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. Author manuscript; available in PMC: 2014 May 20.
Published in final edited form as: J Leukoc Biol. 2001 Dec;70(6):969–976.

Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants

Barry Weinberger *, Debra L Laskin †, Thomas M Mariano ‡, Vasanthi R Sunil †, Christina J DeCoste †, Diane E Heck †, Carol R Gardner †, Jeffrey D Laskin ‡
PMCID: PMC4027972  NIHMSID: NIHMS575008  PMID: 11739560

Abstract

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-κB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-κB subunit and IκB-α, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-κB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Keywords: neonates, chemotaxis, NF-κB

INTRODUCTION

Chemoattractants are defined by their ability to induce directed migration of responsive cells toward sites of tissue injury. Several distinct classes of neutrophil chemoattractants have been identified, including N-formyl-methionyl-leucyl-phenylalanine (fMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), and platelet-activating factor (PAF). Despite normal binding of these chemoattractants to neonatal neutrophils, their ability to induce cellular responsiveness is impaired in neonatal cells relative to adult cells [1–5]. Neutrophils from newborns are also not primed effectively by bacterial lipopolysaccharide (LPS) during inflammatory responses [6, 7], and chemoattractant-induced membrane depolarization, calcium transport, and sugar uptake are diminished in these cells [5, 8]. It has been suggested that impaired responsiveness in neonatal neutrophils may be related to decreased expression or to down-regulation of cell-adhesion molecules, such as CD11b, CD14, and L-selectin [2, 9]. Defects in neutrophil chemotaxis and activation render human neonates uniquely susceptible to bacterial and fungal infections and have been associated with the pathogenesis of conditions such as infant respiratory distress syndrome [5, 10].

The biochemical signaling pathways leading to chemotaxis in neonatal cells have not been elucidated. In adult cells, fMLP binding is associated with a rapid increase in intracellular calcium [11]. This is followed by activation of protein kinases, including members of the mitogen-activated protein (MAP) kinase family and nuclear translocation of nuclear factor κB (NF-κB) [12, 13]. To analyze potential mechanisms underlying impaired chemotactic responsiveness in neonates, we compared the responses of adult and neonatal cells with fMLP and a unique chemotactic antibody, DL1.2, which activates distinct signaling pathways in neutrophils. We speculate that aberrations in signaling mechanisms contribute to defects in chemotaxis in neonatal cells.

MATERIALS AND METHODS

Reagents

LPS (serotype 0128:B12), fMLP, and Hank’s balanced salt solution (HBSS) were obtained from Sigma Chemical Co. (St. Louis, MO). BAY11-7085 and wortmannin were from Calbiochem (La Jolla, CA), and LY 294002 was from Biomol (Plymouth Meeting, PA). Fluo-4 was purchased from Molecular Probes (Eugene, OR). Mouse monoclonal antibody (mAb) to p42/44 MAP kinase was obtained from Zymed (S. San Francisco, CA), and rabbit polyclonal antibody to the doubly phosphorylated, active form of human p38 MAP kinase was from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to the p50 and p65 NF-κB subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse mAb to the fMLP receptor was from BD PharMingen (San Diego, CA). Horseradish peroxidase (HRP) and fluorescein-labeled goat anti-mouse or sheep anti-rabbit immunoglobulin (Ig)G secondary antibodies were obtained from BD Transduction Laboratories (Lexington, KY) and New England Biolabs. Rabbit IgG and mouse IgG1 controls were purchased from Santa Cruz Biotechnology and Coulter Immunotech (Miami, FL). Pertussis toxin was from Gibco BRL Life Technologies (San Diego, CA).

Cell isolation

Human neutrophils were isolated from heparinized umbilical cord blood from healthy, full-term infants or from venous blood from healthy adults. Cells were separated by dextran sedimentation and Ficoll density gradient centrifugation as previously described [14]. These studies were approved by the Institutional Review Board of St. Peter’s University Hospital (New Brunswick, NJ).

DL1.2 antibody

We have previously described a mouse mAb, L12.2, which is chemotactic for human neutrophils [15]. Using agarose and checkerboard assays, we confirmed that the response to this antibody involves directed migration (chemotaxis) of the cells [15]. The antibody used in the present studies was derived from cells that were recloned three times by serial dilution from this hybridoma and designated DL1.2. The hybridoma was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum. In dose-response experiments, we found that maximal chemotactic activity was observed with a 1:3 dilution of concentrated hybridoma culture supernatants, which corresponded to 100 ng/ml protein, and this concentration was used in our studies. An irrelevant mouse anti-human mAb, W6/32 [16], prepared from hybridoma culture supernatants containing 10% serum (as described for DL1.2), was used as a control.

