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Soluble Fas Ligand Is Chemotactic for Human Neutrophilic Polymorphonuclear Leukocytes¹

Department of Internal Medicine, University of Genova Medical School, Genova, Italy

	Abstract

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It has been recently shown that Fas ligand (FasL) expression on islet ß grafts results in neutrophilic infiltration and graft rejection. In this study, we show that human recombinant soluble FasL is endowed with potent chemotactic properties toward human neutrophilic polymorphonuclear leukocytes (neutrophils) at concentrations incapable of inducing cell apoptosis. Furthermore, neutrophils exposed to soluble FasL did not display detectable change of intracellular Ca²⁺ and did not undergo superoxide production or exocytosis of primary and secondary granules. Our results show that FasL is a potent chemoattractant for human neutrophils without evoking their secretory responses. This finding suggests a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.

	Introduction

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Fas ligand (FasL)³ is a 40-kDa type II integral membrane protein belonging to the TNF/nerve growth factor family including TNF, nerve growth factor, lymphotoxin, CD40L, CD30L, CD27L, and TNF-related apoptosis-inducing ligand (TRAIL) 1, 2 . Membrane-bound FasL can be proteolytically cleaved by metalloproteinases generating a soluble (sFasL) and active form of the ligand FasL 3 . The specific receptor for FasL is Fas (CD95, Apo-1), a 45-kDa type I transmembrane protein that is involved in the signaling pathway of apoptosis in a variety of cell types 2, 4 . The Fas/FasL system plays a crucial role in physiological and pathological conditions ranging from the normal tissue homeostasis to tumor escape from the immune surveillance 5 . In particular, it was suggested that FasL expression by cells from the testis and eye may be involved in the maintenance of immune privilege of these tissues by inducing apoptosis of Fas-positive immune effectors 6, 7 . These observations raised the hypothesis that engineered FasL expression may protect allograft from rejection by the immune system of the recipient without inducing host general immunodepression. Indeed, the transplantation of carrier cells expressing FasL was found capable to protect cotransplanted pancreatic islet allograft from rejection 8 . Nevertheless, and quite surprisingly, two more recent reports showed that the transgenic expression of FasL fails to protect grafted islet ß cells from rejection 9, 10 . The islet rejection was T and B cell independent but mediated by locally recruited neutrophilic polymorphonuclear leukocytes (neutrophils) 9, 10 . Furthermore, in a different experimental model, Seino and coworkers 11 reported that locally produced FasL causes the neutrophil-mediated rejection of Fas-negative tumor cells. These data suggest that FasL expressed by transplanted cells may directly or indirectly evoke a neutrophilic inflammatory host response against the graft, but the molecular mechanism governing neutrophil recruitment by FasL-transfected cells is still not elucidated. It has been suggested that FasL could act directly on neutrophils inducing their recruitment 11 , but it is possible that locally produced FasL acts on surrounding stromal cells to induce the production of IL-8 or other chemoattractants active on neutrophils. In this regard, it has been shown that FasL induces the expression of IL-8 in synoviocytes and in a colon carcinoma cell line 12, 13 . The present results support the hypothesis that sFasL is endowed with intrinsic chemoattractant properties toward neutrophils, suggesting for the ligand a proinflammatory function besides the well known proapoptotic activity.

	Materials and Methods

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Materials

HBSS without phenol red (ICN Biomed, Milan, Italy) mixed with Dulbecco’s PBS (ICN Biomed) (HBSS:PBS = 3:1) containing 1 mg/ml BSA (Sigma, Milan, Italy) was used as incubation medium, unless otherwise stated. sFasL was from Alexis (San Diego, CA). Anti-human Fas mAb ZB4, Biomolecular Probes, was from Società Italiana Chimici (Rome, Italy). Rabbit anti-human lactoferrin IgG fraction and peroxidase-conjugated rabbit anti-human lactoferrin IgG fraction were from United States Biochemical (Cleveland, OH). FMLP, cytochalasin B, human lactoferrin, O-dianisidine, ferrycytochrome c (type VI, horse heart), superoxide dismutase (SOD; type I, bovine blood), N-ethyl-maleimide, propidium iodide, ethidium bromide, fluorescein diacetate, and ß-nicotinamide adenine dinucleotide-reduced form were from Sigma. Annexin V-FITC Kit was purchased from Boehringer Ingelheim (Heidelberg, Germany).

