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Originally published online as doi:10.1189/jlb.0507302 on February 1, 2008

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(Journal of Leukocyte Biology. 2008;83:1201-1206.)
© 2008 by Society for Leukocyte Biology

PLUNC is a secreted product of neutrophil granules

Jennifer A. Bartlett*,{dagger}, Benjamin J. Hicks{dagger}, Jamie M. Schlomann{ddagger}, Shyam Ramachandran§, William M. Nauseef{ddagger},|| and Paul B. McCray, Jr.*,{dagger},§,1

* Departments of Pediatrics and
|| Internal Medicine,
{ddagger} Inflammation and
§ Genetics Ph.D. Programs,
{dagger} Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

1Correspondence: 240F EMRB, Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. E-mail: paul-mccray{at}uiowa.edu


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ABSTRACT
 
Airway epithelia and neutrophils are frequently recruited to release host defense factors in response to a variety of pulmonary pathogens. One abundant product of airway epithelia is palate, lung, nasal epithelium clone (PLUNC), a proposed innate immune protein expressed in submucosal glands and surface airway epithelia. In this study, we report the expression of PLUNC in human neutrophils, a previously unrecognized source of this protein. Immunoblots performed on polymorphonuclear cell (PMN) lysates and PMN subcellular fractions indicated that PLUNC was present in the specific granules of the neutrophil. Furthermore, secretion assays demonstrated that PLUNC protein was released by neutrophils upon stimulation with secretogogues, including formyl methionyl leucyl phenylalanine and the calcium ionophore A23187. Although recombinant PLUNC protein failed to exhibit antibacterial activity in our studies, its storage and secretion by a professional phagocytic cell support the hypothesis that PLUNC participates in an aspect of the inflammatory response that contributes to host defense. These studies suggest that PLUNC expression is less restricted than previously believed, and highlight new avenues of research for the study of PLUNC function.

Key Words: innate immunity • endotoxin • inflammation • BPI • LBP


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INTRODUCTION
 
In multicellular organisms, innate immunity provides the first line of defense against pathogenic microorganisms. The innate immune system consists of a large array of polypeptides and proteins, including pattern recognition receptors, signaling molecules, inflammatory mediators, and antimicrobials (reviewed in ref. [1 ]), which act in concert to sense and respond to threats from invading microbes. Such molecules are often produced in regions of the body that interface with the environment, such as the airway and gastric mucosa and the skin. In many cases, antimicrobial peptides and proteins are secreted by epithelial cells into the extracellular spaces. Host-defense proteins are also produced and stored in the granules of inflammatory cells such as neutrophils and macrophages, ensuring that these factors can be delivered to and released at sites of infection by circulating leukocytes.

Currently, there is growing interest in the proposed innate immune molecule known as palate, lung, nasal epithelial clone (PLUNC). As its name suggests, PLUNC is a specific secretory product of the respiratory tract and airways, and early studies describe PLUNC expression predominantly in the oral and nasal regions as well as the respiratory tract [2 3 4 5 6 ]. Consistent with this observation, cultured human tracheobronchial epithelia secrete PLUNC [7 , 8 ], and PLUNC protein is detected in a number of oral and respiratory fluids including saliva [8 , 9 ] and nasal lavage fluid [10 11 12 13 14 15 ]. In the lung, in situ hybridization and immunohistochemical studies localize PLUNC expression to the respiratory epithelium and the submucosal glands of the trachea and bronchi [7 , 8 , 16 ]. Proteomic studies indicate that PLUNC is a glycosylated protein with a molecular weight of ~25 kDa, for which as many as eight isoforms can be identified in nasal lavage fluid [13 ].

The human PLUNC gene and its paralogs form a cluster within a 300-kb region on chromosome 20q11.2 [3 ]. The 3'-downstream region contains genes encoding the acute-phase proteins bactericidal/permeability-increasing protein (BPI) and the LPS-binding protein (LBP), members of a larger superfamily known as the lipid transfer (LT)/LBP family. These LT/LBP genes share a conserved exonic structure with PLUNC and the other members of the gene cluster, suggesting that the PLUNC protein may be structurally related to BPI and LBP. LBP is a circulating glycoprotein secreted by the liver [17 ], whereas BPI is found mainly in the primary granules of the neutrophil [18 ]. Both molecules bind the Gram-negative cell-wall component LPS; in addition, BPI displays direct antimicrobial activity against Gram-negative bacteria [18 ]. As reviewed in ref. [19 ], BPI and LBP coordinate to mediate inflammatory responses to invasion by Gram-negative bacteria through effects on cell signaling, as well as direct microbicidal activity in the case of BPI.

