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(Journal of Leukocyte Biology. 2001;69:289-296.)
© 2001 by Society for Leukocyte Biology

Colostral neutrophils express Fc{alpha} receptors (CD89) lacking {gamma} chain association and mediate noninflammatory properties of secretory IgA

Adenilda C. Honorio-França*, Pierre Launay{dagger}, Magda M. S. Carneiro-Sampaio* and Renato C. Monteiro{ddagger},{dagger}

* Department of Immunology of Instituto de Ciências Biomédicas, and
{ddagger} Division of Rheumatology, University of São Paulo, SP, Brazil
{dagger} Institut National de la Santé et de la Recherche Médicale, Unité 25, Hôpital Necker, Paris, France

Correspondence: R. C. Monteiro, INSERM U25, Hôpital Necker, 161, rue de Sèvres, 75743 Paris, France. E-mail: monteiro{at}necker.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colostrum plays an important role in protecting newborn infants against acute gastrointestinal and respiratory infections. IgA antibodies have been considered the major effector component; however, the role of their receptors on colostral phagocytes, especially neutrophils, has not been studied. Here, we demonstrate that CD15+ colostrum neutrophils express IgA Fc receptors (Fc{alpha}R, CD89) at levels similar to those of blood neutrophils. Most colostral cells (70%) bear secretory IgA (SIgA) on their surface (and intracellularly), whereas blood cells do not. The Fc{alpha}R on colostral neutrophils was identified as the a.1 isoform with a similar molecular mass (55–75 kDa) as that identified for blood neutrophils. Removal of N-linked carbohydrates revealed a major protein core of 32 kDa for both cell types. In contrast, co-immunoprecipitation and immunoblot experiments using a mild detergent, digitonin, revealed a lack of {gamma} chain association with Fc{alpha}R ({gamma}-less) exclusively on colostral neutrophils. The functional role of these {gamma}-less Fc{alpha}R cells was evaluated by measuring superoxide release and killing of SIgA-coated enteropathogenic E. coli. No increase in superoxide release was observed in colostral cells compared with blood neutrophils, whereas optimal release was obtained with PMA stimulation. Furthermore, despite similar bacterial phagocytosis index between both cell types, IgA-mediated bacterial-killing was not detectable with colostral neutrophils, whereas killing was detectable on blood cells. These results reveal exclusive expression of {gamma}-less Fc{alpha}R on colostral neutrophils associated with receptor hyperoccupation by IgA and with low, bacterial-killing activity, which suggest that this receptor may mediate noninflammatory effects of SIgA.

Key Words: human milk • IgA • EPEC • SOD • PMA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human colostrum confers protection against gastrointestinal and respiratory infections in infants [1 , 2 ]. This protective, anti-infectious role is mediated mainly by immunoglobulin (Ig)A antibodies with anti-bacterial and anti-viral activity [3 4 5 6 7 8 9 ]. In colostrum, IgA is present mainly in secretory (S) forms consisting of a dimer that is held together by the joining chain, whereas serum IgA is present mainly in monomeric forms [4 ]. In addition to antibodies and soluble, nonspecific, anti-infectious factors (e.g., lysozyme, lactoferrin, and oligosaccharides), human colostrum contains large amounts of viable leukocytes (109 cells/ml during the first days of lactation). The main cell lineages in this secretion (>80%) consist of phagocyte populations, including many macrophages and neutrophils that contain intracellular IgA [10 , 11 ]. Recently, we demonstrated that colostrum mononuclear phagocytes (MN) are able to kill enteropathogenic Escherichia coli (EPEC) after opsonization with SIgA [12 ]. These cells express IgA receptors (Fc{alpha}R; CD89), and their bactericidal activity against SIgA-coated bacteria was reduced significantly when MN cells were preincubated with My43, an anti-CD89 mAb [12 , 13 ]. Superoxide release was also mediated by Fc{alpha}R [12 ].