Neutrophilic polymorphonuclear leukocyte preparation

Heparinized venous blood (10 U/ml heparin) was obtained from healthy volunteers (20- to 37-yr-old) after informed consent. Neutrophilic polymorphonuclear leukocytes (neutrophils) were prepared by dextran sedimentation, followed by centrifugation (400 x g, 30 min) on a Ficoll-Hypaque density gradient, as previously described 14 . Contaminating erythrocytes were removed by hypotonic lysis 14 . Neutrophils resuspended in incubation medium were >97% pure, as determined by morphologic analysis of Giemsa-stained cytopreps. Cell viability was determined by ethidium bromide-fluorescein diacetate test 15 and was >98%.

Boyden chamber migration assay

Neutrophil locomotion was studied using the leading front method, as previously described 16 . Tests were conducted in duplicate using blind well chambers (NeuroProbe, Cabin John, MA) with a 3-µm pore size cellulose ester filter (SSWPO 1300, lot no. H7PM21937; Millipore, Milan, Italy) separating the cells (4 x 10⁵) from the chemoattractant. After incubation at 37°C for 45 min, the filters were removed, fixed in ethanol, stained with Harris haematoxylin, dehydrated, cleared with xylene, and mounted in Eukitt (Kindler, GmbH, Freiburg, Germany). Duplicate chambers were run in each case and the distance (µm) travelled by the leading front of cells was measured at x400 magnification. Five randomly chosen fields were read for each filter. In some experiments, neutrophils were preincubated with the 50-µg/ml anti-Fas mAb ZB4 or an isotype-matched mAb of irrelevant specificity for 30 min at 4°C and washed. Thereafter, the migration assays were conducted as described above.

Collagen invasion assay

Type I collagen solution was from Sigma (catalogue C4580, lot 126H4667). Gels were prepared according to Sigma protocol. Briefly, 800 µl collagen solution were mixed with 100 µl 10x Earle’s buffered saline and 100 µl reconstitution buffer (2.2% sodium bicarbonate in 0.8 N sodium hydroxide) to restore the collagen solution to physiological pH and osmolarity. Thereafter, collagen solution (final gel concentration, 0.88 mg/ml) was allowed to gel in 24-well plates in the absence or presence of the chemoattractants: 0.1 nM sFasL or 10 nM FMLP in duplicate. After gelification, cells (8 x 10⁵/well) were overlaid on the gel surface and incubated at 37°C for 90 min. At the end of incubation, gels were fixed for 30 min with 2.5% glutaraldehyde and cell migration was measured as the distance between the top of the gel and the plane in which the two faster cells invading the gel were in focus (x100 original magnification) 16 .

Checkerboard analysis

Assays of cell migration with different doses of FasL (0, 0.03, and 0.1 nM) both in the upper and bottom chamber were also performed. The results of these experiments were collected in checkerboard form by which chemokinesis (i.e., change in the extent of random locomotion) and chemotaxis (i.e., change in the directional response to the stimulus) were calculated according to Zigmond and Hirsch 17 , as previously described 16 . In particular, this method allows the calculation of distance that the leading front of neutrophils, moving in a chemoattractant gradient, is expected to cover if the only variable would be the rate of random locomotion 17 . This calculation assumes that the cells are moving randomly in the gradient without sensing the source of the stimulus 17 . Thus, the hypothetical chemoattractant exerts a true chemotactic influence on cells moving in a positive gradient when the experimental values are greater than the calculated ones 17 .

Lactoferrin release assay

The release of lactoferrin by neutrophils was measured by a modification of the ELISA described by Metcalf et al. 18 , using 10⁶ neutrophils preincubated (5 min, 37°C) with 5 µg/ml cytochalasin B and incubated with appropriate doses of sFasL or FMLP (15 min, 37°C, 0.4 ml final volume ). Then, microtiter plates (Falcon, Becton Dickinson, Oxnard, CA), coated with 1 µg/ml of rabbit anti-human lactoferrin IgG, were incubated (90 min, 37°C) with appropriate dilutions of supernatants or purified human lactoferrin. After washing with PBS/Tween 20, peroxidase-conjugated rabbit anti-human IgG were added to each well in appropriate concentrations. After incubation (90 min, 37°C), the plates were washed with PBS/Tween, the peroxidase-substrate solution was added to each well, and the reaction was read at 490 nm on a microtiter plate reader (Titertek TwinReader Plus; Flow Laboratories, McLean, VA). The amounts of lactoferrin in supernatants were calculated on the basis of lactoferrin standards determined in parallel assay. The results were expressed as the number of micrograms of lactoferrin released by 10⁶ neutrophils.