Immunofluorescence and flow cytometry

Neutrophils (1.5×106/ml) were incubated for 60 min at room temperature with DL1.2, anti-fMLP receptor antibody (1:1000), or isotypic IgG1 control anti-body; washed (300 g, 5 min); and then incubated with FITC-labeled goat anti-mouse IgG. After 30 min, the cells were analyzed on a Coulter EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL). For analysis of p65 and IκB-α expression, neutrophils were fixed with 0.1% paraformaldehyde, permeabilized with lysophosphatidylcholine (4 μM), and incubated overnight with a 1:500 dilution of anti-p65, anti-IκB-α, or IgG control. Cells were then washed and incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (1:500, 30 min), and equal numbers of cells (104) were analyzed by flow cytometry. Fluorescence histograms were analyzed by Overton’s cumulative subtraction routine of the Coulter Cytologic Software program. We found that expression of the DL1.2 antigen, like the L12.2 parent clone [15], was limited to neutrophils (unpublished results).

Measurement of neutrophil chemotaxis

The modified Boyden chamber technique [17] was used to assay chemotaxis of neutrophils through Millipore filters. For our studies, we used a 48-well microchemotaxis chamber (Neuro Probe, Pleasanton, CA). DL1.2 (100 ng/mL), fMLP (5×10−8 M), HBSS, or antibody control was placed in each well of the lower chamber. A 5 μm pore-size polycarbonate filter was then placed over the wells, and the upper chamber was set into place. The neutrophil suspension [50 μl; 1×105 cells in HBSS containing 0.5% bovine serum albumin (BSA) and 2.4 mg/ml HEPES, pH 7.2] was added to each well. After incubation for 45 min at 37°C, the filter containing adhered, migrated neutrophils was removed and stained with Wright-Giemsa. Chemotaxis was quantified as the number of cells that migrated through the filter in 10 oil immersion fields. Data are presented as the mean ± SE of four experiments. In some experiments, cells were incubated at room temperature with BAY11-7085 (10 μM, 1 h), LY 294002 (50 μM, 5 min), wortmannin (100 nM, 10 min), pertussis toxin (1 μg/ml, 30 min), cycloheximide (10 μM, 30 min), or actinomycin D (5 μg/ml, 30 min) prior to analysis of chemotactic responsiveness.

Western blotting

Neutrophils were suspended in phosphate-buffered saline (1×106/ml) containing CaCl2 (1.8 μM) with and without fMLP, DL1.2, 12-O-tetradecanoylphorbol 13-acetate (TPA; 170 nM), or an irrelevant antibody control. The cells were incubated for 5 min at 37°C and then solubilized in buffer containing 50 mM HEPES, pH 7.4, 10 mM KCl, 1 mM ethylenediaminetetraacetate (EDTA), 1 mM dithiothreitol (DTT), 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 10 μg/ml aprotinin, and 1% Triton X-100. After 10 min on ice, lysates were centrifuged (4000 g, 5 min), and protein concentrations in the supernatants were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL) with BSA as the standard. Aliquots of supernatants containing 10 μg protein were fractionated on 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gels and electroblotted onto 0.45 μm nitrocellulose paper (Bio-Rad, Hercules, CA) at 250 mA for 4 h in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad) in 25 mM Tris, pH 8.3, 192 mM glycine, 20% (v/v) methanol [18, 19]. Blots were then incubated with DL1.2 (100 ng/ml), mouse mAb to p42/44 MAP kinase (1:500), or rabbit polyclonal anti-phosphorylated human p38 MAP kinase (1:250). After washing, the blots were incubated with goat anti-mouse IgG (1:2000) or sheep anti-rabbit IgG (1:1000) HRP-conjugated antibodies (BD Transduction Laboratories and New England Biolabs) for 1 h at room temperature. Proteins were detected using a Renaissance Western Blot Chemiluminescence Reagent Plus kit (NEN Life Sciences, Boston, MA). Treatment of the lysates with 2-mercaptoethanol did not alter the apparent molecular mass of the DL1.2 antigen in the gels.

