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Originally published online as doi:10.1189/jlb.0304182 on July 26, 2004

Published online before print July 26, 2004
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(Journal of Leukocyte Biology. 2004;76:812-819.)
© 2004 by Society for Leukocyte Biology

Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes

Jonathan M. Swartz*, Jonas Byström*, Kimberly D. Dyer*, Takeaki Nitto*, Thomas A. Wynn{dagger} and Helene F. Rosenberg*,1

* Laboratories of Allergic Diseases and
{dagger} Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

1Correspondence: Building 10, Room 11N104, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD, 20892. E-mail: hrosenberg{at}niaid.nih.gov


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ABSTRACT
 
Plasminogen activator inhibitor-2 (PAI-2) as a potential eosinophil protein was inferred from our gene microarray study of mouse eosinophilopoiesis. Here, we detect 47 kDa intracellular and ~60 kDa secretory forms of PAI-2 in purified human eosinophil extracts. PAI-2 is present at variable concentrations in eosinophil lysates, ranging from 30 to 444 ng/106 cells, with a mean of 182 ng/106 cells from 10 normal donors, which is the highest per-cell concentration among all leukocyte subtypes evaluated. Enzymatic assay confirmed that eosinophil-derived PAI-2 is biologically active and inhibits activation of its preferred substrate, urokinase. Immunohistochemical and immunogold staining demonstrated PAI-2 localization in eosinophil-specific granules. Immunoreactive PAI-2 was detected in extracellular deposits in and around the eosinophil-enriched granuloma tissue encapsulating the parasitic egg in livers of wild-type mice infected with the helminthic parasite Schistosoma mansoni. Among the possibilities, we consider a role for eosinophil-derived PAI-2 in inflammation and remodeling associated with parasitic infection as well as allergic airways disease, respiratory virus infection, and host responses to tumors and metastasis in vivo.

Key Words: eosinophils • proteases • protease inhibitor • secretory proteins • serpin


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INTRODUCTION
 
Eosinophils are granule-rich leukocytes present in the peripheral blood and tissues [1 2 3 ]. Much of our understanding of eosinophil effector function is currently in flux, including the long-standing notion that eosinophils provide host defense against helminthic parasites [4 5 6 ] and that they are crucial mediators of allergic airways disease [7 8 ]. Even less well understood are observations relating to eosinophil infiltration into malignant tumor tissue [9 10 ] and in skin transplant rejection [11 12 ] and eosinophil function in relation to respiratory virus infection [13 14 15 ].

Eosinophils synthesize and secrete a variety of proinflammatory mediators, including lipids, cytokines, and cationic granule proteins (reviewed in ref. [1 ]). Historically, major focus has been placed on the unique cationic granule proteins, which in human eosinophils, include major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN). As a group, these proteins function as broad-spectrum cytotoxins in vitro [1 16 ]. More recently, specific receptors for MBP have been identified on human neutrophils, suggesting a basis for intercellular cross-talk as a more subtle function for MBP [17 ]. ECP and EDN are RNase A family RNases, which have provided impetus for the study of their role in antiviral host defense [15 ].

We have recently documented the transcriptional events relating to eosinophilopoiesis in mouse bone marrow via a four-way, subtractive gene microarray approach [18 ]. Among our findings, we detected a sevenfold, interleukin (IL)-5-dependent increase in expression of the transcript encoding plasminogen activator inhibitor 2 (PAI-2), following a profile indistinguishable from those displayed by the other major eosinophil granule protein transcripts in mice. Looking back at previous microarray studies, we find increased expression of PAI-2 transcripts in association with other examples of eosinophilia, including those in lung [19 ] and in liver [20 ].