Myeloperoxidase release assay

The myeloperoxidase release by neutrophils was assessed as previously described 19 . Neutrophils were incubated in the same conditions of lactoferrin release assay. The myeloperoxidase activity in supernatants was determined by using 0.167 mg/ml O-dianisidine and 0.1 mmol/l hydrogen peroxide in 50 mmol/l phosphate buffer (pH 6.0). One unit of enzyme activity was defined as that oxidizing 1 µmol of O-dianisidine/min/25°C (OD₅₅₀, extinction coefficient 11.3 mM^-1 cm^-1).

Superoxide anion release assay

The release of superoxide anion was studied by using a modification of the method of Babior et al. 20 . as previously described 19 . Briefly, neutrophils (5 x 10⁵) were incubated (20 min, 37°C, 0.5 ml final volume) with 80 µM ferricytochrome c in the absence or presence of 300 U/ml superoxide dismutase. The reactions were then stopped by adding 2 ml of ice-cold 1 mM N-ethyl-maleimide and the superoxide production was determined in the supernatants from the OD₅₅₀ of samples without SOD minus OD₅₅₀ of samples with SOD using a extinction coefficient of 2.1 x 10^-4 M^-1 cm^-1.

Intracellular Ca²⁺ ([Ca²⁺]_i) determination

Neutrophils (2.5 x 10⁶) were loaded with 2 µM fura-2 AM in 10 mM HBSS-HEPES (pH 7.4) for 30 min at 37°C with a final 0.5 ml. volume Then, cell suspension was diluted 10-fold with HBSS-HEPES, incubated for 30 min at 37°C, washed twice, and resuspended in HBSS-HEPES. Fluorescence changes before and after the addition of FasL or FMLP were monitored with Perkin-Elmer LS3 spectrofluorometer at an excitation wavelength of 338 nm and an emission wavelength of 510 nm.

Assessment of neutrophil survival

Neutrophils (2 x 10⁶/ml) were incubated for 12 h in tissue culture tubes (17 x 100 mm; Falcon, Becton Dickinson) in medium supplemented with 10% autologous serum at 37°C in a 5% CO₂ atmosphere (0.5 ml final volume). Cell viability measured as integrity of membrane was assessed by ethidium bromide-fluorescein diacetate test according to Dankberg and Perdinsky 15 as previously described 22 . Briefly, cells (4 x 10⁴/100 µl) harvested from culture tubes were mixed with 50 µl of staining solution (2 µg/ml fluorescein diacetate, 4 µg/ml ethidium bromide in HBSS and incubated for 10 min at room temperature. Thereafter, a drop of cell suspension was placed on a slide, sealed with a coverslip, and analyzed under UV light in a dark field illumination. Neutrophils with intact membrane (i.e., viable cells) appeared as green fluorescent cells, whereas neutrophils with damaged and ethidium bromide-permeable membrane (i.e., necrotic cells) displayed a fluorescent red nucleus. Neutrophil viability was also assessed by the determination of lactate dehydrogenase activity in neutrophil culture supernatants according to Beutler 21 . Cytocentrifuged cell preparations were fixed and stained with May-Grünwald-Giemsa. Thereafter, cytopreps were read blindly by two independent observers by oil immersion light microscopic examination of at least 500 cells/slide (x1000 magnifications). Cells showing apoptotic morphology were identified according to the typical criteria, as previously described 22 . Immunofluorescence analysis of Annexin V binding was performed following the manufacturer’s instruction with minor changes. Briefly, cells were washed and resuspended in 100 µl isotonic binding buffer. Then, 3 µl Annexin V-FITC was added, and after a 15-min incubation, cells were washed and resuspended in ice-cold PBS supplemented with 3% FCS and 0.1% sodium azide. Flow cytometry analysis was performed on an EPICS XL flow cytometer (Coulter, Hialeah, FL).

Statistical analysis

Data were expressed as mean ± SEM. Differences were determined by the nonparametric Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. Differences were accepted as significant when p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neutrophil locomotion and sFasL

