to whole influenza A nucleoprotein in goats (1:4,000) (a generous gift of R. Webster, Memphis, TN) was added. Blots were incubated with one of these antisera for 4 h at room temperature, and then washed three times in PBS and exposed to a 1:2,000 dilution of commericially available alkaline phosphatase-labeled antibody generated against goat IgG (Calbiochem) or rabbit IgG (Promega), diluted in blocking buffer. Blots were incubated with this secondary antibody for 3 h at room temperature and then washed three times in PBS, alkaline phosphatase substrate (Western Blue substrate; Promega) was added, and membranes were allowed to develop.

Northern Blotting

Northern blots were performed with mRNA harvested from unstimulated U937 cells. U937 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI with 10% fetal calf serum. Cells were separated from supernatant and mRNA was isolated from 2 × 10⁸ cells, using a commercially available kit (FastTrack; InVitrogen). One and 2 µg of mRNA, suspended in a solution containing dimethyl sulfoxide, glyoxal, and 0.5 M sodium phosphate (pH 7.0), were loaded onto a 1% agarose gel (in 0.01 M sodium phosphate) and electrophoresed for 4-6 h at 60 V. RNA markers were run in parallel to establish the size of detected transcripts in the sample. Material on the gel was transferred to nylon membranes by capillary action and was probed with a combination of two degenerate oligonucleotides specific for the last seven amino acids of the peptide sequence (GCRTTRTCNCCRAARAAGTA and GGRTTRTCNCCRAARAAAA), labeled with ³²P using T4 polynucleotide kinase (22). The combination of equal volumes of each labeled probe was added to achieve a concentration of 4 × 10⁶ cpm/ml. Membranes were first prehybridized for 3 h at 26° C in hybridization solution containing 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaPO₄, and 1 mM EDTA; pH 7.7), 2× Denhardt's solution, denatured and sheared salmon sperm DNA (100 µg/ml), and 0.5% (sodium dodecyl sulfate (SDS), and then the probe was added in fresh hybridization solution and membranes were hybridized overnight at 26° C. The next day membranes were washed at room temperature in 2× SSC (saline- sodium citrate) with 0.05% SDS and then once in 2× SSC with 0.1% SDS at 26° C. Each membrane was then covered with plastic wrap and exposed to film overnight.

Immunoaffinity Chromatography

Immunoglobulin was purified from anti-SNIP rabbit antiserum with a protein G column (MabTrap G II kit; Pharmacia Biotech, Piscataway, NJ) and coupled to cyanogen bromide-activated sepharose. Purified immunoglobulin was dialyzed in coupling buffer and added to 1.5 g of activated Sepharose that had been swelled in 1 mM HCl. The mixture was rocked overnight at 4° C and then spun down at 1,000 rpm and washed three times in coupling buffer. Coupling was then terminated by adding 25 ml of Tris-HCl, pH 8.0, to the gel and the mixture was allowed to stand for 2 h at room temperature. The gel was then washed three times with 0.1 M acetate buffer plus 0.5 M NaCl (pH 8.0), alternating with 0.1 M Tris-HCl plus 0.5 M NaCl (pH 8.0) (total of six washes), and poured into a column and allowed to settle. U937 cells (1 × 10⁹) were cultured in RPMI without serum overnight at 37° C after which supernatant was separated from cells by centrifugation and then applied to the column. The column was washed with 10 column volumes of wash buffer and eluted with 5 column volumes of elution buffer (50 mM triethylamine, 150 mM NaCl, 2 mM MgCl₂, 1% n-octyl -D-glucopyranoside, pH 11). The eluted material was concentrated with a 3,000-Da exclusionary membrane (Centricon; Amicon, Danvers, MA). Concentrated material was run on a 7.5% SDS-polyacrylamide gel and then stained with Comassie blue.