The realization that PLUNC, BPI, and LBP are related products of the same gene cluster has led to the suggestion that PLUNC may perform roles in host inflammatory responses that are similar or related to those of BPI and/or LBP. There is currently little functional evidence for antimicrobial or immunomodulatory activities by the PLUNC protein; however, several reports have established intriguing connections between inflammation and PLUNC expression. For instance, proteomic studies demonstrate that PLUNC levels in nasal lavage fluid from smokers and in subjects exposed to airway irritants are decreased with respect to that observed in healthy control subjects [12 ]. Subsequent studies indicated that levels of six PLUNC isoforms are lower in nasal lavage fluid from patients with seasonal allergic rhinitis than in controls [11 ]. In contrast, PLUNC expression increases in the nasal respiratory epithelium of rats after olfactory bulbectomy [20 ], and PLUNC mRNA levels are elevated in the lungs of patients with chronic obstructive pulmonary disease [8 ].

In light of this association between altered PLUNC expression and airway inflammation, we sought to more fully characterize the expression pattern of the PLUNC protein. Specifically, we hypothesized that the altered PLUNC levels observed in inflamed airways might be attributed to its delivery by recruited neutrophils in addition to resident airway epithelial cells. In this study, we use immunoblotting to localize PLUNC expression to the specific granules of the neutrophil and demonstrate that PLUNC secretion is a feature of neutrophil activation. Additionally, we test the hypothesis that PLUNC plays a role in the host inflammatory response through direct bacterial killing.


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MATERIALS AND METHODS
 
Culture of human airway epithelia (HAE)
Secretions from primary cultures of HAE were used as a source of native PLUNC protein in these studies. Polarized HAE were prepared from trachea and bronchi by enzymatic dispersion using established methods [21 ]. Briefly, epithelial cells were dissociated and seeded onto collagen-coated, semipermeable membranes with a 0.4-µm pore size (Millicell-HA; surface area, 0.6 cm2, Millipore Corp., Bedford, MA, USA). Cells were maintained in 2% Ultroser G medium at 37°C with 5% CO2. Twenty-four hours after seeding, the mucosal medium was removed, and the cells were allowed to grow at the air-liquid interface. Only well-differentiated cultures (>2 weeks old) were used in these studies. The epithelia used in this study were derived from a patient with interstitial lung disease. The presence of tight junctions was confirmed by transepithelial resistance using a volt-ohm meter (World Precision Instruments, Sarasota, FL, USA; resistance, >500 {Omega}·cm2). The Institutional Review Board of the University of Iowa (Iowa City, IA, USA) approved this study.

Isolation and fractionation of human neutrophils
Polymorphonuclear cells (PMN) were isolated from fresh, heparinized, venous blood, according to a protocol approved by the Institutional Review Board of the University of Iowa. Cells were isolated using dextran sedimentation and separation on a Ficoll-Hypaque gradient, followed by hypotonic lysis to remove erythrocytes. Purified PMN were resuspended in HBSS without Ca2+ and Mg2+ and quantitated by hemocytometer. Neutrophil fractionation was performed as described in ref. [22 ]. Briefly, PMN were treated with the protease inhibitor diisopropylfluorophosphate at a 1 mM final concentration, followed by resuspension in relaxation buffer (10 mM PIPES, 100 mM KCl, 3 mM NaCl, and 3.5 mM MgCl2, pH=7.3) containing 1 mM ATP(Na)2. Cells were disrupted by nitrogen cavitation and centrifuged to remove nuclei. In all subsequent steps, PMN were resuspended and washed in relaxation buffer containing 1.25 mM EGTA. Postnuclear supernatant was layered on a discontinuous Percoll gradient and centrifuged at 48,400 g for 15 min at 4°C to separate the cytosolic, plasma membrane, primary, and specific granule compartments. Subcellular fractions were then removed and spun at 288 x g for 15 min at 4°C to remove Percoll. The cytosolic and granular fractions were washed in relaxation buffer containing EGTA and centrifuged again to remove any remaining Percoll. Subcellular fractions were left in relaxation buffer containing EGTA for subsequent immunoblotting experiments.