As shown in Fig. 1, sFasL induced a significant locomotory response in normal human neutrophils showing the bell-shaped dose-response curve characteristic of chemoattractants. The magnitude of neutrophil migration induced by 0.1 nM sFasL was similar to that generated by the classic neutrophilic chemotactic factor FMLP at 10 nM both in the classical Boyden chamber assay with nitrocellulose filter (Fig. 2, open bars) and in the collagen gel invasion assay (Fig. 2, filled bars). Finally, neutrophil pretreatment with the antagonistic anti-Fas mAb ZB4, but not with an isotype-matched control mAb, inhibited the sFasL capacity of inducing neutrophil locomotion (Fig. 3). These data suggest that sFasL is indeed capable of inducing neutrophil migration by a mechanism specifically dependent on its interaction with Fas.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Locomotory response of human neutrophils to recombinant human sFasL. After a 45-min incubation, net migration was determined after subtracting spontaneous migration, i.e., the distance travelled by neutrophils in the absence of sFasL from the distance travelled by neutrophils toward the ligand. Data are expressed as mean ± 1 SEM (n = 4). Migration in the presence of 0.1 nM sFasL vs control (i.e., migration in absence of sFasL): p < 0.01. Migration in the presence of 1 nM sFasL vs control (i.e., migration in absence of sFasL): p < 0.05. Kruskal-Wallis nonparametric ANOVA test, followed by Dunn’s Multiple Comparison test.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Locomotory responses of human neutrophils in the collagen gel invasion assay and Boyden chamber assay. Neutrophils were incubated in the absence or presence of 0.1 nM recombinant human sFasL or 10 nM FMLP for 90 min (collagen gel invasion assay, filled bars) or 45 min (Boyden chamber assay, open bars). Data are expressed as mean ± 1 SEM, n = 5 (filled bars), and n = 9 (open bars). Migration in the presence sFasL vs control (i.e., migration in absence of sFasL): p < 0.05 (filled bars) and p < 0.01 (open bars). Migration in the presence FMLP vs control (i.e., migration in the absence of FMLP): p < 0.05 (black bars) and p < 0.001 (open bars). Kruskal-Wallis nonparametric ANOVA test, followed by Dunn’s Multiple Comparison test.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Effect of anti-Fas mAb ZB4 on the neutrophil locomotory response to recombinant human sFasL. Neutrophils were pretreated with 50 µg/ml anti-Fas mAb ZB4 or an irrelevant control mAb and subsequently exposed to 0.1 nM sFasL. Data are expressed as mean ± 1 SEM (n = 3). Migration of neutrophil pretreated with ZB4 mAb in presence of sFasL vs control (i.e., migration in the absence of sFasL): p > 0.05. Migration of neutrophil pretreated with the irrelevant control mAb in the presence of sFasL vs control (i.e., migration in the absence of sFasL): p < 0.05. Kruskal-Wallis nonparametric ANOVA test, followed by Dunn’s Multiple Comparison test.

Cell migration in response to a soluble compound can be related to two different locomotory mechanisms. The first one, defined as chemokinesis, is the reaction by which the extent of cell random locomotion is enhanced by a compound in the environment. On the other hand, the directional locomotion along the gradient of the solute in the environment is called chemotaxis 23 . In other words, only chemotaxis is characterized by the directional cell locomotion toward the source of the stimulus and is commonly associated with the recruitment of neutrophils at sites of inflammation 24 . To investigate if the sFasL-dependent neutrophil locomotion can be attributed to a sFasL-induced true chemotaxis, experiments were conducted using the classical checkerboard analysis. As shown in Table I, sFasL was found to stimulate both the rate of cell locomotion and true chemotaxis as suggested by the following findings: 1) cell migration augmented when the absolute concentration of sFasL was increased in absence of a concentration gradient (values along the diagonal from the upper left to the lower right); and 2) neutrophil migration along the gradients (values along the line above the diagonal) was greater than the calculated distance migrated if the cell would have moved randomly in the gradient (values in parentheses).

We have investigated the possibility that neutrophil/sFasL interaction can lead to full cell activation evaluated as superoxide anion production and degranulation. As shown in Table II, sFasL did not elicit azurophilic granule (myeloperoxidase) and specific granule (lactoferrin) exocytosis and did not trigger the neutrophil respiratory burst even at 100-fold concentration used in the migratory assays. It is noteworthy that after a 12-h incubation sFasL did not affect neutrophil survival evaluated as cell viability, lactate dehydrogenase activity in cell culture supernatants, microscopic evaluation of the percentage of neutrophils displaying the typical morphological features of apoptosis (Table III), and flow cytometer analysis of Annexin V-FITC binding (Fig. 4).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Flow cytometer analysis of binding of Annexin V-FITC. Neutrophils were incubated in the absence or presence of 0.1 nM recombinant human sFasL for 12 h. After a 15-min incubation with Annexin V-FITC, flow cytometer analysis was conducted. A representative experiment of the two performed is shown.

Classical chemotactic factors elicit a rapid and transient elevation of [Ca²⁺]_i in neutrophils 25 . Therefore, we monitored [Ca²⁺]_i in Fura-2 loaded cells after triggering with the chemotactic peptide FMLP or sFasL. As shown in Fig. 5, FMLP induced a rapid increase in [Ca²⁺]_i, whereas no [Ca²⁺]_i mobilization was observed in sFasL-stimulated neutrophils. No [Ca²⁺]_i increment was observed increasing the concentrations of sFasL, or prolonging the exposure of neutrophils to the ligand (data not shown).

