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. 2005 May;115(1):108–117. doi: 10.1111/j.1365-2567.2005.02131.x

Neutrophil differentiated HL-60 cells model Mac-1 (CD11b/CD18)-independent neutrophil transepithelial migration

Svetlana O Carrigan 1, Amy L Weppler 1, Andrew C Issekutz 1,2,3, Andrew W Stadnyk 1,2
PMCID: PMC1782134  PMID: 15819703

Abstract

During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially β2 integrin Mac-1 (CD11b/CD18)-independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac-1-independent transepithelial migration proceeds independently of prior cell activation through Mac-1. We took two approaches, one using blocking Fab′ fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL-60 cell line which is Mac-1 deficient to further study neutrophil/epithelial cell interaction. Anti-CD18 Fab′ minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (≤25% inhibition) on the migration to C5a. Upon incubation with dimethylsulphoxide, HL-60 cells differentiated and up-regulated CD11b expression and migrated to C5a and n-formyl methionyl leucyl phenylalanine in a similar manner to peripheral blood neutrophils. In contrast, CD11b expression was minimal on HL-60 cells differentiated with dibutytyl cAMP to a neutrophil-like phenotype. These cells, however, readily migrated across both intestinal and lung epithelial monolayers in response to C5a. We conclude that Mac-1-independent transepithelial migration does not require prior activation of cells via Mac-1 ligation because HL-60 cells lacking Mac-1 (CD11b/CD18) expression migrate effectively. HL-60 cells differentiated with dbcAMP should greatly assist in the search for the Mac-1-independent ligands for neutrophil migration across epithelium.

Keywords: intestinal epithelium, lung epithelium, dibutyryl cAMP, DMSO-differentiated HL-60

Introduction

Granulocyte migration through the intestinal epithelium and into the lumen correlates with disease activity in inflammatory bowel disease (IBD) patients.1–6 Neutrophilic granulocyte retention within intestinal crypts is believed to contribute to the development of crypt abscesses, a typical feature of active IBD.7–9 Therefore, preventing neutrophil transepithelial migration may be one means of preventing excessive intestinal inflammation.

Migration of human peripheral blood neutrophils across model intestinal epithelial monolayers has been shown to involve CD11/CD18, also known as the β2 integrins. For example, migration in response to the chemoattractant n-formyl methionyl leucyl phenylalanine (fMLP) is completely blocked by anti-β2 integrin or anti-αM (CD11b), but not by anti-αL (CD11a) monoclonal antibodies (mAb).10 Although the exact mechanisms of neutrophil Mac-1-dependent interactions with intestinal epithelia remain elusive, epithelial surface fucosylated proteoglycans contribute to Mac-1-dependent interactions.11 The precise subcellular location and identity of these adhesive interactions remain to be determined. In addition to Mac-1-dependent events, we recently showed that Mac-1-independent neutrophil transepithelial migration occurs in response to other chemoattractants. A substantial amount of migration to chemoattractants C5a, interleukin-8 (IL-8) and leukotriene B4 (LTB4) occurred despite the presence of anti-CD18 or anti-CD11b antibody, with C5a being the most potent inducer of Mac-1-independent migration. We also demonstrated that the β2 integrin-independent mechanism(s) operates only in the physiological, basolateral-to-apical direction of migration, illustrating the specificity of these interactions.12 The ligands used in Mac-1-independent migration are unknown as are roles they may have in addition to migration. There is a possibility that Mac-1-independent migration may be an event critical but subsequent to the Mac-1-dependent interaction. For example, activation of β2 integrins by ligands or antibodies can induce the up-regulation of β1 integrins.13 One means to study Mac-1-independent mechanisms is to employ Mac-1 deficient cells. Leucocytes of patients with the severe form of leucocyte adhesion deficiency (LAD) lack expression of β2 integrins,14 but such patients are extremely rare. Mac-1-deficient mouse neutrophils are available; however, they still have other limitations as short-lived cells. A human neutrophil-like cell line, on the other hand, would not have such restrictions.