Immunoblots
Dr. Philip Whitney at the University of Miami School of Medicine (Miami, FL, USA) [7 ] generously provided rabbit polyclonal PLUNC antiserum. To collect secretions for immunoblotting experiments, the apical surfaces of polarized HAE were rinsed sequentially with PBS containing Complete Mini protease inhibitor (Roche Applied Science, Indianapolis, IN, USA). Samples were collected in a total volume of ~200 µL per 24-well plate and stored at 4°C until use. For SDS-PAGE, 15 µL airway epithelial wash was loaded per lane. Samples were electrophoresed through 4–20% gradient Tris-HCl gels (BioRad Laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes, followed by blocking overnight in TBS-Tween containing 5% powdered milk. Membranes were incubated with PLUNC antiserum diluted 1:1000 or 1:5000 in TBS-Tween for 1.5 h. Following four 5-min washes in TBS-Tween, membranes were probed with HRP-conjugated goat anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at 1:20,000 for 1 h. Another series of five TBS-Tween washes was performed, and bands were detected using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). For immunoblots of PMN secretions, myeloperoxidase (MPO) was detected using a polyclonal anti-neutrophil MPO antibody raised in rabbit (Sigma-Aldrich) at a 1:4000 dilution. Lactoferrin was detected using a rabbit polyclonal antibody (Abcam, Inc., Cambridge, MA, USA) at a 1:10,000 dilution.

For electrophoresis of PMN proteins, cells were centrifuged and resuspended in SDS-PAGE loading buffer, followed by brief sonication. Cell lysates were then loaded onto gels in a total volume of 20 µL. To prepare PMN secretions for electrophoresis, samples were concentrated using Centricon centrifugal filter units (YM-10; molecular weight cutoff=10,000 daltons, Millipore Corp.) and mixed with 2x SDS-PAGE-loading buffer prior to gel loading.

Bacterial expression of recombinant PLUNC (rPLUNC)-6xHis
The cDNA for human PLUNC [National Center for Biotechnology Information (NCBI) accession number NM_016583] was cloned into the plasmid vector pMAL-c2x (New England Biolabs, Ipswich, MA, USA) for the expression of a fusion protein containing an N-terminal maltose-binding protein (MBP) tag and a C-terminal 6xHis tag. Full-length fusion protein was expressed in the Escherichia coli strain BL21 Star (DE3, Gibco, Invitrogen Corp., Carlsbad, CA, USA), and the protein was purified by passing over amylose resin (New England Biolabs). The crude protein preparation was further purified by passing over a nickel resin column (Ni Sepharose 6 Fast Flow, GE Healthcare Biosciences Corp., Piscataway, NJ, USA), followed by removal of the MBP tag by cleavage using Factor Xa protease (New England Biolabs). All steps of the purification were carried out in buffers containing 20 mM Tris, 50 mM NaCl, pH 7.3.

Neutrophil secretion assay
PMN were resuspended in RPMI buffer (Gibco, Invitrogen Corp.) containing 10% FBS (Hyclone, Logan, UT, USA), 30 µg/mL kanamycin, 100 mM nonessential amino acids, 100 mM HEPES, and 100 mM sodium pyruvate. Reactions were performed in a total volume of 1 mL, at a final concentration of 10 x 106 PMN/mL. To stimulate degranulation, cells were incubated with 1 µM formyl methionyl leucyl phenylalanine (fMLF; Sigma-Aldrich) or 1 µM A23187 (Sigma-Aldrich). Cells were tumbled for 20 min at 37°C and then moved to an ice-water bath to stop granule release. Samples were centrifuged at 2000 x gfor 10 min to pellet the PMN, and the supernatants were removed to separate tubes. Supernatants containing secreted granule contents were frozen at –20°C until immunoblotting.