PAI-2 is a member of the ovalbumin serpin (serine protease inhibitor) family and was first isolated from placental tissue as an inhibitor of the protease urokinase {also known as urinary plasminogen activator (u-PA); [21 22 23 24 ]}. In addition to placental tissue, PAI-2 (47 kDa cytosolic and/or ~60 kDa secretory forms) has been detected in monocytes/macrophages and in fibroblasts and fibroblast derivatives, keratinocytes, and endothelial cells, in the latter, in response to proinflammatory stimuli. u-PA and the related tissue plasminogen activator catalyze the conversion of inactive plasminogen to proteolytically active plasmin, which degrades polymeric fibrin that comprises the extracellular matrix (ECM). Recent work with PAI-2 and pregnancy has focused on its balance with plasminogen activators and their shared role in tissue remodeling [25 ]. Tumor progression and metastasis are other areas in which a role for PAI-2 has been explored [26 27 28 ].

Given the profound and protean roles of plasmin and plasminogen in mammalian biology, PAI-2 gene-deleted mice are intriguing for their apparent lack of phenotype [29 ]. PAI-2–/– mice develop normally in utero and thereafter, have normal reproductive capacity, exhibit normal monocyte recruitment and wound healing, and have normal responses to endotoxin and bacterial challenge.

In this work, we build on our findings from gene array experiments and demonstrate that human eosinophils contain immunoreactive, biologically active PAI-2 in greater concentration per cell than resting peripheral blood monocytes. The presence of PAI-2 in eosinophils prompts an interesting discussion regarding the role of this protease inhibitor in the various states characterized by eosinophilic inflammation and tissue remodeling in vivo.


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MATERIALS AND METHODS
 
Cells, cell culture, and mice
The AML.14 3D10 cell line was maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS; Cambrex, East Rutherford, NJ), 2 mM L-glutamine (Invitrogen), and 50 µg/mL penicillin and streptomycin (Invitrogen). Venous blood was collected from healthy human volunteers as per protocol in heparinized tubes and diluted 1:1 with Hanks’ balanced saline solution. Peripheral blood mononuclear cells (PBMCs) and granulocytes were separated by Ficoll-Hypaque gradient centrifugation (lymphocyte separation medium, Mediatech, Herndon, VA). Granulocytes were further processed by erythrocyte lysis (associated tyrosine kinase lysing buffer, Cambrex Bio Science, Walkersville, MD), and eosinophils were isolated by magnetic cell sorter CD16 microbead-negative selection (Miltenyi Biotec, Auburn, CA) as per standard methodology. CD16-positive neutrophils were obtained by flushing the column after removal from the magnetic field. The cells were washed and resuspended in buffer, and purity of all eosinophil samples was ≥95% with PBMCs as the primary contaminant. Peripheral blood monocytes were isolated using the monocyte isolation kit II (Miltenyi Biotec) as per the manufacturer’s instructions. The neutrophil and monocyte purities were ≥98%, except for one monocyte sample with a purity of 92%. Liver sections were from wild-type C57Black/6 and IL-5–/– mice (C57Black/6 background) infected with cercariae of Schistosoma mansoni as described [18 20 ], as per National Institutes of Health (NIH; Bethesda, MD) ASP LPD-16E.

Cell lysates
Isolated leukocytes (0.2–3.1x106 cells) or AML3D10 cells (106 cells) were resuspended in 0.25–1.0 mL buffer [phosphate-buffered saline (PBS) with 0.5% bovine serum albumin (BSA) and 1 mM EDTA] and were flash-frozen at –80°C. After thawing, the mixture was sonicated briefly and subjected to brief centrifugation (13,000 g for 5 min) at 4°C. The supernatants were divided into aliquots and stored at –80°C.

Quantitative analysis of bone marrow transcripts
Total RNA was extracted from bone marrow of wild-type and IL-5–/– gene-deleted mice—uninfected and 8 weeks after percutaneous exposure to cercariae of S. mansoni. Three µL cDNA used in the original gene array analysis [18 ] were subjected to quantitative polymerase chain reaction (Q-PCR) using the SYBR green dye (ABI, Foster City, CA) in an Applied Biosystems 7700 PRISM instrument as per the manufacturer’s instructions. Primer sequences were selected as intron-spanning with sequences as follows: ß-actin: 5'-CTCCTTAATGTCACGCACGATTTC-3', 5'-TGGTACGACCAGAGGCATACAG-3', 180 bp; PAI-2: 5'-AACAAAGGTGAAATCCCAAACCTG-3', 5'-CATCATCTGGACAGGTATGCTCTC-3', 177 bp. Tenfold dilutions of plasmids with each of the cloned PCR products (vector PCR 2.1, Invitrogen) were used in duplicate, in amounts ranging from 1 x 106 to 1 x 103 copies of the gene to provide a standard curve. The PCR profile used was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Visual analysis of the Q-PCR products on polyacrylamide gel showed a single band of expected size for each transcript, and Q-PCR reactions using reverse transcriptase (RT)-negative controls showed no band (data not shown). Quantitative results were evaluated based on the standard curve using Sequence Detector version 1.7 (ABI). Ratios of PAI-2 to ß-actin were calculated in Excel (Microsoft).