From among the human cell lines, the HL-60 cell line has been widely used to study various leucocyte functions.15 These cells can be differentiated into neutrophil-like, monocyte-like or eosinophil-like cells depending on the differentiation method used.16,17 Neutrophil-like cells are generated upon dimethyl sulphoxide (DMSO)18, dibutyryl cAMP (dbcAMP)19 or all-trans-retinoic acid treatment.20 DMSO and dbcAMP-differentiated cells are able to produce superoxide and reduce nitroblue tetrazolium18,19,21 and lack monocyte markers such as non-specific esterase activity22,23 features that are characteristic of neutrophils. These models have proven to be useful for studying neutrophil functions such as oxidative burst, adhesion, chemotaxis and migration. Neutrophil-like retinoic acid-differentiated HL-60 cells have been examined for migration in response to fMLP across lung epithelium24 and eosinophil-like differentiated HL-60 cells have been utilized in studies of migration in response to fMLP or bacterial invasion of intestinal epithelium.25 In neither study was Mac-1-dependency of migration assessed. Interestingly, Mac-1 expression on HL-60 cells is minimal26 and does not increase upon dbcAMP differentiation,22 which led us to hypothesize that if C5a induces migration of dbcAMP dHL-60 cells across intestinal epithelium, this would occur via Mac-1-independent mechanisms. Indeed, using Mac-1-deficient neutrophil-like dHL-60 cells we confirmed the existence of Mac-1-independent mechanisms of neutrophil transepithelial migration and conclude that these mechanisms can exist independently of Mac-1 activation on dHL-60 cells and indeed on normal neutrophils.

Materials and methods

Reagents and antibodies

The chemical compounds rhC5a, fMLP, DMSO and dbcAMP were purchased from Sigma Chemical Company (Oakville, ON, Canada). Mouse anti-human β2 (CD18) integrin mAb clone 60.3 (immunoglobulin G2a; IgG2a) was a gift from Bristol-Myers Squibb (Seattle, WA) and clone IB4 (IgG2a) was from the American Type Culture Collection (ATCC; Bethesda, MD). Fab′ fragments were prepared by papain digestion of mAb IB4. Mouse anti-human major histocompatibility complex (MHC) class I (clone W6/32, IgG2a, ATCC) was used as a neutrophil-binding isotype control. Mouse anti-human Mac-1 mAb clone 2LPM19C (IgG1) was a gift from Dr K. Pulford (Oxford, UK). Cy-Chrome-conjugated mouse anti-human Mac-1 (CD11b) mAb (IgG1, clone ICRF44) and Cy5-conjugated donkey anti-mouse IgG were obtained from BD PharMingen (San Diego, CA). Fluoroscein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG were purchased from Chemicon International (Cedarlane Laboratories, Hornby, ON, Canada). Mouse IgG1 negative control antibody was from DAKO (Glostrup, Denmark) and mouse IgG2a negative control was from Cymbus Biotechnology (Chandlers Ford, UK).

Epithelial cell culture

The T84 intestinal epithelial cell line and A549 alveolar epithelial cell line were purchased from ATCC. T84 cells were cultured in 1 : 1 HAM F12/Dulbecco's modified Eagle's minimal essential medium (DMEM), containing in final concentrations: 5% newborn calf serum, 15 mm HEPES, 50 U/ml penicillin and 50 µg/ml streptomycin (Life Technologies, Burlington, ON, Canada), while A549 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mm l-glutamine, 50 µm mercaptoethanol, 1 mm sodium pyruvate, 50 U/ml penicillin, 54 U/ml streptomycin, 45 µg/ml heparin, 50 µg/ml ascorbic acid (Sigma, St. Louis, MO). Inverted T84 monolayers were grown on collagen-coated Transwell™ (Costar, Corning Inc., New York, NY) filters as described previously.12 A549 were seeded on the undersurface of 0·33-cm2 polyester Transwell™ filters (3 µm pore size) coated with type IV collagen at a concentration of 105 cells per filter. Cells were allowed to adhere, then placed upright into the 24-well plates and grown for 4–5 days in RPMI-1640 to confluency. Monolayer permeability was measured one day prior to the experiment using 125I conjugated to human serum albumin (HSA), as described elsewhere.12 Monolayers were used if HSA diffusion was less than 2%.