Fc{alpha}R (CD89) are expressed on the surface of blood neutrophils, eosinophils, and monocytes/macrophages [14 15 16 17 18 ]. Fc{alpha}R are type I transmembrane molecules, distinct from the asialoglycoprotein or polymeric IgA receptors that are encoded by a single gene located on chromosome 19 [16 , 19 ]. Fc{alpha}R exists as at least two isoforms (a.1 and a.2) that are expressed differentially by blood monocytes and alveolar macrophages [18 ]. CD89 are heterogeneously glycosylated proteins with a molecular mass ranging from 55 to 100 kDa, which binds monomeric and polymeric IgA1 and IgA2 antibodies and SIgA, at the boundary between the C{alpha}2 and C{alpha}3 domains [15 , 17 , 20 ]. Fc{alpha}R is a low-affinity receptor (Ka~106 M-1) that differs from other FcR in its IgA binding site, which has been located in the first extracellular domain [21 , 22 ]. It has been shown that Fc{alpha}R is associated with the {gamma} subunits [23 24 25 ]. Recently, we demonstrated that Fc{alpha}R are expressed, associated or not, with {gamma} chains on blood monocytes and neutrophils [26 ]. Studies of mutant Fc{alpha}R expressed in transfectants revealed that Fc{alpha}R-{gamma}2 mediates downstream signaling events including cytokine release, calcium influx, exocytosis, endocytosis, and antigen degradation [25 , 26 ], and the {gamma}-less Fc{alpha}R mediates protection against degradation of serum IgA by an endocytosis/recycling mechanism [26 ]. Activation of {gamma}-associated Fc{alpha}R induces recruitment and phosphorylation of tyrosine kinases including lyn, syk, and Btk [27 , 28 ].

Little is known about the functional activities of colostral polymorphonuclear phagocytes in the newborn gut. Some studies have shown that colostral neutrophils present lower phagocytic and bactericidal activities than do blood neutrophils [29 ]. We wondered if Fc{alpha}R could exist and function on colostral polymorphonuclear cells. Here, we show that Fc{alpha}R is expressed by colostral neutrophils but lacks the {gamma} association and presents low bacteria-killing opsonized with SIgA. {gamma}-less Fc{alpha}R bear SIgA, which may be important in transport and protection of neonate gut from inflammatory mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
After obtaining informed consent, approximately 15 ml colostrum and 10 ml blood were collected from 120 clinically healthy women 18–35 years of age at the Hospital Universitário (São Paulo, Brazil), between 48 and 72 h postpartum [12 ]. These patients lived in an endemic area for EPEC infection, and all presented high levels of anti-EPEC antibodies in serum and colostrum [30 ]. Twenty other samples were collected from healthy women in the Hôpital Bon Secours (Paris). Blood was collected from 30 volunteer donors 18–35 years of age and living in the same endemic area in São Paulo. Serum from a 4-year-old patient with X-linked agammaglobulinemia (before IVIG treatment) was also studied (0.66 g/ml IgG and undetectable IgM and IgA).

Separation of colostral and blood cells
Colostrum was separated into three distinct phases: the cell pellet, an intermediate aqueous phase, and a lipid-containing supernatant, as described [12 ]. Cells were separated by a Ficoll-Paque gradient (Pharmacia, Upsala, Sweden). This procedure resulted in 95% pure polymorphonuclear cell preparations as analyzed by light microscopy. Eosinophils were rarely seen. Purified neutrophils were resuspended independently in serum-free medium 199 at a final concentration of 2 x 106 cells/ml. The heparinized blood was obtained from 30 volunteer donors 18–35 years of age, fractionated by Ficoll-Paque centrifugation, and subjected to dextran sedimentation [15 ]. Neutrophil purity was over 95%, as determined by morphologic criteria. Purified polymorphonuclear neutrophil (PMN) phagocytes were resuspended independently in serum-free medium 199 and washed twice. The resulting PMN phagocyte suspensions were adjusted to 2 x 106 cells/ml. Most studies were done with colostral and blood neutrophils from the same donor. The human monocytic cell line U937 was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin.