Western blotting
Eosinophil extracts were prepared using ice-cold 1% Triton X-100 in 50 mM Tris, pH 8.0. An equal volume of Tris-glycine-sodium dodecyl sulfate (SDS) sample buffer (Invitrogen) was added to the extract for a final concentration of 5.25 x 107 cell equivalents/mL. AML.14 3D10 extracts were prepared from 8.25 x 107 cell equivalents/mL. Sample (20 µL) was loaded into each lane, and SDS-polycarylamide gel electrophoresis was performed on 14% Tris-glycine gels (Invitrogen). Blots were probed with one of three primary antibodies: murine monoclonal antibody to human PAI-2 (hPAI-2; American Diagnostica, Stamford, CT) at a working concentration of 3 µg/mL, rabbit polyclonal antibody to hPAI-2 (Abraxis, Warminster, PA) at 5 µg/mL, or goat polyclonal antibody (A-19), which detects hPAI-2 and mouse PAI-2 (mPAI-2; Santa Cruz Biotechnology, Santa Cruz, CA), at 2 µg/mL. The secondary antibodies were 1:1000 dilutions of alkaline phosphatase-conjugated goat anti-mouse, goat anti-rabbit immunoglobulin G (IgG), or rabbit anti-goat IgG, respectively (BioRad, Richmond, CA). Blots were developed using 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitroblue tetrazolium (NBT) color development solution (BioRad) for 5–10 min.

Activity assay
A colorimetric assay was used to measure the inhibitory activity of eosinophil-derived PAI-2 for one of its characterized targets, human u-PA [30 ]. A 50-µL vol of high molecular weight u-PA (American Diagnostica) diluted in 0.5% polyethylene glycol (Sigma-Aldrich, St. Louis, MO) at a concentration of 26.25 IU/mL and 50 µL eosinophil lysate or recombinant PAI-2 (2.5 ng, Abraxis) was incubated together for 20 min at 37°C. Following incubation, 350 µL activity assay buffer (50 mM Tris, 34 mM NaCl, pH 9.1) and 50 µL colorimetric substrate Spectrozyme UK (American Diagnostica) were added, and the combination was incubated for 1 h at 37°C. Appropriate controls, including no u-PA and no inhibitor, were performed and included as shown. Samples were evaluated spectrophotometrically at 405 nm. Statistical significance was assessed using the two-tailed t-test.

Immunodepletion
Three 60 µL samples of eosinophil lysate were incubated for 1 h at room temperature with one of the following: 3 µL rabbit polyclonal anti-hPAI-2 antibody + 1.5 µL PBS, 4.5 µL control antibody, or 4.5 µL PBS. After 1 h, 5 µL Protein G agarose (Boehringer Manheim, Indianapolis, IN) was added, and each was incubated at room temperature for an additional hour. Samples were centrifuged at 12,000 gfor 10 min at 4°C to remove immunoprecipitated proteins, and activity was assessed as described earlier.

Degranulation assay
Isolated eosinophils were resuspended in RPMI 1640 (Invitrogen) containing 10% heat-inactivated FBS (Cambrex), 2 mM L-glutamine (Invitrogen), and 50 µg/mL penicillin and streptomycin (Invitrogen) at 8.3 x 106 cells/mL. The 1 mL aliquots were pretreated with 5 µg/mL cytochalasin B (CB; Sigma-Aldrich) for 5 min at 37°C as described [31 ] or an equal volume of control diluent [dimethyl sulfoxide (DMSO)], followed by phorbol myristate acetate (PMA; Sigma-Aldrich) at a concentration of 10 ng/mL or diluent control (DMSO) for 30 min at 37°C. Aliquots were centrifuged, and supernatants were frozen for quantitation by enzyme-linked immunosorbent assay (ELISA).