Neutrophil labelling and isolation

Neutrophil isolation was as described previously.12 Following labelling with sodium chromate (Inline graphicCrO4; Amersham, Oakville, ON), cells from the 58%/72% discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient interface were collected, washed three times in Ca2+- and Mg2+-free Tyrode's solution, resuspended in DMEM supplemented with 5 mg/ml pyrogen-free HSA, 15 mm HEPES, and counted. Neutrophils were >95% pure by Crystal Violet dye staining and >98% viable by Trypan blue dye exclusion.

HL-60 cell culture, differentiation and labelling

The HL-60 cell line was obtained from ATCC and maintained in Iscove's modified Dulbecco's medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 µg/ml streptomycin at 37° with 5% CO2. Cells were differentiated into the granulocyte lineage with DMSO or dibutyryl cyclic AMP; 5 × 105 cells/ml were incubated with 1·2% DMSO for 5 days or with 0·5 mm dbcAMP for 2 days. After incubation the cells were washed, resuspended in Ca2+- and Mg2+-free Tyrode's buffer, and incubated with Inline graphicCrO4 for 30 min at 37°. After labelling, cells were washed three times in Ca2+- and Mg2+-free Tyrode's buffer, resuspended in DMEM with 5 mg/ml HSA and 15 mm HEPES, and counted. Cells were ≥90% viable prior to the migration assay.

Neutrophil and HL-60 cell migration assays

After isolation, neutrophils were resuspended in HSA/HEPES-containing DMEM at a concentration of 106 cells/ml. For antibody treatments, cells were incubated with 30 µg/ml anti-β2 intact antibody or Fab′ for 20 min at room temperature. Neutrophils or dHL-60 cells (105 in 100 µl) were added to the upper chamber of each Transwell. Migration was induced by placing chemoattractant in 600 µl of DMEM/HSA/HEPES in the well. Cells were allowed to migrate for 70 min (across bare filters) or 2 hr (across epithelial monolayers). We titrated the concentration of chemoattractants for both bare filters and epithelial monolayers to achieve the best rate of migration and observed plateaus within the first hour of migration across bare filters and after 1·5 hr across monolayers (data not shown and,12). The optimal C5a concentrations for inducing migration were 10−9 m, 10−8 m and 2 × 10−9 m with bare filters, T84 monolayers and A549 monolayers, respectively, with both neutrophils and HL-60 cells. For fMLP, 10−8 m and 10−7 m was optimal with bare filters and T84 monolayers, respectively, for neutrophils, and 10−9 m and 10−8 m, respectively, for HL-60 cells. Following migration, neutrophils or dHL-60 cells remaining in the Transwell™ upper chamber (non-migrated fraction) were collected, while cells from the lower chamber (migrated fraction) were lysed with 1% Triton-X-100 and collected separately. Finally, monolayer-associated neutrophils or dHL-60 cells were lysed with 0·2 m NaOH. Radioactivity (c.p.m.) in non-migrated, migrated, and monolayer-associated fractions was determined separately, using a Wizard™ 3′′ 1480 automatic γ counter (Wallac, Turku, Finland). We routinely recovered greater than 85% of the applied 51Cr labelled cells when all three fractions were combined. The percent of migrated cells was then calculated as: c.p.m. of the migrated fraction × 100%/c.p.m. of the cells added to the upper chamber at the beginning of the migration assay. Triplicate wells for each treatment were used and data are reported as the mean ± SD of a representative experiment, or the mean percentage migration of three independent experiments ± SEM where specified.