Antibodies
Mouse monoclonal antibodies (mAbs) used were A77 (IgG1{kappa}) mAb specific for Fc{alpha}R [31 ], 32.2 (IgG1{kappa}) mAb specific for Fc{gamma}RI [CD 64; American Type Culture Collection (ATCC), Rockville, MD], IV.3 (IgG2b) mAb specific for Fc{gamma}RII (CD 32; ATCC), 3G8 (IgG1{kappa}) mAb specific for Fc{gamma}RIII (CD16), and an irrelevant IgG1{kappa} control mAb (clone 7.1 anti-GST protein). Fluorescein isothiocyanate (FITC)-conjugated anti-CD15 and phycoerythrin (PE)-labeled anti-CD89 (clone A59) were purchased from PharMingen (San Diego, CA). Rabbit anti-mouse Ig (RAM) antibodies were obtained from rabbits immunized with an IgG1{kappa} (clone A59). F(ab')2 fragments of A77, IgG1{kappa}, and RAM IgG fractions were prepared by pepsin digestion (Sigma Chemical Co., St. Louis, MO) as previously described [32 ] and purified on (diethylamino)ethyl (DEAE) columns. Complete digestion and F(ab')2 purity were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit anti-serum specific for the {gamma}-chain was kindly provided by Dr. U. Blank (Pasteur Institute, Paris, France) and used as described [27 ]. FITC-conjugated goat Ab specific for mouse (GAM) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG were purchased from Southern Biotechnology Associates (Birmingham, AL). Human SIgA was purified from a defatted colostrum pool by affinity chromatography on CNBr-Sepharose-4B (Sigma) bound to sheep anti-human IgA as described previously [12 ]. The purified IgA preparation was also tested by immunoelectrophoresis using goat anti-human IgG and IgM antisera as described [12 ]. IgG and IgM were undetectable in the preparation. Mean levels of total IgG, IgM, and IgA in colostrum were 0.15, 0.4, and 7.5 mg/ml, respectively. Colostrum and sera contained IgA and IgG anti-EPEC antibody activities, respectively, as described previously [30 ].

Immunofluorescence and flow cytometry
Cells were preincubated with 10 µl human IgG (10 mg/ml) for 20 min on ice to mask Fc{gamma}R [31 ] before the incubation with 10 µl PE-labeled A59 mAb anti-Fc{alpha}R (0.1 mg/ml) and 10 µl FITC-conjugated, anti-CD15 mAb for 30 min at 4°C in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and 0.1% sodium azide. PE-labeled, irrelevant IgG1 mAb was used as negative control. After incubation, the cells were washed twice in PBS containing 5% BSA and 0.1% sodium azide. For blood cells, erythrocytes were lysed using a lysis solution (Becton Dickinson, Rutherford, NJ). For detection of IgA on the surface of PMN phagocytes, colostrum cells were washed extensively and then stained with a F(ab')2 fragment of anti-human IgA conjugated to FITC (Southern Biothenology Associates) for 30 min at 4°C. In all experiments, the cells were analyzed by flow cytometry (FACScalibur, Becton Dickinson). For internalization of surface-bound IgA, colostral cells were washed and incubated for different time periods before staining with PE-conjugated, goat, anti-human IgA Ab (Southern Biotechnology Associates) as described [32 ]. Blood cells were incubated with polymeric IgA1 (0.5 mg/ml) for 30 min at 4°C prior to endocytosis assay.

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted by the acid-phenol procedure, and cDNA synthesis was performed as described previously [18 ]. PCR was done with 2 µl cDNA, adding 20 pmol sense primer from the 5' leader region (GGG CTC GAG CAC GAT GGA CCC CAA ACA GAC C) and 20 pmol antisense primer from the 3' noncoding region (GGG GGA TCC TCC TCT CTG CCT TCA CC) of U937 Fc{alpha}R cDNA, 5 U Taq polymerase (Gibco-BRL, Grand Island, NY), 0.2 mM each dNTP (Promega, Madison, WI), and PCR buffer to a final vol of 50 µl [18 ]. In a thermal cycler, the mix then underwent 30 cycles of denaturation at 94°C for 45 sec, with annealing at 62°C for 45 sec and extension at 75°C for 1 min.