Binding of SNIP to PMNs

PMNs were diluted to 10 × 10⁶/ml in HBSS plus 0.1% BSA. Cells in 400 µl were added to tubes and buffer or various concentrations of unlabeled SNIP (1,000-fold excess of subsequently added fluoresceinated SNIP) were added. The mixtures were incubated for 30 min at 4° C and then varying concentrations of fluoresceinated SNIP were added and the mixtures were incubated for an additional 2 h at 4° C. At the end of the incubation cells were washed at 4° C in PBS and then resuspended in 400 µl of PBS plus 1% Triton to lyse cells. This material was added to 2 ml of PBS and fluorescence was determined (excitation, 490 nm; emission, 516 nm). The level of fluorescence generated by a known concentration of fluoresceinated SNIP was also determined and a standard curve was generated from these values. Binding of SNIP in the presence of excess unlabeled SNIP was subtracted from binding in the presence of buffer alone to obtain specific binding. A Scatchard plot (23) was also generated to further assess features of SNIP binding sites.

Binding of SNIP by Proteins in PMN Lysates and Purification of SNIP-binding Protein

PMNs were isolated and resuspended at 50 × 10⁶/ml in lysis buffer (1% Triton X, 20 mM Tris [pH 8.0], 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM sodium orthovanadate, 0.15 M aprotinin) and rocked for 1 h at 4° C, and then centrifuged at 12,000 × g for 10 min and supernatant representing lysed cellular material was saved. Lysates (8 µl) were incubated with 38 µl of 100 mM SNIP or buffer at 37° C for 30 min and then 100 µM biotinylated or buffer was added. The mixture was incubated for 1 h at 37° C and then an equal volume of 2× nondenaturing sample buffer was added and 30 µl was separated on a 12% nondenaturing polyacrylamide gel, using nondenaturing electrophoresis buffer. Subsequently material separated on the gel was transferred to a nitrocellulose membrane, blocked overnight at 4° C in PBS plus 5% low-fat milk, incubated in alkaline phosphatase-labeled streptavidin (2 µg/ml) in PBS plus 5% milk, washed three times in PBS, and developed with alkaline phosphatase substrate (Western Blue substrate, Promega).

Biotinylated SNIP was then used to construct an affinity column. Ten milligrams of biotinylated SNIP was added to a resin containing avidin in a total volume of 15 ml. The mixture was rocked overnight at 4° C and then placed in a column and allowed to sediment. Phosphate-buffered saline (25 ml) was run through the column and then 8 ml of neutrophil lysates was loaded and run through the column at 0.4 ml/ min. The column was then washed with 25 ml of PBS, after which bound material was eluted with elution buffer (citric acid at pH 3.0) into tubes containing 2 M Tris-HCl to neutralize the pH. Eluted material was concentrated 5-fold and then run on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane for Western blotting. Blots were probed with antibodies to three receptors associated with neutrophil apoptosis, including interleukin 10, Fas, and CR3, all at 1:1,000 dilutions, followed by administration of appropriate secondary antibodies labeled with alkaline phosphatase and detection with alkaline phosphatase substrate.

Statistics

Data were stored and analyzed with a software package (SPSS, Chicago, IL) and a Gateway P5-133 computer. If differences in means for multiple groups were involved, those differences were assessed by analysis of variance (ANOVA) and individual group differences were assessed by the Neuman-Keels test. Data for in vivo studies were not normally distributed, so differences between multiple groups were assessed by a Friedman test. All data represented are means ± SEM. p Values < 0.05 were considered significant (24).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of Preexposure to Peptide on Nasal Inflammation

In vivo nasal exposure to interleukin 8 resulted in a time- dependent increase in total nasal lavage cells and in lavage neutrophils. Preexposure to SNIP attenuated this increase, most significantly at 3 h after beginning of exposure (Figure 2A and 2B). Pretreatment with SNIP resulted in a significant reduction in lavage protein content at the 4-h time point (Figure 2C). There was a trend toward a time-dependent increase in nasal lavage MPO levels after IL-8 exposure but this was not significant (Figure 2D) and SNIP did not significantly affect these levels.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Effect of peptide preexposure on nucleated cells (A), neutrophils (B), protein (C ) and myeloperoxidase (D) content in nasal lavage. Squares indicate values during baseline exposure (without SNIP), triangles show values with SNIP pretreatment (n = 7, mean ± SEM, *p < 0.05 versus baseline exposure).