Glycosidase treatment

Deglycosylation reactions were carried out using a GlycoPro kit (ProZyme, San Leandro, CA). Briefly, 15 μg neutrophil proteins were diluted to 35 μl with distilled water. Sodium phosphate buffer (10 μl of 0.25 M; pH 7.0) and 2.5 μl denaturing solution (2% SDS, 1 M 2-mercaptoethanol) were then added. After boiling for 5 min and cooling to room temperature, 2.5 μl of 15% Triton X-100 and 0.5 μl glycosidase(s) [2500 U/ml peptide-N-glycosidase F; 1 U/ml endo-O-glycosidase; 2.5 U/ml sialidase A; or 2 μl proteinase K (10 mg/ml)] were added. The samples were then incubated for 6 h at 37°C, and one-half of each reaction was analyzed by Western blotting as described above. Aliquots of fetuin (15 μg) were incubated under the same conditions and served as enzyme controls. These products were stained with Coomassie Brilliant Blue after electrophoresis.

Analysis of intracellular-free calcium

Neutrophils suspended in HBSS containing 0.5% BSA and 2.4 mg/ml HEPES, pH 7.2 (1×106/ml), were incubated for 30 min (37°C) with Fluo-4 (5 μg/ml). The cells were then stimulated with fMLP, DL1.2, or control and analyzed by flow cytometry 0.5–5 min later. Fluo-4 exhibits characteristic fluorescence, which is proportional to the binding of the dye to intracellular calcium [20].

NF-κB nuclear-binding activity

An electrophoretic mobility shift assay (EMSA) was used to detect nuclear binding of activated NF-κB. Neutrophils were suspended in HBSS (1.5×106/ml) containing 0.1% BSA and 2.4 mg/ml HEPES, pH 7.2. Following incubation with fMLP, DL1.2, or control for 45 min, nuclear extracts were prepared [21]. Briefly, neutrophils were centrifuged for 15 s in a microfuge and then resuspended in 400 μl cold buffer [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF)]. After 15 min on ice, 25 μl of a 10% solution of Nonidet P-40 (NP-40) was added, and the tube was mixed on a vortex for 10 s. The lysate was then centrifuged for 30 s in a microfuge, and the nuclear pellet was resuspended in 50 μl ice-cold buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and placed on a rocker platform at 4°C for 15 min. The sample was then microcentrifuged for 5 min at 4°C, and the resulting soluble nuclear extract was collected for analysis.

A double-stranded probe containing an NF-κB binding site (5′-AGT TGA GGG GAC CGC CCG CGG CCC GT-3′; Integrated DNA Technologies, Skokie, IL) was labeled with (α-32P)-dCTP. For binding reactions, nuclear extracts (50 μg protein) were incubated for 15 min at room temperature with labeled probe in buffer containing 10 mM Tris-HCl, pH 7.4, 40 mM NaCl, 10 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% NP-40, 4% glycerol, and 0.1 μg/μl poly(dI-dC) · poly(dI-dC) (Pharmacia Biotech, Piscataway, NJ). For competitive binding assays, nuclear extracts were incubated with a 100-fold excess unlabeled probe together with a 32P-labeled probe. Supershift assays were performed by incubation of the samples with polyclonal antibodies to p50 or p65 (1 μg/60 μl reaction volume) for 1 h prior to addition of the labeled probe. Samples were electrophoresed on 4% nondenaturing polyacrylamide gels in buffer (0.04 M Tris-acetate, 0.001 M EDTA, pH 8.0) at 15 V/cm. Gels were then dried and autoradiographed.

RESULTS

Chemotactic response of adult and neonatal neutrophils to fMLP and DL1.2

At an optimal concentration (100 ng/ml), DL1.2 exhibited neutrophil chemotactic activity that was comparable to fMLP (Fig. 1). Chemotactic activity was not observed with W6/32, an irrelevant anti-human control mAb, demonstrating that the response was specific for DL1.2. These findings are similar to those previously demonstrated using the parental clone L12.2 [15]. Neutrophils from neonates were found to be significantly less responsive to DL1.2, as well as fMLP, when compared with adult cells (Fig. 1). To determine if this was because of decreased expression of the antigen recognized by DL1.2 or the fMLP receptor, we analyzed the cells by flow cytometry. In adult and neonatal neutrophils, binding of DL1.2 and antibody to the fMLP receptor was homogeneous (Fig. 2). Whereas binding of anti-fMLP receptor antibody was similar in adult and neonatal cells (Fig. 2), DL1.2 binding was 10- to 20-fold greater in neutrophils from neonates than in neutrophils from adults.