ELISA
PAI-2 concentrations in cell lysates or degranulation supernatants were measured using a hPAI-2 ELISA kit (Abraxis). Protocols were followed as described in kit instructions: lower detectable limit, 10 pg PAI-2/mL.

Immunohistochemistry
Cytospin preparations of eosinophils were fixed for 10 min in Parafix (1.3 M depolymerized formaldehyde solution, Molecular Histology, Gaithersburg, MD) and were permeabilized in 0.5% Triton X-100 for 10 min. Cells were probed using the Immunopure ABC alkaline phosphatase rabbit IgG staining kit (Pierce, Rockford, IL) with the rabbit polyclonal antibody to hPAI-2 (Abraxis) at a working concentration of 20 µg/mL. Liver sections from uninfected or infected (S. mansoni, t=8 weeks) mice were fixed in 10% formaldehyde in PBS, dehydrated, and embedded in paraffin. After the liver sections were deparaffinized, rehydrated, and permeabilized with 0.5% Triton X-100, they were incubated with 10% normal donkey serum in PBS for 1 h at 37°C. One section was incubated with a 1:100 dilution of the goat polyclonal (A-19) anti-PAI-2 antibody (cross-reacts with mouse PAI-2; Santa Cruz Biotechnology) in PBS containing 0.1% normal donkey serum for 1 h at 37°C, and the other slide received the same incubation and treatment without the primary antibody. After washing threee times in PBS, the sections were incubated in a 1:200 dilution of alkaline phosphatase-conjugated anti-goat IgG (BioRad) in 0.1% donkey serum in PBS for 1 h at 37°C and then washed in PBS. The slides were developed in 1-Step NBT/BCIP (Pierce) for 8 min. Immunogold localization was performed on formalin-fixed eosinophils using the polyclonal rabbit anti-hPAI-2 antibody (Abraxis) and gold-conjugated goat anti-rabbit IgG secondary antibody (Paragon Bioservices, Baltimore, MD).

Relative expression of transcripts encoding PAI-2
Total RNA was extracted from peripheral blood eosinophils and AML.14 3D10 cells (RNAzol, Tel-test, Friendship, TX). RNA (2 µg) was subjected to DNase I treatment (Invitrogen) and reverse-transcribed using a First Strand cDNA synthesis kit for RT-PCR (avian myloblastosis virus; Roche Diagnostics, Indianapolis, IN). cDNA (2–4 µL) was subjected to Taqman (Q-PCR) using custom primers and probe to hPAI-2 (GenBank #NM002575) and glyceraldehyde 3-phosphate dehydrogenase (ABI) using an Applied Biosystems 7700 PRISM instrument per the manufacturer’s instructions. The PAI-2 forward and reverse primer sequences are as follows: 5'-GCAGATCCAGAAGGGTAGTTATCC-3' and 5'-AGAGCGGAAGGATGAATGGATTTT-3'. The Fam reporter probe sequence is 5'-TTTTGCAGGCACAAGCT-3'.

Statistical analysis
Differences between samples or conditions were assessed for significance using two-tailed t-tests and confirmed with the Mann-Whitney U-test for nonparametric data.