Cell culture and expression of rPLUNC-6xHis in mammalian cells
The cDNA for C-terminally His-tagged human PLUNC was cloned into the plasmid vector pacAd5 CMV K-N pA, provided by the Gene Transfer Vector Core at the University of Iowa [23 ]. This is a mammalian expression vector that possesses a CMV promoter driving strong expression in most mammalian cell types. For PLUNC expression studies, 293T cells were maintained in 10 cm culture plates in the presence of DMEM (Gibco, Invitrogen Corp.), supplemented with 10% FBS and 1% penicillin-streptomycin (Gibco, Invitrogen Corp.). Prior to transfection, plates were seeded at a density of 1 x 106 cells per plate and incubated at 37°C with 5% CO2 until cells reached ≥90% confluency. Transfections were performed using Lipofectamine 2000 transfection reagent (Gibco, Invitrogen Corp.) in the presence of Opti-MEM (Gibco, Invitrogen Corp.), according to the manufacturer’s recommendations. Cells were transfected with 24 µg plasmid per plate of the PLUNC expression vector (pAd5-PLUNC-6xHis) or empty plasmid control (pAd5-empty) and incubated for 4–6 h at 37°C. The media were then replaced with fresh, warmed DMEM and incubated for 48 h at 37°C. To harvest secreted PLUNC-6xHis, cell supernatants were collected after 48 h and centrifuged at 1000 RPM to sediment cell debris. The resulting supernatants were then removed and stored at 4°C for further analysis. The presence of PLUNC-6xHis in the 293T cell supernatants was verified by immunoblot. To prepare the supernatants for use in antimicrobial studies, samples were prepared in 10 mM sodium phosphate buffer and concentrated approximately tenfold. Buffer exchange and concentration were achieved using Amicon Ultra-15 centrifugal filter units (molecular weight cutoff=10,000 daltons, Millipore Corp.).

Antimicrobial assays
Antimicrobial assays were performed using conventional CFU-based methods, as described [24 ]. Briefly, bacteria were grown at 37°C in Luria-Bertani (LB) broth until reaching log phase, and log-phase bacteria were then pelleted and resuspended in 10 mM sodium phosphate buffer (pH 7.4) to a concentration of 5 x 107 cells/mL. For each condition, samples were diluted to the desired concentration in 10 mM sodium phosphate buffer and then incubated with 1.0 x 106 bacteria in a total volume of 100 µL. Reaction mixtures were incubated for 3 h at 37°C. To assess bacterial viability, serial dilutions of the reaction mixtures were plated on LB agar, and colonies were counted after overnight growth at 37°C. Recombinant human β-defensin 3 (HBD3) used in these studies was obtained from PeproTech Inc. (Rocky Hill, NJ, USA).


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RESULTS
 
To screen for PLUNC expression in an inflammatory cell type, we asked whether PLUNC is present in neutrophils. As shown in Figure 1 A , we detected PLUNC in PMN lysates by immunoblot, confirming that PLUNC was a product of the neutrophil. On these blots, the PLUNC protein ran as a single immunoreactive band of ~21 kDa, in keeping with earlier work characterizing the PLUNC protein in airway secretions [7 ]. To determine more precisely where PLUNC was localized in the neutrophil, we performed immunoblots on subcellular fractions isolated from PMN (Fig. 1B) . These studies revealed that PLUNC was associated with the specific granules of the neutrophil and absent from the primary granules, the plasma membrane, and the cytoplasmic compartment.


Figure 1
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  		Figure 1. PLUNC expression in neutrophils. (A) Immunoblot demonstrating the presence of PLUNC in PMN lysate. Increasing amounts of His-tagged rPLUNC protein were electrophoresed and blotted, as described in Materials and Methods, as a positive control for the antibody. Lane 4 contains PMN lysate (~1.4x106 cell equivalents), which exhibited a single immunoreactive band at the appropriate size for the PLUNC protein. Apical wash (25 µL) from primary cultures of well-differentiated HAE was included as an additional positive control. The apparent size discrepancy between the native PLUNC and rPLUNC proteins is a result of the presence of the 6xHis epitope tag at the C terminus of the recombinant protein. Results are representative of immunoblots performed on PMN isolated from multiple donors (n=3). (B) Immunoblot performed on PMN subcellular fractions. PLUNC protein was absent from the cytosolic (C), plasma membrane (PM), and azurophilic granule (AG) compartments but was readily detected in the specific granule (SG) fraction (~2.7x106 cell equivalents loaded per lane) from purified human PMN. Apical wash (25 µL) from a primary culture of HAE was included as a positive control for the antibody. Results are representative of immunoblots performed on PMN fractions isolated from multiple donors (n=3).

The specific granules of the neutrophil contain a number of proteins that may be secreted into the extracellular space when the cells are activated by bacteria or other inflammatory stimuli. Therefore, we hypothesized that neutrophils secrete PLUNC protein under conditions that normally trigger degranulation. To test this, we performed assays in which PMN were stimulated with fMLF or the calcium ionophore A23187 and the resulting secretions collected for immunoblotting. As shown in Figure 2 , PLUNC levels were increased significantly in PMN secretions after stimulation with both secretogogues, indicating that PMN release PLUNC in response to inflammatory stimuli. This rapid appearance of PLUNC in the PMN secretions was accompanied by parallel release of the primary granule marker protein MPO and the specific granule marker lactoferrin. Taken together, these results suggest that PLUNC secretion is strongly tied to degranulation and is likely to be a general feature of neutrophil activation in response to a variety of stimuli.