Fig. 1.

Fig. 1

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Comparison of chemotactic responsiveness of adult and neonatal neutrophils. Chemotaxis of neutrophils toward fMLP, DL1.2, or an irrelevant control antibody (W6/32) was assayed using microwell chambers as described in Materials and Methods. Each bar is the mean ± SE of four experiments. *, Significantly different (P<0.05) from adult neutrophils.

Fig. 2.

Fig. 2

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Flow cytometric analysis of DL1.2 antigen and fMLP receptor expression in adult and neonatal neutrophils. Cells were incubated for 60 min at room temperature with DL1.2, anti-fMLP receptor antibody, or IgG1 control. Neutrophils were then washed and incubated with FITC-labeled goat anti-mouse IgG. After 30 min, the cells were analyzed by flow cytometry. Each curve represents an individual donor sample. Four to six representative donors are shown.

In further studies, we characterized the antigen recognized by DL1.2 in adult and neonatal cells. Western blot analysis revealed that DL1.2 recognized a protein on adult neutrophils with an apparent molecular mass of approximately 120 kDa (Fig. 3). It is interesting that in neonates, we consistently observed that the apparent molecular mass of the antigen was 2–3 kDa greater than in adults. Protease treatment of lysates from adult and neonatal neutrophils, prior to Western blot analysis, completely abrogated DL1.2 binding, and digestion with N-glycosidase F resulted in the appearance of three distinct lower molecular-mass proteins (Mr=25 kDa, 28 kDa, and 55 kDa; Fig. 3). The 2–3 kDa difference in the apparent molecular mass of the DL1.2 antigen between adult and neo-natal cells persisted in the largest protein (Mr=55 kDa) after N-glycosidase F digestion. In contrast, treatment of neutrophil lysates with sialidase or O-glycosidase had no major effects on the molecular mass of the DL1.2 antigen in adult or neonatal neutrophils.

Fig. 3.

Fig. 3

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Comparison of the DL1.2 antigen in adult and neonatal cells. Extracts from neonatal (lanes 1, 3, 7, 9, 11, and 13) or adult (lanes 2, 4, 5, 6, 8, 10, and 12) neutrophils were treated with buffer only (lanes 1–4, 6, 7), proteinase K (lane 5), N-glycosidase F (lanes 8 and 9), sialidase (lanes 10 and 11), or N-glycosidase F, endo-O-glycosidase, and sialidase (lanes 12 and 13). The proteins (7.5 μg/lane) were then analyzed by Western blotting as described in Materials and Methods. The arrow indicates the 120 kDa band of the DL1.2 antigen. One representative blot of three is shown.

Chemoattractant-induced signaling in adult and neonatal neutrophils

In our next series of studies, we compared biochemical signaling pathways induced by fMLP and DL1.2 in adult and neonatal neutrophils. Consistent with previous studies [11], we found that fMLP caused a rapid and transient increase in intracellular calcium levels in neutrophils from adults, which peaked within 30 s (Fig. 4). Although fMLP also induced increases in intracellular calcium in neonatal neutrophils, the response of these cells was reduced. Moreover, there appeared to be a subpopulation of neonatal cells that did not mobilize calcium after fMLP stimulation. In contrast to fMLP, DL1.2 had no effect on intracellular calcium levels in adult or neonatal neutrophils.

Fig. 4.

Fig. 4

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Chemoattractant-induced calcium mobilization in adult and neonatal neutrophils. Cells from adults or neonates loaded with Fluo-4 were stimulated with fMLP or DL1.2. Samples (104 neutrophils) were analyzed by flow cytometry 30 s, 1 min, 3 min, or 5 min later. One representative histogram for adults and neonatal cells is shown (n=3–4).

Phosphatidylinositol-3-kinase (PI3K) catalyzes the formation of phosphoinositides, which play key roles in fMLP-induced neutrophil activation and in transmembrane calcium flux [22]. In adult and neonatal neutrophils, chemotaxis induced by DL1.2 and fMLP was strongly inhibited by wortmannin and LY 240092, two agents that block PI3K (Table 1) [22, 23]. No major differences were noted between the cells. Similarly, in adult and neonatal cells, chemotaxis induced by DL1.2 and fMLP was inhibited by pertussis toxin, which is known to block G protein-coupled receptor signaling pathways [24]. Chemotaxis was also inhibited by the RNA and protein synthesis inhibitors actinomycin D and cycloheximide (Table 1).