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RESULTS
 
Expression of transcripts encoding PAI-2 parallels bone marrow eosinophilia
The full details of the gene four-way subtractive gene microarray experiment are included in our previous publication [18 ]. Briefly, total bone marrow RNA from strain and age-matched wild-type and IL-5 gene-deleted mice—control and mice infected with the helminthic parasite S. mansoni (t=8 weeks)—were subjected to gene microarray, and transcripts were compared for relative expression levels. As anticipated, the expression of transcripts encoding the eosinophil-specific granule proteins mMBP-1 and mMBP-2, mEPO, and mouse eosinophil-associated RNases 1 and 2 (mEar-1 and mEar-2) paralleled the development of bone marrow eosinophilia (Fig. 1A ). Transcripts encoding PAI-2, a member of the serpin ovalbumin family of serine protease inhibitors, also followed this profile. Normalizing the expression levels in lane 1 (bone marrow RNA from IL-5–/– uninfected mice) to 1.0 results in a calculated, approximate sevenfold increase in expression of PAI-2 transcript in bone marrow of wild-type S. mansoni-infected mice. These data were confirmed by Q-RT-PCR, which yielded PAI-2/actin ratios as shown in Figure 1B . Normalization as described for Figure 1A results in a calculated 9.6-fold increase in expression of PAI-2 transcripts in bone marrow from wild-type S. mansoni-infected mice (*, P<0.005).



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  		Figure 1. Gene microarray expression profiles of mouse eosinophil granule proteins. (A) Expression profile of transcripts encoding PAI -2 (overdrawn white line) relative to those of the known eosinophil granule protein genes, including mEPO, mMBP-1 and mMBP-2, and mEar-1 and mEar-2 as per a four-way subtractive gene microarray experiment described in text [18 ]. (B) Confirmation of gene microarray results by Q-PCR determination of relative transcript levels. Ratios of PAI-2 to actin are as shown by solid bars. Fold increases were calculated by normalizing the ratio determined for uninfected IL-5–/– mice to 1.0 and determining relative levels as shown. (A and B) n = 3 mice per condition. *, P < 0.005, compared with all other conditions. WT, Wild-type.

Immunohistochemical and immunogold localization of PAI-2 in human eosinophils
Immunoreactive PAI-2 was detected in isolated human eosinophils using rabbit polyclonal anti-hPAI-2 antiserum and alkaline phosphatase-conjugated secondary antibody staining (Fig. 2A ). No staining was observed in the absence of primary antibody (Fig. 2B) . Staining was detected in the cytoplasm, with a punctate, discrete quality, suggesting granule localization (Fig. 2C) . Subcellular localization within the granule compartment was confirmed by electron microscopy and immunogold staining (Fig. 3A , inset). Nuclear staining is also observed with this method (Fig. 3A) . No gold particles were detected within specific granules or in the nucleus in the absence of primary antibody (Fig. 3B) .



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  		Figure 2. Immunohistochemical staining of human eosinophils. (A) Detection of PAI-2 in human eosinophils with primary rabbit anti-PAI-2 antibody followed by alkaline phosphatase-conjugated goat anti-rabbit IgG and developing reagents; original magnification, 40x. (B) No staining was detected in the absence of primary antibody; same magnification as above. (C) Area within box in A magnified to highlight granular staining pattern. Images shown are representative of ~100 eosinophils evaluated for each staining procedure.



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  		Figure 3. Immunogold localization of PAI-2 in a human eosinophil. (A) Labeling performed with rabbit polyclonal anti-hPAI-2 followed by gold-conjugated goat anti-rabbit IgG demonstrating subcellular localization in the nucleus (N), as well as in the electron dense-specific granules (inset, see arrows). Original magnification, 3 x 104; inset, 7 x 104. (B) Negative control (original magnification, 3x104); no staining detected in the absence of primary antibody (inset, 5x104). Images shown are representative of five eosinophils evaluated for each staining procedure.

Characterization of eosinophil and eosinophil cell line-derived PAI-2
Two forms (47 kDa and ~60 kDa) of PAI-2 protein were detected in extracts derived from human eosinophils using two different antibodies (Fig. 4 ). Using a murine monoclonal anti-hPAI-2 antibody, immunoreactive bands at 47 kDa were detected. The rabbit polyclonal anti-hPAI-2 antibody detected the 47-kDa and the 60-kDa forms of PAI-2 in the identical sample. The 47-kDa form was detected in the eosinophilic AML.14 3D10 cell line [32 ] probed with the murine monoclonal anti-hPAI-2 antibody. Although mRNA transcripts encoding PAI-2 were readily detectable in isolated peripheral blood eosinophils and AML.14 3D10 cells by Q-RT-PCR methods, there was no discernible change in PAI-2 expression in response to cytokines (IL-5) or cytokines and biochemical modulators (butryric acid), respectively (data not shown).