Flow cytometry

Neutrophils were isolated as described above and resuspended at a concentration of 106 cells/ml in DMEM/HSA/HEPES. Where specified, cells were treated with intact or Fab′ anti-β2 antibody for 20 min at room temperature then washed to eliminate excess antibody. The HL-60 cells were washed once with Ca2+- and Mg2+-free Tyrode's buffer. Cells were incubated with 10−8 m fMLP or 10−9 m C5a or without chemoattractant for 30 min at 37°, rapidly cooled, washed with ice-cold Hank's balanced salt solution (HBSS; Invitrogen) containing 0·5% bovine serum albumin (BSA) and 0·1% NaN3 (Sigma), and resuspended in ice-cold HBSS/BSA/NaN3 at 5 × 106 cells/ml. A 10 µl aliquot of Cy-Chrome-conjugated anti-CD11b antibody, 10 µg/ml anti-CD18 antibody, 10 µg/ml mouse IgG1, or 10 µg/ml mouse IgG2a was added to separate tubes containing 5 × 105 cells each (in 100 µl), and incubated for 30 min on ice in the dark. Cells were then washed with cold HBSS/0·5% BSA/0·1% NaN3. Cells incubated with unconjugated primary antibodies were further treated with either FITC-conjugated or Cy5-conjugated donkey anti-mouse IgG. Following washing, cells were fixed in 1% paraformaldehyde. Fluorescent cells were counted using a Becton-Dickinson FACS Calibur flow cytometer and analysed with WinList 5·0 software (Verity Software House, Inc., Topsham, ME).

Statistical analysis

Data was analysed using one way anova followed by a post-hoc Tukey test. Statistical analyses were conducted using SPSS Version 10.

Results

Neutrophil migration to fMLP and C5a across acellular filters is inhibited by anti-β2 antibodies

We have previously reported that in the presence of anti-CD18 or anti-CD11b mAb neutrophil migration across inverted T84 monolayers persists in response to the chemoattractants C5a (∼60% of the migration to C5a in absence of the antibody), IL-8 (25%) and LTB4 (25%). Neutrophil migration in the presence of antibody was detected as early as 30 min after the addition of chemoattractant and plateaued at 2 hr.12 In contrast, migration to fMLP was completely inhibited by anti-β2 integrin or anti-CD11b mAb suggesting that the β2 integrin-dependent component of neutrophil transintestinal epithelial migration to this stimulus is Mac-1-dependent. Inhibition of neutrophil migration across matrix-coated filters by anti-CD11b mAb had been shown by others27 and because our inverted monolayers are also grown on matrix coated filters, we wanted to determine whether similar inhibition of migration occurs in the absence of epithelial cells. Indeed, neutrophil migration to C5a across either collagen-coated or acellular filters was partially inhibited and migration was inhibited to fMLP in the presence of mAb to the level of background migration in absence of chemoattractant (Fig. 1a). Neutrophil migration in the presence of binding isotype-matched control mAb was not inhibited (Fig. 1a, insert). Substituting filters from a different manufacturer or material (polycarbonate versus polyester), eliminating albumin from the migration medium or employing a different clone of anti-β2 mAb did not affect the degree of inhibition caused by anti-β2 integrin mAb with bare filters (data not shown). Anti-CD11b mAb, 2LPM19c also reduced neutrophil migration across bare filters (data not shown).

Figure 1.

Figure 1

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Effect of anti-β2 integrin antibody on neutrophil migration and activation. (a) Effect of intact anti-β2 integrin antibody on neutrophil migration across bare filters and T84 monolayers. Freshly isolated neutrophils were induced to migrate across bare filters or inverted T84 monolayers. Black bars: migration without added mAb, grey bars: migration in the presence of 30 µg/ml anti-β2 antibody. Migration across T84 monolayers in the absence of chemoattractant was routinely less than 2%. The figure shows a representative of over five experiments across bare filters and over 20 experiments across T84 monolayers. Each bar is the mean of migration from three wells ± SD. Insert: Neutrophil migration across bare filters and T84 monolayers was assessed in the presence of binding isotype control anti-MHC class I antibody W6/32 (IgG2a). Black bars: migration without added mAb, grey bars: migration in the presence of W6/32 antibody. Each bar is the mean of migration from three wells ± SD. (b) Effect of intact anti-β2 integrin antibody on neutrophil Mac-1 expression. Neutrophils were treated with anti-β2integrin mAb or Fab′ fragments for 20 min at room temperature or left untreated. Bars indicate percentage increase in CD11b mean fluorescent intensity following intact antibody (n = 4) or Fab′ (n = 2) treatment of neutrophils relative to no anti-β2 integrin mAb or Fab′ control. Bars represent the mean ± SD. Both IB4 and 60.3 anti-β2 intact mAb were tested with similar results. (c) Effect of Fab′ fragments of anti-β2 integrin mAb on neutrophil migration across bare filters and T84 monolayers. Migration was performed as in (a), except that Fab′ fragments were used instead of intact antibody. This experiment was repeated twice with similar results. Each bar is the mean of migration from three wells ± SD.