TABLE 1.

Effects of Inhibitors on Neutrophil Chemotaxis

Chemotaxis (% Control)
Adults Neonates
fMLP
 Control 100 100
 Wortmannin 35.0 ± 2.9* 31.8 ± 1.6*
 LY 294002 25.9 ± 3.4* 24.2 ± 1.1*
 BAY11-7085 40.7 ± 4.4* 50.7 ± 3.5*
 Pertussis toxin 67.6 ± 5.7* 46.7 ± 13.5*
 Cycloheximide 63.4 ± 8.2* 70.0 ± 4.0*
 Actinomycin D 34.2 ± 9.6* 35.7 ± 19.2*
DL1.2
 Control 100 100
 Wortmannin 29.4 ± 2.1* 38.6 ± 2.0*
 LY 294002 27.3 ± 1.9* 34.2 ± 1.5*
 BAY11-7085 25.0 ± 3.5* 56.5 ± 3.4*
 Pertussis toxin 12.0 ± 12.5* 62.0 ± 4.9*
 Cycloheximide 46.9 ± 10.5* 61.0 ± 2.0*
 Actinomycin D 12.0 ± 19.5* 58.3 ± 5.4*
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Neutrophils from adults or neonates were preincubated at room temperature with buffer control, wortmannin (100 nM, 10 min), LY 240092 (50 μM, 5 min), BAY11-7085 (10 μM, 1 hr), pertussis toxin (1 μg/ml, 30 min), cycloheximide (10 μM, 30 min), or actinomycin D (5 μg/ml, 30 min). Chemotaxis toward fMLP or DL1.2 was then assessed in microwell chambers. Values represent the percentage of control chemotaxis in the absence of inhibitors (mean±SE, n=6–12).

*

Significantly different (P<0.05) from control.

Recent studies have implicated MAP kinases in chemoattractant-induced signaling in adult neutrophils [25–27]. Western blot analysis showed that fMLP-induced activation of p42/44 and p38 MAP kinases was similar in adult and neonatal neutrophils (Fig. 5, and unpublished results). Thus, in both cell types, activation, as indicated by decreased electrophoretic mobility, was evident within 5 min. In contrast to fMLP, DL1.2 had no effect on p42/44 or p38 MAP kinase in either cell type.

Fig. 5.

Fig. 5

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Chemoattractant-induced activation of p42/44 and p38 MAP kinases. Neutrophils from adults were incubated for 5 min at 37°C with medium control (lanes 1 and 6), DL1.2 (lanes 3 and 8), fMLP (lanes 4 and 9), an irrelevant antibody control (lanes 5 and 10), or TPA (lanes 2 and 7), which was used as a positive control [51]. Cellular extracts (10 μg/lane) were analyzed by Western blotting. One representative blot of three is shown.

The transcription factor NF-κB is a downstream signaling molecule known to be activated by chemoattractants [12, 13]. We found that fMLP, as well as DL1.2, rapidly induced nuclear translocation of NF-κB proteins in adults and neonates (Fig. 6). Supershift assays revealed that the NF-κB complex contained p50 and p65 subunits. Preincubation of nuclear extracts with excess unlabeled probe competitively reduced binding of the labeled probe, demonstrating its specificity. In adult and neonatal neutrophils, BAY11-7085, which blocks NF-κB activation [28], significantly reduced chemotaxis in response to fMLP, as well as to DL1.2 (Table 1). As observed with chemotaxis, activation of NF-κB by fMLP and DL1.2 was reduced in neonatal relative to adult neutrophils (Fig. 6). This was correlated with reduced constitutive expression of NF-κB p65 and IκB-α protein in neonatal neutrophils. Moreover, whereas treatment of adult cells with fMLP or DL1.2 resulted in decreased expression of p65 and IκB-α, this response was diminished in cells from neonates (Fig. 7).

Fig. 6.