View larger version (10K): [in this window] [in a new window]   FIGURE 5. Effect of recombinant human sFasL or 10 nM FMLP on intracellular [Ca2+] levels in human neutrophils. Neutrophils (2.5 x 105) were loaded with Fura-2 AM and fluorescence changes (DF) were monitored before and after exposure to 0.1 nM sFasL or 10 nM FMLP. The downward arrow indicates the stimulus addition. Results represent one of three experiments that yielded similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The molecular mechanisms governing sFasL-dependent chemotactic activity are presently unknown. In their study, Seino et al. 28 suggest that the signaling components recruited by sFasL for its chemotactic properties seems to be independent from the known death domain interacting molecules. Here, we show that neutrophils exposed to sFasL did not display detectable [Ca²⁺]_i mobilization. Stimulation of neutrophils by classical chemoattractants such as FMLP, IL-8, and C5a results in a rapid and transient rise in [Ca²⁺]_i, and indeed this metabolic response is considered a hallmark of chemoattractant-triggered neutrophil activation 25 . Nevertheless, the role of [Ca²⁺]_i elevation in neutrophil chemotaxis is a matter of debate. In fact, protrusive surface activity, gelsolin-actin complexes, and net actin assembly can occur in the absence of Ca²⁺ transient rise 30 . Furthermore, extracellular and intracellular Ca²⁺ chelators do not block neutrophil migration in response to chemotactic factors 31, 32 . Accordingly, Fabbri et al. 33 have recently shown that chemotaxis, unlike superoxide anion production, do not depend on [Ca²⁺]_i enhancement in human neutrophils. In this regard, Haines et al. 34 observed that Substance P and TGF-ß1 induce neutrophil chemotaxis without increasing [Ca²⁺]_i levels and without activating oxidative metabolism or granule exocytosis. They postulated that chemoattractants can be classified in two functional groups 34 . In particular, they distinguished "classical chemoattractants," i.e., FMLP, C5a, and IL-8, which also evoke secretory responses such as superoxide anion release or lysosomal degranulation, from "pure chemoattractants," which comprise a group of substances, such as Substance P 34, 35 and TGF-ß 29, 34 , as well as fibrinopeptide B 36 , devoid of secretagogue properties. Accordingly, we did not observe a detectable superoxide production or exocytosis of primary and secondary granules in neutrophils exposed to sFasL. Thus, the present results show that sFasL is a potent chemoattractant for human neutrophils but is incapable of evoking their secretory responses. This is a novel observation that helps to better define the recently identified new class of "pure chemoattractants" comprizing sFasL as well as substance P, fibrinopeptide B, and TGF-ß1 34 .

Chemotactic factors from bacteria and cytokines from various cell types generate a network of cellular interactions resulting in neutrophil recruitment at sites of inflammation 37 . Indeed, neutrophils themselves may amplify the inflammatory process by their ability to release different cytokines and chemokines, particularly IL-8 38 , platelet-activating factor, and leukotriene B₄ 39, 40 . The ability of sFasL to induce neutrophil migration, as described in this paper, suggests another paracrine mechanism for neutrophil recruitment at inflammatory sites. In fact, neutrophils, which express constitutively detectable membrane-bound FasL, can release a biologically active 30-kDa form of sFasL, possibly shed by a specific neutrophil-derived metalloproteinase 41 . In other words, this finding may represent a rapid way to locally increase the tissue concentration of chemotactic factors, thereby favoring secondary waves of neutrophil recruitment. In this regard, Hashimoto and coworkers 42 have recently reported that high concentrations of sFasL are detectable in synovial fluids from patients with active rheumatoid arthritis, usually characterized by neutrophilic articular effusion.

In conclusion, our results showing sFasL chemoattractant properties toward human neutrophils suggest a novel proinflammatory function for this ligand and may help to clarify the mechanism governing FasL-mediated graft rejection, thereby offering rational bases for controlling and modulating FasL-based immunotherapies.

	Footnotes

 
¹ This work was supported by Grant "Programma di Ricerca Scientifica di interesse nazionale prot.9706117821_002" (to F.D.).

² Address correspondence and reprint requests to Dr. Luciano Ottonello, Semeiotica Medica 2, Dipartimento di Medicina Interna, Viale Benedetto XV 6, I-16132 Genova, Italy. E-mail address:

³ Abbreviations used in this paper: L, ligand; s, soluble; SOD, superoxide dismutase; [Ca²⁺]_i, intracellular Ca²⁺.

Received for publication September 30, 1998. Accepted for publication December 7, 1998.

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