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Monocytes from Wiskott-Aldrich Patients Display Reduced Chemotaxis and Lack of Cell Polarization in Response to Monocyte Chemoattractant Protein-1 and Formyl-Methionyl-Leucyl-Phenylalanine¹

Raffaele Badolato^2,*,§, Silvano Sozzani^||, Fabio Malacarne^¶, Susanna Bresciani^*, Maurilia Fiorini^*, Alessandro Borsatti^||, Alberto Albertini, Alberto Mantovani, Alberto G. Ugazio^* and Luigi D. Notarangelo^*

^* Clinica Pediatrica, Cattedra di Chimica, and Sezione di Patologia Generale ed Immunologia, Universita’ di Brescia, Brescia, Italy; ^§ Dipartimento di Patologia e Medicina Sperimentale e Clinica, Universita’ di Udine, Udine, Italy; ^¶ Servizio di Immunologia Clinica, Spedali Civili, Brescia, Italy; and ^|| Istituto Ricerche Farmacologiche "Mario Negri," Milan, Italy

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by trombocytopenia, eczema, and progressive decline of the immune function. In addition, lymphocytes and platelets from WAS patients have morphologic abnormalities. Since chemokines may induce morphologic changes and migration of leukocytes, we investigated the monocyte response to chemoattractants in cells from WAS patients with an identified mutation in the WAS protein gene. Here, we report that monocytes derived from four patients with molecularly defined typical WAS have a severely impaired migration in response to FMLP and to the chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 compared with normal donors. Conversely, neither MCP-1 binding to monocytes nor induction of the respiratory burst by MCP-1 and FMLP is significantly different between WAS patients and normal donors. Within a few minutes of stimulation, monocytes respond to chemokines with increased expression of adhesion molecules and with morphologic changes such as cell polarization. Although up-regulation of CD11b/CD18 expression following stimulation with FMLP or MCP-1 is preserved in WAS patients, cell polarization is dramatically decreased. Staining of F-actin by FITC-phalloidin in monocytes stimulated with chemoattractants shows F-actin to have a rounded shape in WAS patients, as opposed to the polymorphic distribution of F-actin in the polarized monocytes from healthy donors. These results suggest that WAS protein is involved in the monocyte response to the chemokines MCP-1 and macrophage inflammatory protein-1.

	Introduction

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Wiskott-Aldrich syndrome (WAS)³ is an X-linked recessive disorder, characterized by severe thrombocytopenia and eczema, associated with increased susceptibility to pyogenic and opportunistic infections (1). The immune abnormalities of WAS include impaired delayed-type hypersensitivity (DTH) to multiple Ags, progressive decline of T cell number and functions, and reduced Ig production in response to polysaccharide Ags (2, 3). The genetic defect responsible for WAS has been unraveled in 1994, when positional cloning allowed identification of the gene mutated in WAS patients (4); this gene encodes for an intracellular protein of about 60 kDa, termed WAS protein (WASp), that is expressed in T lymphocytes, monocytes, platelets, and B cell lines (5, 6). However, the pathophysiology of WAS has remained largely elusive even following cloning of the WASP gene.

Abnormalities of cytoskeleton organization, with paucity of leukocyte microvilli, have been reported as a hallmark of the disease and may contribute to the lymphoid and platelet disturbances (1, 7, 8); recent reports have indicated that WASp binds to several intracellular proteins containing SH3 domains, such as Cdc42 (a ras superfamily member), p47^phox (a cytosolic subunit of NADPH oxidase), and Fyn (a Src family member), that are all involved in the regulation of leukocyte functions and motility (9, 10, 11, 12, 13, 14, 15).

Leukocytes migrate in response to concentration gradients of chemoattractants, such as bacterial products, complement components, and a new class of cytokines recently identified and called chemokines (16, 17). These chemotactic factors induce leukocyte infiltration into inflamed tissues such as inflamed joints of rheumatic patients, granulomatous reactions, or DTH responses (18, 19). Stimulation by chemotactic factors results in changes in leukocyte morphology, cell polarization and adhesion, and induction of respiratory burst and of leukocyte degranulation and thereby activation of leukocyte antibacterial properties (17, 20, 21). In the present report we investigated whether monocyte activation in response to this new class of chemoattractants is affected in WAS patients and how these defects might relate to the biologic role of WASp. A selective defect in chemotaxis, cell polarization, and F-actin distribution was observed in response to all the chemokines tested, whereas other chemokine-induced responses (e.g., activation of the respiratory burst and up-regulation of CD11b/CD18 expression) were not affected.