Fig. 6

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Chemoattractant-induced NF-κB nuclear-binding activity. Neonatal or adult neutrophils were incubated with fMLP, DL1.2, TPA (170 nM), LPS (100 ng/ml), or medium control (unstimulated) for 15 min. Nuclear extracts (50 μg protein/lane) were analyzed by an electrophoretic mobility shift assay. Supershift assays were performed in DL1.2-stimulated cells from adults using control medium (−) or polyclonal antibodies to p50 or p65 proteins. To demonstrate specificity, nuclear extracts from DL1.2-stimulated neutrophils were incubated with (Cold Comp.) or without (−) 100-fold excess of unlabeled, double-stranded probe in addition to the 32P-labeled probe. One representative blot of two is shown.

Fig. 7.

Fig. 7

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Flow cytometric analysis of NF-κB p65 and IκB-α expression in adult and neonatal neutrophils. Freshly isolated neutrophils were fixed and permeabilized (top panels) or incubated for 60 min with fMLP, DL1.2, or an irrelevant antibody (control) prior to fixation and permeabilization (bottom panels). Cells were then incubated overnight with anti-p65 or anti-IκB-α antibodies, or IgG control, washed, incubated with FITC-labeled goat anti-rabbit IgG (30 min), and analyzed by flow cytometry. Studies were performed on cells from three adults and three neonates. Data from one representative donor are shown.

DISCUSSION

The accumulation of neutrophils at sites of tissue injury is mediated by chemotactic factors released as part of the inflammatory response. This process is known to be impaired in neutrophils from neonates [5, 8]. The present studies were aimed at elucidating alterations in biochemical signaling pathways that may underlie defects in neonatal chemotaxis. Consistent with previous studies [29], we found that fMLP receptor expression was similar in adult and neonatal neutrophils. In contrast, neonatal neutrophils expressed 10- to 20-fold more of the chemotactic antigen, which binds DL1.2 when compared with adult neutrophils. This may be a result of maturational changes in sugar or protein components of the antigen that cause alterations in its affinity and/or accessibility to the antibody. These findings indicate that reduced responsiveness of neonatal cells is not a result of diminished binding of chemoattractants. Biochemical analysis of the DL1.2 antigen revealed that it is a glycoprotein with an apparent molecular mass of 120 kDa. This is significantly larger than chemotactic receptors for fMLP, PAF, IL-8, or LTB4, demonstrating that it is unique [30–33]. It is interesting that the DL1.2 antigen appeared to be 2–3 kDa larger in neonatal cells when compared with adult cells, suggesting that the antigen is developmentally linked. Similar maturational changes in expression of CD11b, CD14, CD35 (CR1), CD55, and immunoglobulin receptors have been described previously [2, 6, 34, 35]. Digestion of neutrophil proteins with glycosidases showed that the DL1.2 antigen contained N-linked sugars but not sufficient mannose residues or O-linked sugars to affect the mobility of the antigen on polyacrylamide gels. Under our conditions, N-glycosidase F digestion of the DL1.2 antigen yielded three immunoreactive peptides ranging in molecular weight from 24 to 55 kDa. The small difference in molecular weight of the DL1.2 antigen in neonatal and adult cells persisted in the larger (55 kDa) fragment, possibly because of differences in the size of the protein and/or other post-translational modifications.

One of the earliest biochemical responses observed in neutrophils following fMLP binding is mobilization of intracellular calcium [36, 37]. We found that adult and neonatal neutrophils differed with respect to fMLP-induced calcium mobilization. Whereas intracellular calcium levels increased rapidly after treatment of adult and neonatal neutrophils with fMLP, this response was attenuated in cells from neonates. Moreover, in neonatal cells, a subpopulation was identified that was unresponsive to fMLP. It is possible that decreased calcium mobilization contributes to impaired chemotaxis of neonatal cells to fMLP. In contrast to fMLP, DL1.2 did not induce calcium mobilization in adult or neonatal neutrophils, suggesting that calcium does not play a role in DL1.2-induced chemotaxis. These findings are consistent with recent studies of other inflammatory mediators that induce neutrophil chemotaxis without altering intracellular calcium, including transforming growth factor-β1 and soluble Fas ligand [38, 39]. Thus, it appears that the requirement for calcium in neutrophil chemotaxis is dependent on the chemoattractant and that impaired calcium mobilization, by itself, is not sufficient to account for decreased chemotaxis in neonatal cells.