Effect of Synthetic Peptide on PMN Chemotaxis

In vitro exposure of PMNs to SNIP resulted in reduced chemotaxis in response to 3 nM interleukin 8 at all concentrations tested (Table 1) when compared with chemotaxis in response to control peptide (200 µg/ml). The median inhibitory concentration (IC₅₀) for the peptide was less than 1 µM, as all the concentrations of peptide examined reduced the distance migrated by more than 50% compared with control peptide. When nitrocellulose membranes were soaked in fibrinogen, a CR3 ligand, or BSA, SNIP inhibition of chemotaxis in response to IL-8 was most profound when cells migrated through the fibrinogen matrix (percentage of control peptide exposure was 9.11 ± 5.8% for SNIP-exposed PMNs migrating on fibrinogen matrix compared with 63.64 ± 17% on BSA matrix, p = 0.02) (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Chemotaxis in response to IL-8 (micrometers migrated in response to IL-8 minus distance migrated in response to buffer alone) of PMNs exposed to SNIP (shaded columns) or control peptide with reverse SNIP sequence (solid columns), using nitrocellulose membranes soaked with fibrinogen or BSA (10 µg/ml) (n = 6, *p < 0.05 versus cells exposed to control peptide and versus SNIP-exposed PMN migrating on BSA matrix).

Effects of SNIP on PMN Apoptosis

Exposure to SNIP for 2 h caused an increase in the percentage of apoptotic PMNs that stained positive for low molecular weight double-stranded DNA fragments (Figure 4). In addition, DNA from PMNs exposed to SNIP showed evidence of ladder formation (Figure 5), indicating degradation, whereas exposure to control peptide did not induce this phenomenon.

View larger version (24K): [in this window] [in a new window]   Figure 4.   Representative photomicrograph of monolayer containing PMNs exposed to buffer or SNIP (100 µg/ml) for 2 h (A). Low molecular weight DNA fragments are demonstrated (arrows) by darkly staining whole or partial nuclei. Cells are counterstained with hematoxylin and eosin. (B) Quantitation of the percentage of positive cells for five separate experiments (*p < 0.05 versus control).

View larger version (98K): [in this window] [in a new window]   Figure 5.   Effect of SNIP on in vitro DNA fragmentation documented by ladder formation. DNA ladder was detected by a procedure that employs polymerase chain reaction to increase sensitivity. (A) Standards; (B) neutrophils treated with SNIP (50 µg/ml) for 45 min; (C ) PMNs treated with control peptide with reversed SNIP sequence; (D) control thymus DNA.

Binding of SNIP to PMNs and PMN Lysates

PMNs contain specific binding sites for SNIP. Addition of increasing concentrations of fluoresceinated SNIP caused progressive increase in cell-associated fluorescence, which was blocked by the presence of excess unlabeled SNIP and saturated at high concentrations of peptide (Figure 6). Receptors saturated at an average of 650,000 binding sites per PMN (average of four preparations; range, 176,000-1,589,000 receptors/ PMN). SNIP also specifically bound to proteins in PMN lysates. Biotinylated SNIP also bound to specific proteins in PMN lysates (Figure 7).

View larger version (17K): [in this window] [in a new window]   Figure 6.   Specific binding of SNIP to neutrophils. Neutrophils were incubated with the indicated concentrations of fluoresceinated SNIP for 2 h at 4° C in the presence or absence of a 1,000-fold excess of unlabeled SNIP. The amount of SNIP bound to neutrophils was calculated by comparison with a standard curve and binding in the presence of excess unlabeled SNIP was subtracted from total binding to obtain specific binding. Results are from four separate experiments (mean ± SEM). Inset: Scatchard analysis of binding.

View larger version (57K): [in this window] [in a new window]   Figure 7.   Specific binding of biotinylated SNIP to proteins in neutrophil lysates. Biotinylated SNIP was incubated with neutrophil lysates in the presence (A) or absence (B) of excess unlabeled SNIP for 1 h at 37° C and the mixture was subjected to nondenaturing PAGE, transferred to a nitrocellulose, and probed with strepavidin-alkaline phophatase. The filter was developed with alkaline phosphatase substrate to detect binding proteins. Migration of standards is indicated.

Partial Identification of SNIP-binding Proteins

A monoclonal antibody to the CD11b component of CR3 detected a protein of appropriate molecular weight on Western blots of material eluted from the affinity column containing biotinylated SNIP (Figure 8). Antibodies to interleukin 10 or Fas did not detect any molecules in this eluted material (data not shown). When the primary antibody to CD11b was omitted no molecules were detected in the eluted material (Figure 8) on Western blotting, suggesting the detected molecule was CD11b and not a nonspecific effect.