Figure 2
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  		Figure 2. PLUNC is secreted by activated neutrophils. Purified PMN were stimulated with fMLF (1 µM) or A23187 (1 µM) for 20 min, and the resulting secretions were analyzed by immunoblot. On a representative blot, rPLUNC-6xHis (300 ng) and HAE wash serve as positive controls to verify the specificity of the PLUNC antiserum. Little to no PLUNC protein was observed in secretions from naïve PMN (unstim.); these levels increased after treatment with fMLF or A23187, indicating that PLUNC release was triggered by secretogogue stimulation. Immunoblots were also performed to probe for parallel secretion of neutrophil granule proteins, specifically, the azurophilic granule protein MPO and the specific granule protein lactoferrin. Levels of MPO and lactoferrin increased in the PMN secretions upon treatment with fMLF and A23187, demonstrating that degranulation took place. Each lane represents secretions from ~1.6 x 106 cells. Results are representative of immunoblots performed on secretions from multiple experiments (n=3).

The presence of PLUNC in the PMN-specific granules suggests a host-defense function. We hypothesized that, like the related neutrophil granule protein BPI, PLUNC might possess antimicrobial properties. To address this hypothesis, we evaluated the antimicrobial activities of rPLUNC protein expressed in bacteria and in mammalian cells. In Figure 3 A , we expressed His-tagged rPLUNC protein in E. coli and performed killing assays on a panel of bacteria, including E. coli DH5-{alpha}, Pseudomonas aeruginosa PAO1, and the Gram-positive Listeria monocytogenes. Following incubation of these bacteria with PLUNC-6xHis, there was no apparent effect on bacterial survival. In contrast, the antimicrobial peptide HBD3 exhibited several logs of killing. To address the possibility that PLUNC expressed in E. coli might be rendered inactive as a result of the limitations of producing recombinant proteins in a bacterial system, we additionally expressed His-tagged rPLUNC protein in the human kidney epithelial cell line 293T. Using this material, we performed similar killing assays using the same panel of bacteria (Fig. 3B) . Incubation of these bacteria with 293T cell supernatants containing secreted PLUNC-6xHis also had no significant effect on bacterial survival. Taken together, these results led us to conclude that PLUNC alone is not an effective antimicrobial agent against this panel of organisms.


Figure 3
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  		Figure 3. PLUNC fails to exhibit antimicrobial activity in bacterial killing assays. Antimicrobial assays were performed to test for activity against E. coli DH5-{alpha}, P. aeruginosa PA01, and L. monocytogenes. (A) Bacterially expressed His-tagged PLUNC at 100 µg/mL was incubated for 3 h with 1.0 x 106 of each bacterium, and serial dilutions of surviving bacteria were plated to assess bacterial viability. Numbers of CFUs recovered with PLUNC-treated bacteria were not significantly different from those observed for bacteria incubated in buffer alone, suggesting that PLUNC is not antimicrobial under these conditions. The broad-spectrum antimicrobial peptide HBD3 (10 µg/mL) served as a positive control for killing activity (averaged results of three experiments). (B) His-tagged rPLUNC was expressed in 293T cells, and cell supernatants containing secreted PLUNC were harvested two days after transfection. Supernatants were buffer-exchanged and concentrated as described in Materials and Methods, and the samples from PLUNC-expressing cells and from mock-transfected control cells were incubated for 3 h with 1.0 x 106 of each bacterium. Similar to the findings for bacterially derived PLUNC protein, PLUNC expressed in mammalian cells exhibited no significant killing activity against the organisms tested. Data represent the averaged results of three separate experiments.


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DISCUSSION
 
Here we present the first evidence for expression of PLUNC by a cell of hematopoietic lineage. Using immunoblotting, we readily detected PLUNC protein in the specific granules of resting PMN. Furthermore, PLUNC was released by neutrophils upon stimulation by the secretogogues fMLF and A23187, suggesting that PLUNC release is likely to occur during neutrophil responses to inflammatory stimuli. Although the function of the PLUNC protein is currently unknown, it has been proposed to be an innate immune molecule on the basis of its homology to BPI and LBP, as well as its documented expression at sites of frequent microbial exposure such as the respiratory epithelium. Therefore, the finding that PLUNC is produced by a professional phagocyte, in addition to its previously recognized epithelial expression, is intriguing, as it provides suggestive evidence that the PLUNC protein may contribute to host defense through direct antimicrobial activity, endotoxin binding, or other functions. Such an expression pattern is observed for numerous innate immune molecules, including the {alpha}-defensins, the human cathelicidin LL-37/human cathelicidin antimicrobial protein 18, BPI, lactoferrin, lysozyme, and neutrophil gelatinase [25 ], each of which exerts effects in multiple tissue sites where the host interfaces with its environment.