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  		Figure 4. Western blot analysis of human eosinophil extracts (8x105 cell equivalents/lane) probed with mouse monoclonal anti-PAI-2 (lane 1) and rabbit polyclonal anti-PAI-2 (lane 2) in which the 47-kDa and/or the ~60-kDa secretory forms of PAI-2 can be detected, respectively. In lane 3, extract of cells of the human eosinophil AML.14 3D10 cell line probed with the mouse monoclonal anti-PAI-2, in which the 47-kDa form of PAI-2 is detected. Blots are representative of n = 2 replicates of each sample.

PAI-2 content in eosinophils and other hematopoietic cell lysates
The PAI-2 levels of isolated peripheral blood eosinophils, neutrophils, and monocytes were measured with whole cell lysates using a highly specific sandwich ELISA (Fig. 5 ). Eosinophils contain 182 ± 44 ng/106 cells (mean±SEM; range, 30–444 ng/106 cells; n=10 samples); monocytes, 39 ± 7 ng/106 cells (range, 24–56 ng/106 cells; n=4 samples); neutrophils, 0.3 ± 0.2 ng/106 cells (range, 0.01–0.7 ng/106 cells; n=3 samples). We also tested a lysate of the AML.14 3D10 cell line, which contained 18.0 ± 4.9 ng PAI-2/106 cells. Although monocytes are a significant, physiologic source of PAI-2 [33 ], our data indicate that resting monocytes contain only 39 ± 7 ng/106 cells and that eosinophils on average contain significantly more of this protease inhibitor on a per-cell basis (*P<0.02).



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  		Figure 5. Concentration of PAI-2 in lysates from leukocyte populations. Eosinophils (n=10), monocytes (n=4), and neutrophils (n=3). Horizontal bars denote sample means ± SEM as shown.

Degranulation of PAI-2 from stimulated eosinophils
PAI-2 levels were measured in supernatants from eosinophils induced to degranulate with CB/PMA using the sandwich ELISA. Treatment of isolated eosinophils in buffer (PBS with 0.5% BSA and 1 mM EDTA) with CB/PMA (see Materials and Methods) resulted in 33 ± 0.22% of the total available PAI-2; diluent alone (DMSO), 26 ± 0.55% (*P<0.01, n=6)

Eosinophil-derived PAI-2 is biologically active
The u-PA activity after incubation with eosinophil lysates decreases by 40%, similar to that observed after incubation with recombinant PAI-2 (Fig. 6A ). Urokinase is a known target for PAI-2, and these data demonstrate that the human eosinophil lysates contain significant u-PA inhibitory activity when compared with no lysate control (*P<0.01). After immunodepletion, there was no longer any inhibition of u-PA activity (Fig. 6B) . Inhibitory activity in the lysate sample treated with an irrelevant antibody control remained unaffected. This confirms that the inhibitory activity of the eosinophil lysate was solely a result of biologically active PAI-2.



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  		Figure 6. Biological activity of PAI-2 in lysates of human eosinophils. (A) Lysates from human eosinophils (lane 2, 50 µl , ~105 cell equivalents, combined data from six lysates, each performed in triplicate) inhibit urokinase activation by uPA, analogous to that mediated by recombinant PAI-2 (rPAI-2; 2.5 ng into the reaction, lane 3). Statistically significant differences from the no-lysate control (lane 1, n=1, performed in triplicate). *, P < 0.01. (B). Inhibition of urokinase activity is abolished by immunodepletion of eosinophil lysate with rabbit polyclonal anti-PAI-2 (lane 4, n=1, performed in triplicate) but not with an irrelevant antibody control (ctrl; lane 3, n=1, performed in triplicate). Statistically significant differences from the no-antibody or anti-PAI-2 (lanes 1 and 4, respectively). **, P < 0.005.