Cell iodination, immunoprecipitation, and immunoblotting
Cell (0.7–1x107)-surface iodination with Na125I (1 mCi; Amersham Corp., Arlington Heights, IL) was carried out by the lactoperoxidase method [33 ]. For immunoprecipitation of Fc{alpha}R, cells (107/ml) were lysed for 30 min at 4°C in PBS containing 0.5% Nonidet P-40 (NP-40) or 1% digitonin (Aldrich Chemical Co., Milwaukee, WI), 0.02% sodium azide, 1% aprotinin, 1 mM diisopropylfluorophosphate, 5 mM iodoacetamide, and 1 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 14,000 g for 30 min to remove insoluble materials, cleared lysates were immunodepleted of Fc{gamma}R using human IgG and 32.2, IV.3, and 3G8 mAbs and precipitated with test mAb as described previously [31 ]. Bound materials were treated or not treated with N-glycanase (Genzyme, Cambridge, MA), and samples were subsequently prepared for SDS-PAGE [34 ]. For immunoblotting, immunoprecipitated proteins were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose Hybond-C (Amersham) filter as described previously [27 ]. The blots were incubated in blocking buffer composed of 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl containing 3% BSA, and 0.1% Tween 20 and then incubated with anti-{gamma} (1 µg/ml) for 2 h at room temperature. HRP-conjugated, goat anti-rabbit IgG (1:3000 dilution) was used a secondary Ab. Filters were developed using the Enhanced Chemiluminescence (ECL; Amersham) detection system.

Bacterial opsonization
EPEC isolated from stools of an infant with acute diarrhea (serotype 0111:H-, LA+, eae+, EAF+, bfp+) [8 ] was prepared and adjusted to 108 bacteria/ml, as described previously [12 ]. Colostrum supernatants and serum from 10 individuals were collected, pooled, and frozen at -70°C. Immediately before use, colostral and serum aliquots were thawed and mixed with appropriate volumes of bacterial suspension to a final concentration of 2 x 107 bacteria/ml in 10% of the opsonin sources. Another bacterial suspension prepared at the same concentration in medium 199 without opsonin was used as an untreated bacterial control. Both bacterial suspensions were incubated for 30 min at 37°C and used in the bactericidal assays. Using immunofluorescence assay, we observed that close to 100% of bacteria was coated with IgA, as the latter were used in saturating conditions as described [12 ].

Release of superoxide anion
Release of superoxide was measured by determination of cytochrome C (Sigma) reduction as previously described [12 , 35 ]. Briefly, phagocytes and bacteria, opsonized or not, were mixed and incubated for 30 min for phagocytosis. Cells were then resuspended in PBS containing 2.6 mM CaCl2, 2 mM MgCl2, and cytochrome C (2 mg/ml). Phorbol myristate acetate (PMA) stimulation was performed as control at 0.5 µg/ml. After a 60 min incubation at 37°C, the reaction rate was measured by absorbance at 550 nm [35 ]. Results were expressed as percentage of maximal release obtained after incubation of cells with PMA in nmol/O2-. All the experiments were performed in duplicate or triplicate.

Bactericidal assay
The assay was performed as previously described [12 ]. Briefly, equal volumes of bacteria and cell suspensions were mixed and incubated at 37°C for 30 min. Phagocytosis was stopped by incubation on ice. To eliminate extracellular bacteria, the suspensions were centrifuged twice (160 g, 10 min, 4°C), and the cells were resuspended in serum-free medium 199. Bacterial-killing by phagocytes from colostrum or blood was determined using a microbiological plate technique [36 ] and evaluated during 2 h of incubation at 37°C under continuous shaking in the presence or absence of superoxide dismutase (SOD; 140 units) [37 ]. At 0 and 120 min, 0.5 ml bacterium/phagocyte suspensions was taken and analyzed for viability (>95% viable as determined by trypan blue exclusion), and cells were lysed in 0.6 ml of 1% Triton X-100 (Sigma) to release intracellular bacteria. From each tube, 100 µl was taken, and a series of sixfold dilutions were then prepared in tubes containing 900 µl Tryptic Soy Broth (TSB; Difco Labs, Detroit, MI) and plated onto agar Petri dishes. After 18 h at 37°C, the number of colonies was determined. The bactericidal index was calculated as follows: bactericidal index = 1 - (NT/NO) x 100, where NT is the number of colony-forming units at 120 min after phagocytosis, and NO represents the number of colony-forming units at time 0. No bactericidal activity was observed for SIgA, serum, or colostral supernatant pools alone. All experiments were performed in duplicate or triplicate. To verify bacterial internalization and phagocytosis index, cells were stained with an acridine orange method as described [38 ].