Our studies demonstrate that PLUNC is stored in the specific granules of the neutrophil and is secreted upon degranulation. It is interesting to contrast this with the related molecule BPI, which is a product of the azurophilic granule, a subcellular compartment distinctly different from the specific granule [26 ]. The observation that PLUNC and BPI are found in different granular compartments suggests probable differences in the timing of expression of these related LT/LBP family members during neutrophil differentiation and granulogenesis. It also hints at possible differences in the functions performed by the two proteins. Secretion of primary granule proteins such as BPI into phagocytic vacuoles is generally associated with bactericidal or bacteriostatic actions as well as digestion of microbial components, whereas specific granule proteins are associated with an array of functions as diverse as microbial killing and replenishment of cell membrane components involved in neutrophil activation [27 ].

It is often suggested that similar to its relative BPI, PLUNC may play some directly antimicrobial role against invading pathogens in the extracellular milieu. However, our studies with rPLUNC protein, produced using two different methods, have thus far failed to demonstrate an antibacterial function for PLUNC. Whereas these results suggest that PLUNC is not directly microbicidal, we have yet to investigate several possibilities that might explain this negative result. For instance, it is possible that PLUNC may display a fairly narrow spectrum of activity against a relevant oral or airway pathogen that was not tested in these studies. Interestingly, Chu and colleagues [28 ] recently reported that murine rPLUNC inhibited the growth of Mycoplasma pneumoniae, suggesting that further studies involving a wider array of respiratory pathogens may be valuable. Alternatively, it is possible that rather than performing a directly microbicidal role, PLUNC may act synergistically with other antimicrobial agents in the phagosome or have indirect effects on microbes through pathogen-sensing or immunomodulatory mechanisms. Preliminary support for this latter idea comes from the observation that multiple PLUNC isoforms in human nasal lavage fluid can associate with immobilized LPS [13 ]. Further studies will be needed to explore PLUNC binding to LPS or other conserved microbial pattern molecules.

Proteomic studies using two-dimensional gel electrophoresis indicate that as many as eight PLUNC isoforms can be identified in human airway fluids [13 ], which display slight differences in molecular masses and isoelectric points. However, it is unclear whether the same array of isoforms is present in the neutrophil. Based on data available in the GenBank database through the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), two transcript variants have been described for the human PLUNC gene. These variants differ by 55 nucleotides in the 3'-untranslated region, a difference that is not predicted to affect the coding region of the protein. As it therefore seems unlikely that the differences in size and charge observed for airway-derived PLUNC isoforms are a result of alternative splicing, it is instead thought that the different isoforms likely arise from differences in post-translational processing. In support of this, it has been demonstrated that at least some of these isoforms are subject to sialylation [11 ]. It is currently unknown whether these isoform variations have functional consequences for the PLUNC protein. We have not defined specifically which of these isoforms are expressed in PMN, primarily because they are not well-resolved by one-dimensional gel electrophoresis.

In conclusion, we report the novel finding that PLUNC is stored and secreted by the specific granules of PMN. The identification of PLUNC in a phagocytic cell type broadens our understanding of the possible biological functions of this protein, which is primarily recognized as an oral or respiratory epithelial cell secretory product. Our findings reveal that PLUNC expression is more widely distributed than originally appreciated and increase the number of tissues in which PLUNC might potentially carry out its actions. Additionally, the detection of PLUNC in phagocytes adds weight to the hypothesis that PLUNC is a host-defense protein. Additional work is needed to determine the function of the PLUNC protein and its precise role in the neutrophil inflammatory response.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health P50 HL-61234 (P. B. M.) and AI-34879 (W. M. N.). We thank Kevin Leidal for valuable technical assistance. Additionally, we recognize Dario Mizrachi for assistance in the preparation of the PLUNC expression construct. We are grateful to Lokesh Gakhar, Reshma Anthony, and Anthony Fischer for critical review of the manuscript.

Received May 14, 2007; revised December 19, 2007; accepted January 12, 2008.


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