Extracellular deposition of PAI-2 in association with eosinophilic inflammation
Shown in Figure 7 are sections from S. mansoni-infected mouse liver stained with hematoxylin and eosin (Fig. 7A) and immunostained without and with a cross-reacting goat polyclonal anti-PAI-2 antibody (Fig. 7B band 7C , respectively). PAI-2 can be detected in the inflammatory cells in the granuloma and in the tissues immediately surrounding and including the helminth egg (Fig. 7C , arrowheads). No significant staining for PAI-2 could be detected in healthy liver tissue (Fig. 7D) . Western blots of human eosinophil extract and ovalbumin-sensitized and challenged eosinophil-enriched mouse lung homogenate probed with the goat polyclonal anti-PAI-2 antibody permit detection of the 47-kDa and/or ~60-kDa immunoreactive bands to the forms of PAI-2 documenting antibody specificity (Fig. 7E) .



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  		Figure 7. Immunohistochemical staining of PAI-2 deposition at sites of tissue eosinophilia. (A) Hematoxylin and eosin-stained section from liver of infected wild-type mouse showing a S. mansoni egg, surrounded by a granuloma with inflammatory infiltration. (B) No primary antibody control. (C) Primary antibody, goat polyclonal anti PAI-2, with prominent staining of PAI-2 in inflammatory cells in granuloma, in tissue immediately surrounding the schistosome egg, and on egg itself. (D) Liver from uninfected wild-type mouse, with primary antibody, goat polyclonal anti PAI-2, demonstrating no specific detection of PAI-2. Original magnifications, 40x; representative of at least five sections evaluated for each condition. (E) Western blot of eosinophil extract probed with goat polyclonal anti PAI-2 (n=1). Immunoreactive bands of 47 kDa intracellular and ~60 kDa secretory forms of PAI-2 can be detected.


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DISCUSSION
 
In this study, we have identified PAI-2 as a novel constituent of human eosinophils, present at an average concentration of 182 ng/106 cells. We observed significant variation among the 10 normal blood donors tested, with PAI-2 concentrations in eosinophil lysates ranging from 30 to 444 ng/106 cells. Among the possibilities, this may represent normal variation within the blood donor population, skew based on unreported mild allergy or respiratory symptoms, or related and/or unrelated variations in expression of the 47-kDa and ~60-kDa forms, their individual localizations, and accessibility to lysate preparation. The relatively limited range observed for the different monocyte lysates prepared under identical conditions indicates that the variation is a result of true heterogeneity among the eosinophil donors and is not a factor relating to the assay materials. Given that the major eosinophil granule proteins—MBP, EPO, EDN, and ECP—are present in eosinophils at concentrations ranging from 3 to 5 µg/106 cells, we believe that it is fair and reasonable to state that PAI-2 is a novel and significant constituent of human eosinophilic leukocytes.

Several recent reports have explored constituent proteases and proteolytic activities in conjunction with eosinophilic inflammation, specifically the roles played by matrix metalloproteinases [34 35 36 37 ] and protease-activated receptors [38 39 ] in the pathophysiology of allergic airways disease. Although the literature on plasmin–plasminogen activators and eosinophils is not large, there are some intriguing reports suggesting the existence of a plasminogen activator and PAI [40 41 42 43 44 ]. However, this is the first report documenting significant quantities of the specific u-PA inhibitor, PAI-2, in eosinophils, providing impetus for consideration of the role of this protein and by extension, eosinophils in plasminogen–plasmin-mediated tissue destruction and remodeling.

Given several earlier explorations into the role of PAI-2 and neoplasia [9 10 ], it might be interesting to revisit the question of eosinophil infiltration and malignancy from this perspective. Tissue eosinophilia appears infrequently in association with solid tumors, yet several studies have looked into its statistical significance and confirmed that the presence of eosinophils correlates with a good prognosis [45 ] and in some cases, a decreased likelihood of metastasis [46 ]. Elevated levels of PAI-2 found in and around some tumors were also a good prognostic indicator [47 ], believed to be related to its inhibitory activity on u-PA, as inhibition of protease-mediated breakdown of the ECM was believed to inhibit cancer cell migration [48 49 50 ]. However, recent research on the related inhibitor PAI-1 suggests that this model is likely to be overly simplistic [51 52 ].