	Materials and Methods

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Patients

Four patients with molecularly defined WAS were included in this study. Patients were evaluated and treated at the Department of Pediatrics, University of Brescia (Table I). Monocytes constituted 5 to 15% of all leukocytes in the patients studied. As a control, PBMC were obtained, after informed consent was granted, from age-matched subjects that were hospitalized for minor head trauma.

PBMC were purified by Ficoll separation medium (Lympholyte-H, Cedarlane Laboratories, Hormby, Canada) gradient centrifugation as described previously (22). Monocytes constituted 30 to 40% of PBMC as determined by a direct immunofluorescence assay using the mAb CD14 (Dako, Glostrup, Denmark). When indicated, monocytes were purified by Percoll separation medium (Pharmacia Biotech, Uppsala, Sweden) as described previously (23). Cells were cultured in RPMI 1640 (HyQ, HyClone Europe, Cramlignton, U.K.) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l L-glutamine, 20 mmol/l HEPES (Imperial, Andover Hants, U.K.), and 10% heat-inactivated FCS (Boehringer Mannheim, Mannheim, Germany). Human recombinant MCP-1, MIP-1, and RANTES were obtained from Pepro Tech (Rocky Hill, NJ). All reagents and media, tested by The Endotoxin Kit (Sigma, St. Louis, MO), contained endotoxin at levels <12 pg/ml.

Cytokine determination

The MCP-1 concentration was assessed by an ELISA kit purchased from R&D Systems Europe (Abingdon, U.K.), following the manufacturer’s instructions.

Migration assays

Migration of monocytes (1.5 x 10⁶ cells/ml in RPMI 1640 and 1% BSA) was evaluated by a microchamber technique as described previously (24). For monocytes, 5-µm pore size polycarbonate filters (Neuro Probe, Cabin John, MD), were employed. Under the assay conditions employed, only monocyte in PBMC preparations migrated across the filter. At the end of the incubation (90 min), filters were removed, fixed, and stained by Diff-Quick (Harleco, Gibbstown, NJ), and three oil immersion fields were counted in a blind evaluation after coding samples. In each assay, FMLP (Sigma) at a concentration of 10 nM was used as a standard chemoattractant for monocytes.

MCP-1 binding

Biotin-conjugated MCP-1 (R&D Systems, Minneapolis, MN) was used, following manufacturer’s instructions. Briefly, cells (10⁶/100 µl) were washed twice with PBS, resuspended in 50 µl of RPMI 1% BSA, and incubated at 16 to 20°C with 10 µl of biotin-conjugated-MCP-1 (R&D, Minneapolis, MN). After 15 min, 10 µl of avidin-FITC (R&D Systems) was added to each sample, and the incubation was continued at 4°C for 30 min. Cells were then washed with cold medium (RPMI/1% BSA) twice, resuspended in PBS plus 1% paraformaldehyde, and analyzed by a flow cytometer FACScan (Becton Dickinson, San Jose, CA). Binding activity was expressed as the mean channel fluorescence of MCP-1-avidin binding cells (>90% of monocytes were positive for MCP-1 binding).

FACS analysis

Whole blood treated with FMLP was washed and incubated with saturating concentrations of CD11b (Dako) or control mouse-IgG (Dako) for 30 min at 4°C. Cells were washed twice with PBS, resuspended in 100 µl of PBS, and incubated at 4°C for 30 min with 4 µl of FITC-conjugated rabbit anti-mouse IgG (Dako). RBCs were then lysed by incubating the blood with 4 ml of FACS lysing buffer (Becton Dickinson) for 5 min at room temperature. After three washes with PBS, cells were resuspended in PBS plus 1% paraformaldehyde and analyzed with a FACScan (Becton Dickinson). At least 5000 events were acquired, and on the basis of forward and side scatter, the window for monocyte-gated cell was set.