Intact anti-β2 integrin antibodies but not Fab′ fragments induce Mac-1 up-regulation on neutrophils

Fc-mediated events in combination with β2-integrin crosslinking may occur upon treatment with an intact mAb.28 We therefore speculated that intact mAb may have an activating effect on neutrophils and thus non-specifically inhibit migration across acellular filters. To quantify activation we measured Mac-1 surface expression on neutrophils treated with anti-β2 integrin mAb. Figure 1(b) shows that incubation of neutrophils with an intact anti-β2 integrin mAb resulted in Mac-1 up-regulation. On the other hand, incubation with anti-β2 integrin Fab′ fragments resulted in considerably less Mac-1 up-regulation than with intact immunoglobulin (Fig. 1b). Furthermore, the anti-β2 integrin Fab′ inhibited by less than 20% the neutrophil migration to fMLP across bare filters, yet blocked 75% of migration to fMLP across T84 monolayers (Fig. 1c). These data confirmed the β2 integrin dependency of neutrophil transepithelial migration to fMLP while over 75% of migration to C5a remained β2 integrin independent, e.g. in the presence of Fab′ fragments. These results illustrate how engagement of Mac-1 can confound analyses because of its multiple roles in neutrophil function and indicated the necessity to study Mac-1-independent mechanisms in a Mac-1-deficient cell system.

Granulocyte-differentiated HL-60 cell expression of Mac-1

Neutrophils are short-lived cells and once removed from the blood their viability is limited to several hours thereby precluding extensive manipulations such as transfection or expression interference technologies to reduce Mac-1 expression. An immortal cell line such as the HL-60 cells ought to overcome such a shortcoming. Peripheral blood neutrophils express a high level of Mac-1 (CD11b), which was up-regulated upon chemoattractant stimulation (Fig. 2a). Undifferentiated HL-60 cells expressed essentially no CD11b and a very low level of surface CD18 (Fig. 2b), but these cells do not respond chemotactically to either C5a or fMLP (data not shown). Following incubation with DMSO, HL-60 cells expressed CD11b and expression increased following chemoattractant stimulation in a manner similar to blood neutrophils (Fig. 2c). In contrast, dbcAMP-differentiated HL-60 cells expressed CD18 but the vast majority were virtually negative for CD11b and CD11b was not up-regulated in response to either C5a or fMLP (Fig. 2d).

Figure 2.

Figure 2

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Mac-1 expression on neutrophils and HL-60 cells. (a) Mac-1 expression on freshly isolated neutrophils. Purified neutrophils were treated with chemoattractants for 30 min or left untreated (NT), then stained with anti-CD11b or anti-CD18 antibody. The mean fluorescence intensity is shown in the top-right corner of each histogram. One representative experiment is shown of 10 for CD11b and two for CD18. (b) Mac-1 expression on undifferentiated HL-60 cells. HL-60 cells were stained for CD11b or CD18 expression. One representative experiment of four for CD11b and three for CD18 is shown. (c) Mac-1 expression on the surface of DMSO-differentiated HL-60 cells. Cells were differentiated for 5 days with 1·2% DMSO, and then stained for CD11b and CD18. One representative experiment of three is shown. (d) Mac-1 expression on the surface of dbcAMP-differentiated HL-60 cells. Cells were treated with 500 µm dbcAMP for 2 days, washed and stained for CD11b and CD18. One representative experiment of four for CD11b and three for CD18 is shown.

Pattern of differentiated HL-60 cell migration to C5a and fMLP across acellular filters and intestinal epithelial monolayers

We next compared the chemotactic responses of the DMSO and dbcAMP-differentiated HL-60 cells. DMSO differentiated cells responded to both C5a and fMLP across both bare filters and T84 monolayers (Fig. 3a). In contrast, dbcAMP-differentiated cells migrated to C5a as efficiently across epithelial monolayers as across bare filters but migrated poorly in response to fMLP across T84 monolayers (Fig. 3b). The migration response was maximal by the second day of incubation with dbcAMP.