View larger version (55K): [in this window] [in a new window]   Figure 8.   Western blot of material eluted from an affinity column containing SNIP. Blot was probed with a monoclonal antibody to CD11b, a component of CR3 (left). Blot on the right shows a similar blot probed with omission of the primary antibody. Molecular weight markers are noted. Similar blots probed with antibodies to interleukin 10 or Fas did not demonstrate any positive bands.

Identification and Purification of Higher Molecular Weight Proteins with SNIP Sequence

Western blots of bronchoalveolar lavage fluid fractionated on C₁₈ Sep-Pak cartridges showed a 70-kDa protein detected by antiserum to whole nucleoprotein or to the sequence REGSYFFGDNA (Figure 9). An immunoaffinity column containing antibody to the SNIP sequence bound similar-sized protein (70 kDa) in U937 culture supernatant (Figure 10). On Northern blots of U937 mRNA probed with degenerate oligonucleotides specific for the SNIP sequence a prominent 1.8-kb band was noted (Figure 11).

View larger version (35K): [in this window] [in a new window]   Figure 9.   Western blots of bronchoalveolar lavage fluid C18 Sep-Pak extracted and probed with antiserum to whole nucleoprotein produced in goats (A) or to the REGSYFFGDNA sequence produced in rabbits (B). Molecular weight markers are indicated. Arrows indicate protein detected by both antisera.

View larger version (42K): [in this window] [in a new window]   Figure 10.   Coomassie blue-stained 7.5% SDS-polyacrylamide gel showing U937 culture supernatant material that eluted from an immunoaffinity column containing antibody to the REGSYFFGDNA sequence (lane A). Lane B, material eluting from a sham run (column loaded with buffer alone). Molecular weight markers are noted.

View larger version (22K): [in this window] [in a new window]   Figure 11.   Northern blot of U937 mRNA probed with degenerate oligonucleotides. Lane A, 1 µg of loaded mRNA; lane B, 2 µg of loaded mRNA. Migration of RNAs of known size is noted.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The current study demonstrates that an 11-mer synthetic peptide, with the amino acid sequence REGSYFFGDNA, and chemically blocked amino and carboxy terminals, inhibits nasal influx of PMNs and protein exudation induced by interleukin 8 in normal subjects. The natural form of this peptide appears to be a fragment of a larger, 70-kDa protein that is produced by U937 cells and is a product of a 1.8-kb mRNA transcript detected in these cells. The reduction in inflammation mediated by this peptide may occur through effects on PMN programmed cell death, as in vitro exposure to this peptide causes an increase in apoptosis of these cells. Finally, PMNs appear to specifically bind this protein and proteins in PMN lysates are identified that specifically bind SNIP. One SNIP-binding protein is the integrin CR3 or a molecule that coassociates with this integrin.

A previous study (11) has demonstrated that nasal challenge with interleukin 8 causes time-dependent nasal inflammation, quantitated by nasal smears and nasal biopsy. In that study subjects with allergic rhinitis demonstrated a greater increase in inflammatory cells after administration of nasal interleukin 8 compared with normal subjects. However, normal subjects also showed significant increases in PMN and eosinophil numbers after interleukin 8 nebulization. Our study builds on this previous work, using the model to study in vivo effects of an inhibitor peptide. In contrast to this previous study we quantitated inflammation using nasal lavage, which has the advantage of sampling a larger nasal area than nasal scrapings or biopsy. However, as in the previous study by Douglass and colleagues (11), the rise in nasal inflammation induced by interleukin 8 in our study was due primarily to influx of PMNs.

We have previously described the isolation and partial characterization of a peptide present in bronchial lavage of normal subjects (18, 19). Initial studies (18) demonstrated that a low molecular weight aminopeptidase-sensitive molecule present in bronchial lavage from normal humans inhibits PMN chemotaxis and oxidant production. Using methods devised to purify this natural inhibitor molecule, a low molecular mass (< 3.2-kDa) peptide that inhibits PMN chemotaxis to human recombinant C5a was purified and a partial amino acid sequence (REGSYFFGDNA) was determined. The present study demonstrates that this sequence was used to produce the synthetic peptide employed in the present study. Acetylation of the N terminus and amidation of the C terminus were performed to enhance activity by decreasing in vivo proteolytic degradation and subsequently increase peptide stability (25).