Monocyte polarization assay

The polarization assay was performed with purified monocytes in suspension stimulated with chemoattractants as described previously (25). Briefly, purified monocytes were stimulated, in duplicate, with chemoattractants or with medium alone for variable lengths of time (2–10 min) at room temperature in polypropylene tubes. Stimulation was stopped by adding an equal volume of 10% formaldehyde to the medium. After coding samples, at least 200 cells were counted and classified on the basis of their morphology (spherical or head-tail shape) by an independent investigator. Data were expressed as a percentage.

Immunofluorescence staining of F-actin

The monocyte polarization assay in response to FMLP, MCP-1, and MIP-1 was performed as described above. At the end of the incubation period, monocytes were centrifuged over a slide at 700 rpm for 10 min by cytospin. Monocytes adherent to the slide were washed twice with PBS, incubated with 0.165 µM FITC-phalloidin or with a control IgG conjugated to FITC for 30 min at 4°C, and washed. Slides were stained with FITC-phalloidin and then photographed by a fluorescence microscope. Slides stained with FITC-IgG did not display any detectable fluorescence.

Superoxide anion production

Superoxide production by monocytes was measured as superoxide dismutase (SOD; Sigma)-inhibitable cytochrome c reduction by a modified Pick and Mizel method (26). Briefly, monocytes, contained in PBMC preparations were resuspended in medium (Hanks’ containing 5 mM glucose, 0.5 mM calcium chloride, 4 mM sodium azide, and 80 µM cytochrome c; Sigma) and preincubated in 96-well microtiter plates for 5 min at 37°C. Stimulation of cells was performed in triplicate with or without addition of SOD (250 µg/ml) at 37°C for 60 min. At 5-min intervals, the OD at 550 nm of each reaction mixture was determined in an ELISA reader. Superoxide production was calculated from the difference in the ODs at 550 nm between the wells with and without SOD and was converted to nanomoles of superoxide anion per 2 x 10⁵ monocytes.

Statistical analysis

Comparisons between normal donors and WAS patients were performed where indicated using nonparametric analysis for unpaired data.

	Results

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We studied the chemotactic response of monocytes obtained from WAS patients and from normal donors in response to the bacterial product FMLP. This agent, used at a concentration of 10 nM, induced a reduced chemotactic response in monocytes obtained from WAS patients compared with normal donors (Fig. 1). FMLP, like most of chemotactic agonists, including C5a, platelet-activating factor, and chemokines, induces migration of leukocytes by activating G protein-coupled receptors (27).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. Chemotaxis of monocytes in response to FMLP in WAS patients. FMLP (10 nM) or medium alone was placed in the lower wells of a microchemotaxis chamber. Monocytes (1.5 x 106 cells/ml in RPMI 1640 containing 1% BSA) were added to the upper wells. The two wells were separated by a 5-µm pore size polycarbonate filter. After incubation at 37°C in air with 5% CO2 for 90 min, filters were removed, fixed, and stained as previously described (24). The results are expressed as the mean (±SD) number of cells that migrated across the filter in three high power fields (HPF), counted in triplicate, after subtraction of the number of cells that migrated in response to medium alone. Statistical analysis of the chemotactic response to FMLP between WAS patients and normal donors was performed using nonparametrical analysis (Mann-Whitney test).

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Chemotaxis of monocytes in response to MCP-1 in WAS patients. MCP-1 (50 ng/ml) or medium alone was placed in the lower wells of a microchemotaxis chamber. Monocytes (1.5 x 106 cells/ml in RPMI 1640 containing 1% BSA) were added to the upper wells. The results are expressed as the mean (±SD) number of cells that migrated across the filter in three high power fields (HPF), counted in triplicate, after subtraction of the number of cells that migrated in response to medium alone. Statistical analysis of the chemotactic response to MCP-1 between WAS patients and normal donors was performed using nonparametrical analysis (Mann-Whitney test).