Figure 3.

Figure 3

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Migration of granulocyte-differentiated HL-60 cells across bare filters and T84 monolayers. (a) Migration of DMSO-differentiated HL-60 cells. HL-60 cells were differentiated with 1·2% DMSO for 5 days. After washing and 51Cr labelling, 105 HL-60 cells per filter were used in the migration assay. Each bar is the mean of percentage migration ± standard error of the mean (SEM) of three independent experiments. (b) Migration of dbcAMP-differentiated HL-60 cells. HL-60 cells were differentiated with 500 µm dbcAMP for 2 days, washed, labelled with 51Cr and used in the migration assay as described earlier. Each bar is the mean percentage migration of three independent experiments ± SEM.

Similar to neutrophils, DMSO-differentiated HL-60 cell migration to fMLP was significantly blocked by anti-β2 integrin mAb whereas most of migration to C5a occurred despite β2 integrin mAb (Fig. 4a). In contrast, the antibody did not inhibit the migration of dbcAMP dHL-60 cells across T84 monolayers during 30–180 min migration experiments (Fig. 4b and data not shown), indicating that neither Mac-1 nor β2 integrins are required for normal transmigration kinetics. To exclude the possibility that CD11b is up-regulated during transepithelial migration and therefore not accessible to the antibody, we measured CD11b surface expression on postmigrated HL-60 cells and found that it remained low (Fig. 4c). Similarly, the small number (∼5%) of dbcAMP dHL-60 cells migrating across T84 monolayers in response to fMLP remained Mac-1-low and their migration could not be inhibited by anti-CD18 antibody (data not shown).

Figure 4.

Figure 4

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Granulocyte-differentiated HL-60 cells migrate across T84 inverted monolayers. (a) DMSO-differentiated HL-60 cells migrate across T84 monolayers similar to neutrophils. DMSO dHL-60 cells were allowed to migrate across inverted T84 monolayers for 2 hr in response to 10−8 m C5a or 10−7 m fMLP. Black bars: migration to chemoattractant alone, open bars: migration to the chemoattractant in the presence of 30 µg/ml of intact anti-β2 antibody. Each bar is the mean percentage migration of three independent experiments ± SEM. (b) β2 integrin independent migration of dbcAMP-differentiated HL-60 cells in response to C5a. DbcAMP dHL-60 cells were stimulated, with the indicated concentrations of C5a, to migrate across inverted T84 monolayers for 2 hr. Bars are the mean percentage migration ± SEM from five experiments using dbcAMP-differentiated HL-60 cells and 10−8 m C5a as a chemoattractant across T84 monolayers (P = 0·1482). (c) Migration across T84 monolayers does not induce significant Mac-1 up-regulation on dbcAMP-differentiated HL-60 cells. HL-60 cells were differentiated for 2 days, washed and allowed to migrate in response to C5a for 2 hr across inverted T84 monolayers. Transmigrated cells were collected and stained for CD11b expression. Similar results were obtained in three experiments.

Finally, we tested whether the Mac-1-independent mechanism of migration was unique to the T84 colonocyte line or perhaps applied to other epithelia by measuring dbcAMP-differentiated HL-60 cell migration across lung epithelial monolayers. As shown in Fig. 5, Mac-1 deficient HL-60 cells readily migrated across the A549 lung epithelial cell monolayers in response to C5a, even in the presence of the intact anti-β2 integrin antibody. Thus, the capacity of HL-60 cells to migrate using Mac-1- and β2 integrin-independent mechanisms was not unique to intestinal epithelium.

Figure 5.

Figure 5

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Granulocyte-differentiated HL-60 cells migrate across inverted A549 lung epithelial cell monolayers. The migration of dbcAMP dHL-60 cells across A549 human lung adenocarcinoma cells was measured as in Fig. 4(b) using C5a (2 × 10−9) as stimulus in absence or presence of anti-β2 integrin mAb (30 µg/ml). Bars are the mean percentage migration ± SEM from three consecutive experiments (P = 0·1399).