Programmed cell death of PMNs is a process by which these cells can be cleared from sites of inflammation without causing further tissue damage (12). A number of environmental influences have been described to modulate apoptosis in these cells (12). Apoptosis results in characteristic morphologic changes including cellular shrinkage, cytoplasmic vacuolization, and nuclear condensation due to fragmentation of DNA by endonucleases. In some instances cutting of double-stranded internucleosomal DNA results in "ladder" formation on agarose gel electrophoresis, although this does not occur in all instances (26), and it is a relatively insensitive measurement of apoptosis. We could demonstrate such an abnormality in PMNs exposed to SNIP only by using PCR to increase sensitivity. This is probably due to the relatively low percentage of apoptotic cells present after peptide exposure. The in situ assay we used detected a 270% mean increase in apoptotic PMNs after 2 h of exposure to peptide when compared with control. However, even with this increase only 6.8% of PMNs, on average, showed evidence of apoptosis. This relatively low percentage of apoptotic cells may be due to the insensitivity of this assay or because SNIP, or its natural precursor(s), may function as an enhancer of apoptosis induced by other agents, such as tumor necrosis factor. In any event, apoptotic PMNs demonstrate functional deficits and this may be one mechanism by which SNIP or its natural precursors inhibit PMN chemotaxis. PMNs that are undergoing apoptosis show deficient chemotaxis, phagocytosis, degranulation, and oxidant production in response to receptor-dependent stimulation (13). Ability to respond to receptor-independent stimulation, such as that due to phorbol myristate acetate, is at least partially maintained in apopotic PMNs. Our study shows a relationship between inhibition of PMN chemotaxis to interleukin 8 (a receptor-dependent phenomenon) and an increase in the percentage of apoptotic cells caused by the peptide studied. Further studies are needed to conclusively link the two effects of this peptide.

Our studies also suggest PMNs have specific receptors for SNIP. Ligation of a limited number of plasma membrane receptors, including Fas (27), interleukin 10 (28), and tumor necrosis factor (29) receptors and the integrin CR3 (30) on PMNs and other cells is associated with accelerated apoptosis. The molecular weight of one of the SNIP-binding proteins that we detected using biotinylated SNIP was consistent with that of CR3. In addition, CR3 eluted from an affinity column containing SNIP, suggesting that this integrin, or a molecule that coassociates with this integrin, binds SNIP. Finally, chemotaxis studies using nitrocellulose membranes loaded with fibrinogen or BSA showed SNIP inhibition of PMN migration in response to IL-8 was more significant in the presence of fibrinogen than BSA. Because fibrinogen is a CR3 ligand this further supports the notion that SNIP mediates its effects through CR3, possibly through alteration of the normal recycling of this integrin needed for orderly migration.

Components of influenza virus can induce apoptosis in a number of different cell types (31, 32). One study has demonstrated that influenza neuraminidase activates latent transforming growth factor- (32), a molecule that can induce cellular apoptosis. In addition, several studies (33, 34) have demonstrated that influenza components can inhibit PMN functions, including chemotaxis and oxidant production. One of these viral components, the nucleoprotein, shares a span of amino acid sequence that is markedly similar to the currently studied peptide. We have previously demonstrated (20) that purified nucleoprotein can inhibit PMN chemotaxis and oxidant production. To date no published studies have examined the effects of nucleoprotein on PMN apoptosis.

The current study demonstrates that pretreatment with a peptide that is a fragment of a larger cell-derived protein inhibits PMN chemotaxis in vitro, accelerates apoptosis of these cells, and attenuates nasal inflammation in normal humans. Whether this peptide can be effective in the treatment of inflammatory diseases remains to be determined. The observation that subjects with allergic rhinitis have an accentuated response to nebulization of interleukin 8 suggests that this may be a disorder in which this peptide could be effective. Future studies should be directed at the optimal structure of related molecules, designed to maximize the antiinflammatory potential of this molecule, and whether it is effective in various inflammatory states.