View larger version (9K): [in this window] [in a new window]   FIGURE 4. Chemotaxis of monocytes in response to MCP-1, MIP-1, and FMLP in WAS patients: dose response. Increasing concentrations of MCP-1 (10, 50, and 200 ng/ml), MIP-1 (10, 50, and 200 ng/ml), or FMLP (10-7, 10-8, and 10-9 M) were placed in the lower wells of a microchemotaxis chamber. Monocytes (1.5 x 106 cells/ml in RPMI 1640 containing 1% BSA) were added to the upper wells. The results are expressed as the mean (±SD) number of cells that migrated across the filter in three high power fields (HPF), counted in triplicate. Results shown are representative of three independent experiments performed on three patients and three normal subjects. Statistical analysis of the chemotactic response to the chemoattractants was based on nonparametrical analysis (Kruskal Wallis test). Asterisks indicate a significant difference (p < 0.05).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Superoxide anion production of monocytes in response to PMA, FMLP, and MCP-1 in WAS patients. Monocytes (2 x 105 in Hanks’) from three WAS patients or from three normal donors were stimulated with PMA (10 ng/ml), FMLP (10 nM), MCP-1 (50 ng/ml), or medium alone, in triplicate, for 30 min with or without addition of SOD to the medium as described in Materials and Methods. Superoxide anion production is expressed as nanomoles per 200,000 monocytes for 30 min. Results are expressed as the mean (±SE) of three independent experiments performed.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Up-regulation of CD18/CD11b expression on monocytes in WAS patients. Whole blood was incubated for 15 min at 37°C with medium or FMLP (10 nM). Cells were washed and stained with control mouse IgG or CD11b mAbs as described in Materials and Methods. After incubation with rabbit anti-mouse FITC, cells were washed twice, fixed with PBS/1% paraformaldehyde, and analyzed with a flow cytometer. Monocytes were gated on the basis of forward and side scatter and the green fluorescence of at least 5000 events analyzed. The results shown are representative of three independent experiments performed on three patients and three normal subjects. In three WAS patients analyzed, we detected an increase in mean channel fluorescence of 111 ± 9 U, on the average, while normal subjects up-regulated CD11b expression of 99 ± 11 mean channel units. Mean channel values of activated cells were calculated after subtraction of basal levels of CD11b.

View larger version (45K): [in this window] [in a new window]   FIGURE 8. F-actin staining of stimulated monocytes in WAS patients. Monocyte polarization in response to FMLP (10 nM), MCP-1 (50 ng/ml), or MIP-1 (50 ng/ml) was performed as described above for normal donors or WAS patients. Cytospin of the cells was then stained with FITC-phalloin or FITC-IgG as described in Materials and Methods and photographed using a fluorescence microscope. The results shown are representative of two independent experiments performed on two patients and two normal subjects.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In leukocytes, chemoattractants induce actin polymerization, integrin up-regulation, and superoxide anion production through G protein-coupled receptors; these receptors activate a cascade of intracellular events, including changes in cytosolic free calcium and release of phosphoinositides (30). In this signaling pathway, proteins with GTPase activity, such as Rho, Cdc42, and Rac, are involved (14, 27). These proteins belong to the Ras superfamily and determine membrane ruffling and pseudopodia formation in fibroblasts (14). The active form of Cdc42 was found to interact with WASp (9, 10, 11). Following interaction with Cdc42, WASp forms clusters of aggregation of F-actin (9). We found that monocytes from WAS patients, after stimulation with MCP-1 or with FMLP, lack the capability of cell polarization and show a diffuse distribution of F-actin in cells with a rounded shape, in contrast to the accumulation of F-actin in pseudopods as observed in normal donors after stimulation with FMLP, MCP-1, or MIP-1. These results demonstrate that monocytes from WAS patients have a defective cell polarization in response to chemoattractants and suggest that WASp is involved in the regulation of F-actin polymerization in vivo (9). In addition, our results suggest that the defect of cell polarization observed in WAS patients may determine the reduction of leukocyte chemotaxis in response to chemoattractants. A possible explanation for the monocyte defects observed in WAS patients is based on the hypothesis that in normal individuals, following stimulation with chemokines, Cdc42 would interact with WASp and thereby induce F-actin polymerization. A similar role has been postulated for the GTP binding protein Rac in the induction of respiratory burst in response to chemokines in polymorphonuclear cells, but has not been described for Cdc42 (32). The small GTP binding proteins, Rho and Rac, are essential intracellular components of the superoxide anion synthase that translocate to the membrane and activate the enzyme after polymorphonuclear cells stimulation with FMLP or IL-8 (27, 33). We speculate that chemokines such as MCP-1 and MIP-1 might activate Cdc42, and that, in turn, WASp, bound to activated Cdc42, might constitute the anchor for G-actin polymerization.