Discussion

We previously reported that in addition to well-documented β2 (Mac-1)-dependent mechanisms11,29–32 neutrophils are able to use β2 (Mac-1)-independent mechanisms when migrating across intestinal epithelium in the physiological basolateral-to-apical direction in response to the chemoattractants C5a, IL-8 and LTB4·12 Yet, in the course of describing CD18-independent migration, we exposed that neutrophil activation, measured as increased Mac-1 expression, is a confounder when employing intact mAb to β2 integrin, the result of which is inhibition of migration even across acellular filters (Fig. 1a). This is not simply an Fc-mediated event, as isotype-matched mAbs against other neutrophil antigens that ought to similarly promote Fc-binding do not prevent migration in this assay12 and shown in Fig. 1(a), insert. Mac-1 may therefore have dual roles: (1) to activate the cells and (2) to promote migration as an adhesion molecule on migrating neutrophils. In some studies, β2-independent mechanisms were invoked after additional neutrophil activation, including via β2 integrins.13,33 Thus, we could not rule out that in our migration system, activation of neutrophils through Mac-1 was required for the Mac-1-independent migration. We addressed this issue by using minimally activating Fab′ fragments of mAb to β2 integrin. Using this reagent we confirm the β2 integrin-dependency of migration to fMLP and our initial observation of β2 integrin-independent neutrophil migration to C5a (Fig. 1c).

Another way to distinguish these events would be to study neutrophils from patients with the severe form of LAD, whose leucocytes lack surface expression of β2 integrins.14 LAD neutrophils exhibit chemotaxis34 but have defective transendothelial migration35 and transepithelial migration to fMLP.29 However, the use of LAD neutrophils is problematic because such patients are rare and are treated when young by bone marrow transplantation. Therefore, establishing an alternate Mac-1-deficient leucocyte system became an objective for studying Mac-1-independent mechanisms, including Mac-1-independent neutrophil transepithelial migration. The migration of dbcAMP differentiated HL-60, which lack Mac-1, mimics this absolute Mac-1-independent process (Figs 3b and 4b).

We chose to use the HL-60 promyelocytic cell line differentiated along the granulocytic lineage because these cells are more manipulable compared to short-lived neutrophils. Undifferentiated HL-60 cells lack detectable amounts of chemoattractant receptors and Mac-1, which is consistent with the promyelocytic phenotype.36,37 DMSO and dbcAMP have both been used by others to convert HL-60 cells into a neutrophil-like phenotype that is able to reduce nitroblue tetrazolium;18,19,21 that lacks monocyte markers such as non-specific esterase activity22,23 and expresses some chemotattractant receptors.38 Despite some differences between dHL-60 and native neutrophils38–40 these cells are useful when long-term manipulations or large numbers of cells are required for study. Some differences between neutrophils and HL-60 may actually be helpful in studying adhesion molecules involved in neutrophil transepithelial migration. For example, the absence of secondary granules will exclude a number of adhesion molecules as potential candidates in Mac-1-independent migration.40 Mac-1 expression on DMSO-differentiated HL-60 cells has been well documented26,41–43 while Mac-1 expression on dbcAMP-differentiated HL-60 cells has not been well studied. Although some investigators have demonstrated positive staining for CD11b on dbcAMP-differentiated cells, the level of its expression did not differ from the level on undifferentiated HL-60 cells.22 Others showed that CD18 mRNA increased upon dbcAMP differentiation but it was not reported whether this coincided with CD11b expression.44 Our study shows that Mac-1 is essentially absent on undifferentiated HL-60 but expression increases following DMSO treatment (Fig. 2b, c). We have also determined that despite up-regulated expression of CD18 following dbcAMP differentiation, CD11b expression remains extremely low (Fig. 2d). The latter findings allowed us to speculate that dbcAMP dHL-60 cells would necessarily utilize Mac-1-independent mechanisms if they migrate.