Although helminth infection in mammals triggers eosinophilia in the surrounding tissues, there are aspects of the role(s) played by eosinophils that remain unclear [4 5 6 ]. In Figure 7 , we demonstrate immunoreactive PAI-2 in eosinophils and in the tissue adjacent to and on the encapsulated egg. Our work suggests a mechanism by which eosinophils may be involved in the deposition of fibrous tissue, thus promoting and sustaining the characteristic granuloma formation, perhaps serving to limit the spread of egg antigens that elicit inflammation. This mechanism may be explored further in eosinophil-deficient and/or PAI-2-deficient mice.

Tissue remodeling occurs in the lungs of asthmatic individuals, but many of the mechanisms underlying this pathogenic process remain unexplored [53 54 ]. By inhibiting u-PA and blocking the activation of plasmin, PAI-2 localized in eosinophils may contribute to this process by promoting deposition of fibrin and fibrous tissue. In support of this concept, Flood-Page et al. [55 ] and Cho et al. [56 ] demonstrated that eosinophils participate in transforming growth factor-ß-dependent deposition of ECM components. It is intriguing to consider a role for eosinophil-derived PAI-2 in this process. It is interesting that Oh and colleagues [57 58 ] have presented a role for the related protease inhibitor, PAI-1, in mast cells and mast cell-mediated airway remodeling events. The presence of PAIs in two of the major inflammatory cells involved in tissue remodeling invokes issues relating to the beneficial aspects of redundancy of function. Eosinophil-derived PAI-2 may prove to be integral to the tissue-remodeling process and may ultimately serve to explain the role of eosinophils in this and other fibrosis-related [59 ], pathophysiologic states.

Another setting where eosinophils are associated with tissue remodeling is chronic skin allograft rejection. Le Moine and colleagues [12 ] noted that eosinophil infiltration into transplanted tissue is not observed in IL-5–/– mice, and these mice likewise do not develop dermal fibrosis and tissue rejection. It is intriguing to consider the possibility that PAI-2 from the degranulating eosinophils alters the balance in the plasmin/fibrin system, promoting fibrosis and rejection in wild-type mice.

A final thought on PAI-2 function and eosinophils relates to respiratory virus infection. Eosinophil recruitment to the airways has been observed in response to infection with respiratory syncytial virus [60 ], and a role in host defense against this virus has been suggested [15 ]. It is interesting that parallel studies have also considered an antiviral role for PAI-2 through down-regulation of intercellular adhesion molecule 1, a receptor for picornaviruses [61 ], and by inducing expression of antiviral interferons {alpha} and ß in transfected HeLa cells [62 ]. At this point, these studies remain intriguing but difficult to connect to one another.

In summary, PAI-2 as an eosinophil protein was inferred from gene microarray study of mouse eosinophilopoiesis and has emerged as a major protein component of human eosinophilic leukocytes. A significant portion of immunoreactive PAI-2 is located in the eosinophil-specific granules. Although 47 kDa and 60 kDa forms of PAI-2 are present in human eosinophils, at present, we do not have the tools necessary to discern their individual localizations. PAI-2 is isolated in the biologically active form and is the only inhibitor of u-PA detected in eosinophil lysates. Immunoreactive PAI-2 was also detected in mouse tissue eosinophils and deposited extracellularly near the schistosome egg. Future studies with PAI-2–/– mice alone and double gene deletions with various eosinophil-deficient strains of mice will help us to discern the contributions of eosinophil-derived PAI-2 to eosinophil-mediated inflammation and tissue remodeling.


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ACKNOWLEDGEMENTS
 
We are grateful for the helpful input of Dr. Darren Saunders (Garvan Institute of Medical Research, Darlinghurst, Australia) for his assistance with the urokinase activity assays. We also thank Tara Garvey (Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH) and Susanna Navarro (Universitat Autonoma de Barcelona) for their assistance in the completion of this project. We also thank Howard Adams and the staff of the 14BS animal facility for the care of the mice used in this study.

Received March 22, 2004; revised June 23, 2004; accepted July 5, 2004.


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