The decreased chemotactic response of monocytes in WAS patients suggests possible defects of leukocyte recruitment in inflamed tissues in these subjects. Leukocyte migration and homing are induced by local production of chemokines in inflammatory reactions, as observed in dermatitis and in DTH; in these cases, chemokine release in the derma was shown to be required for leukocyte recruitment (34, 35). Our observation that monocytes from WAS children have deficient chemotactic responses to MCP-1 and to other chemokines, but produce normal amounts of MCP-1 when activated by LPS (data not shown) provides a possible explanation for the impaired DTH that is often observed in WAS patients, even when the in vitro lymphocyte proliferative response to mitogens is preserved (1, 3). Furthermore, chemokines such as MIP-1, IL-8, and stromal derived factor-1 are involved in the regulation of chemotaxis and the proliferation of hemopoietic precursors (36, 37). Whether interactions with marrow stromal cells and a proliferative advantage of hemopoietic precursors with intact WASp function account for the nonrandom pattern of X-chromosome inactivation reported in CD34⁺ hemopoietic progenitor cells from WAS carrier females remains to be seen (38).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Chemotaxis of monocytes in response to MIP-1 in WAS patients. MIP-1 (50 ng/ml) or medium alone was placed in the lower wells of a microchemotaxis chamber. Monocytes (1.5 x 106 cells/ml in RPMI 1640 containing 1% BSA) were added to the upper wells. The results are expressed as the mean (±SD) number of cells that migrated across the filter in three high power fields (HPF), counted in triplicate, after subtraction of the number of cells that migrated in response to medium alone. Statistical analysis of the chemotactic response to MIP-1 between WAS patients and normal donors was performed using nonparametrical analysis (Mann-Whitney test).

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Time course of monocyte chemotaxis in WAS patients. MCP-1 (50 ng/ml), FMLP (10 nM), or medium alone was placed in the lower wells of a microchemotaxis chamber. Monocytes (1.5 x 106 cells/ml in RPMI 1640 containing 1% BSA) were added to the upper wells. Cell migration was stopped after 60, 90, or 120 min of incubation as indicated on the x-axis. The results are expressed as the mean (±SD) number of cells that migrated across the filter in three high power fields (HPF), counted in triplicate, after subtraction of the number of cells that migrated in response to medium alone. Results shown are representative of three independent experiments performed on three patients and three normal subjects. Statistical analysis of the chemotactic response to the chemoattractants was based on nonparametrical analysis (Kruskal Wallis test). Asterisks indicate a significant difference (p < 0.05).

	Acknowledgments

	Footnotes

 
¹ This work was supported by Telethon, Rome, Italy (Grant A.42 to L.D.N. and Grant E.635 to S.S.); Consiglio Nazionale delle Ricerche (Grant 97.04210.CT04); the Associazione Italiana per la Ricerca sul Cancro; the 40% Grant (A.M.); and a fellowship from the Association Comitato Promotore TeleThon (to R.B.).

² Address correspondence and reprint requests to Dr. Raffaele Badolato, Clinica Pediatrica, Universita’ di Brescia, c/o Spedali Civili, 25123 Brescia, Italy. E-mail address:

³ Abbreviations used in this paper: WAS, Wiskott-Aldrich syndrome; DTH, delayed-type hypersensitivity; WASp, Wiskott-Aldrich syndrome protein; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; SOD, superoxide dismutase.

Received for publication August 8, 1997. Accepted for publication March 10, 1998.

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