DMSO-differentiated cells express Mac-1 following differentiation, therefore we were not surprised that the migration of DMSO-differentiated HL-60 cells across an intestinal epithelial monolayer included both β2-dependent and independent components (Fig. 4a) similar to peripheral blood neutrophils. In our study, Mac-1-deficient dbcAMP dHL-60 cells also migrated efficiently to C5a, with nearly 40% migrating across acellular filters and epithelial monolayers. Mac-1 remained low on transmigrated neutrophils suggesting that this adhesion molecule is also not involved in the later events of dHL-60 cell transmigration to C5a. Twenty percent of Mac-1-deficient dHL-60 cells migrated across bare filters whereas only 5% migrated across T84 epithelial monolayers in response to fMLP, suggesting that the presence of Mac-1 is a requirement for the cell migration in response to fMLP. A small number of Mac-1-deficient dHL-60 cells were still able to migrate across T84 monolayers in response to fMLP (Fig. 3b) and the migration was not inhibited by anti-CD18 antibody (data not shown). This is consistent with the CD18-independent component of neutrophil migration in response to fMLP shown in Fig. 1(a). We found that dbcAMP-differentiated cells did not respond chemotactically to either IL-8 (10−10−10−8 m) or LTB4 (10−9−10−7 m), therefore we could not assess the Mac-1 dependency of transepithelial migration of these cells in response to IL-8 or LTB4 (data not shown).

Several aspects of neutrophil migration across cellular monolayers in vitro were studied previously using the neutrophil-differentiated HL-60 cell system, including migration across activated endothelial22 and lung epithelial monolayers in response to LTB4 and fMLP24,45, and transintestinal epithelial migration of eosinophil-differentiated HL-60 has been also reported.25 However, in none of these cases was the β2 integrin dependency of cell migration studied. We expand on this knowledge showing that DMSO-differentiated cells migrate across intestinal epithelial cells using both β2 integrin dependent and β2 integrin independent mechanisms, a pattern comparable to that of blood neutrophils. Furthermore we demonstrate that the dbcAMP-differentiated Mac-1 deficient HL-60 cells have a robust chemotactic and transepithelial migration response to C5a. Finally, our findings are not exclusive to migration across intestinal epithelial monolayers, because Mac-1-deficient dbcAMP dHL-60 cells are equally efficient at migrating across A549 lung epithelial monolayers (Fig. 5). The ability of dHL-60 lacking Mac-1 to migrate across epithelial monolayers demonstrates that Mac-1-dependent interactions are not necessary for the Mac-1-independent migration to occur. We also showed that migration was independent of other members of the β2 integrins, because β2 integrin blockade did not affect dbcAMP dHL-60 migration across epithelium (Figs 4b and 5).

The relevance of our findings to IBD lies in the fact that all of the chemoattractants that induce Mac-1-independent neutrophil transepithelial migration have been implicated in IBD. Both IL-8 and LTB4 were found to be elevated in mucosa and were detected in stool of IBD patients46–52 and C5a has been shown to be critical in the development of the trinitrobenzene sulphonic acid colitis in rats.53 It is not known what exactly attracts neutrophils to the gut lumen of IBD patients. Although bacterial products (including formylated peptides) are likely candidates, total bacterial numbers in lumen of IBD patients do not appear to be higher compared to healthy controls.54,55 Alternatively, the numbers of the epithelium-invading bacteria might be higher in such patients.55 Therefore, host responses to the bacterial invasion, including chemoattractants secreted by the host epithelium or generated by complement activation are likely to contribute to the neutrophil recruitment. Considering our results using two different epithelia, β2 integrin-independent mechanisms may be more important than previously appreciated with intestinal epithelium alone. Therefore, the Mac-1 deficient dbcAMP dHL-60 call model defined here is likely to be of considerable utility for the study of Mac-1-independent neutrophil functions including migration across epithelium and possibly other cellular barriers. All future discoveries made using HL-60 cells, however, will have to be subsequently tested with native neutrophils, including neutrophils from patients with inflammatory disease.

Acknowledgments

This study was supported by grants from the Crohn's and Colitis Foundation of Canada (A.W.S.) and Canadian Institutes of Health Research (MT-7684 to A.C.I.). S. Carrigan was supported by a scholarship from the Natural Sciences and Engineering Research Council of Canada. A. Weppler was supported by a scholarship from the Nova Scotia Health Research Foundation. We would like to thank Wendy J. Hughes for her technical assistance.

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