In adult and neonatal neutrophils, chemotaxis induced by fMLP, as well as DL1.2, was blocked by wortmannin and LY 294002, two inhibitors of PI3K. This enzyme catalyzes the phosphorylation of PIP2 to PIP3, a step required for calcium-dependent neutrophil chemotaxis in response to fMLP, IL-8, and PAF [40–42]. The enzymatic products of PI3K induce some of their effects by mobilizing intracellular calcium stores [43, 44]. Our data suggest that PI3K activation is important in DL1.2-induced chemotaxis but that this enzyme exerts its effects in neutrophils through a calcium-independent pathway. It is possible that phosphoinositides alter neutrophil polarization and motility by interacting with actin-binding proteins and inducing cytoskeletal reorganization [45]. Alternatively, PI3K-regulated phospholipid signaling may modulate neutrophil adhesion and motility by activating Akt/protein kinase B [46].

We also found that chemotaxis in response to DL1.2 and fMLP was inhibited by pertussis toxin in adult and neonatal cells, indicating that their activity is linked to G proteins. These are heterotrimeric molecules that function as intermediates in transmembrane signaling pathways initiated by various hormones, neurotransmitters, and chemoattractants [24, 36]. Activation of G proteins increases guanosine 5′-triphosphate (GTP) binding, leading to phosphoinositide hydrolysis, calcium mobilization, and induction of protein kinase C. Our finding that DL1.2-induced chemotaxis is calcium-independent indicates that the signaling pathways initiated by G-protein activation are distinct for different chemoattractants.

Members of the MAP kinase family are also thought to be important in the signaling pathways leading to chemotaxis [25–27]. The p38 MAP kinase and the p42/44 extracellular signal-related kinase (ERK) are distinct proteins that have been identified in neutrophils [47]. We found that both enzymes were activated by fMLP in neonatal and adult cells. Similar effects of fMLP have been shown in adult neutrophils [25–27]. The fact that no major differences were observed between adults and neonates indicates that these MAP kinases are not involved in reduced chemotaxis of neonatal cells to fMLP. In contrast to fMLP, DL1.2 had no effect on p38 or p42/44 MAP kinase activation in adult or neonatal neutrophils. These data support the concept that fMLP and DL1.2-induced chemotaxis are regulated by distinct signaling pathways.

NF-κB is a ubiquitous transcription factor activated by a number of inflammatory mediators, including fMLP [12, 13]. DL1.2, like fMLP, was found to induce nuclear translocation and DNA binding of NF-κB in adult and neonatal neutrophils. Moreover, inhibition of NF-κB activity resulted in decreased DL1.2 as well as fMLP-induced chemotaxis in both cell types. These results suggest that the NF-κB signaling pathway plays an important role in neutrophil chemotaxis. This is consistent with the observation that suppression of NF-κB activation significantly reduced endotoxin-induced neutrophil infiltration into the lung [48, 49]. We also found that NF-κB nuclear binding activity in response to fMLP and DL1.2 was decreased in neonatal neutrophils when compared with adult cells. Expression of the NF-κB p65 subunit and IκB-α proteins was also reduced in neonatal cells. Following treatment with fMLP or DL1.2, levels of these proteins were reduced to a greater extent in adult when compared with neonatal neutrophils. Based on these findings, it appears that impaired responsiveness of neonatal neutrophils to chemoattractants is, at least in part, a result of altered NF-κB signaling. This may be mediated by diminished levels of p65 in neonatal cells and/or to reduced regeneration of transcription factor proteins following chemoattractant-induced activation. The findings that chemotaxis is inhibited by actinomycin D and cycloheximide, which block RNA and protein synthesis, respectively, provide support for this possibility.

The present studies suggest that deficiencies in neonatal neutrophil chemotaxis are a result of aberrations in chemoattractant-induced signaling pathways. Moreover, maturational changes underlying developmental defects in neutrophil-functional activity are specific for the chemoattractant. Thus, whereas reduced NF-κB activity appears to contribute to the diminished responsiveness of neonatal cells to fMLP and DL1.2, defective calcium mobilization is important only in the response to fMLP. The relative inability of neutrophils in newborns to accumulate at sites of bacterial invasion is the most consistent defect described in neonatal sepsis [50, 51]. A more complete understanding of the pathways controlling neutrophil migration and activation may provide insights into new therapeutic approaches for treating infectious and inflammatory diseases in neonates.

Acknowledgments

This work was supported by National Institutes of Health grants ES05022, ES04738, ES06897, and GM34310 and by a grant from the New Jersey Thoracic Society.

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