Statistical analysis
The Student’s t-test was applied to determine differences in superoxide release in the presence of different sources of opsonization. Analyses of variance (ANOVA) were used to compare the bacterial-killing index in the presence of different sources of opsonization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Colostral neutrophils express Fc{alpha}R and carry IgA on their membrane
First, we addressed the expression of Fc{alpha}R (CD89) on colostral polymorphonuclear cells by performing double immunofluorescence staining using anti-CD89 mAb with anti-CD15 or anti-IgA Ab. Figure 1 shows that all colostral polymorphonuclear cells were CD15 bright, indicating that these cells are mainly neutrophils, because eosinophils are known to express low levels of CD15 molecules [17 , 39 ]. All CD15+ colostral neutrophils express CD89 at similar levels as blood neutrophils (Table 1 ). We next examined whether colostrum Fc{alpha}R were occupied by IgA using anti-IgA F(ab')2 fragments. As shown in Figure 1a major subpopulation of colostral CD89+ neutrophils carried high levels of IgA on their cell surfaces (30-fold normal levels) and represented about 70% of total colostral polymorphonuclear cells. In contrast, only one population of blood CD89+ neutrophils bore very low levels of IgA on their cell surface as previously described [32 ]. Cytoplasmic staining of colostral polymorphonuclear cells also showed large amounts of intracellular IgA (unpublished results).



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  		Figure 1. Expression of CD89 bearing IgA on colostral polymorphonuclear cells. Cells were separated by centrifugation over Ficoll-Hypaque density gradients. Viable cells preincubated with excess human IgG to block Fc{gamma}R were stained directly with PE-labeled A59, anti-Fc{alpha}R mAb or with an irrelevant PE-labeled, IgG1 control. After washes, cells were stained with FITC-labeled anti-CD15 and with FITC-labeled F(ab')2 fragments of anti-IgA, as described in Materials and Methods. Two-color immunofluorescence analysis was then carried out by flow cytometry.

 

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  		Table 1. Comparative CD89 Expression on Neutrophils from Blood and Colostrum

 
Colostral neutrophils express the Fc{alpha}R a.1 isoform
Because alveolar macrophages express the spliced variant a.2 isoform of Fc{alpha}R, different than that expressed by blood cells [18 ], first we investigated the Fc{alpha}R molecular nature of colostral neutrophils using RT-PCR. Figure 2A shows that colostrum neutrophils expressed an 896 bp Fc{alpha}R transcript corresponding to the a.1 isoform. To determine the biochemical nature of the Fc{alpha}R expressed by colostral neutrophils, iodinated cell-surface proteins immunoprecipitated by anti-Fc{alpha}R mAb from blood and colostral neutrophils were examined by SDS-PAGE (Fig. 2B) . Fc{alpha}R molecules with an apparent Mr of 55–75 kDa were precipitated specifically from colostral neutrophils by A77 anti-Fc{alpha}R F(ab')2 fragments. When immunoprecitates were digested with N-glycanase, Fc{alpha}R proteins were resolved into a major band of 32 kDa similar to that of blood neutrophils. Similar results were obtained with cells from two additional individuals (unpublished results). These results indicate that colostral and blood neutrophils express the same Fc{alpha}R protein core corresponding to the full-length Fc{alpha}R a.1 isoform [18 ].



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  		Figure 2. Molecular characterization of Fc{alpha}R on colostral neutrophils. (A) Identification of Fc{alpha}R transcript a.1 in colostral neutrophils. The RT-PCR products were separated by 1% agarose gel electrophoresis. (B) Biochemical nature of Fc{alpha}R on neutrophils: Blood (lanes 1, 3, and 4) and colostral (lanes 2, 5, and 6) neutrophils were surface-labeled with Na125I, and the membrane proteins were solubilized using a 0.5% NP-40 lysis buffer as described in Materials and Methods. Lysates were divided into three aliquots and incubated with irrelevant IgG1{kappa} (lanes 1 and 2) or anti-Fc{alpha}R mAbs A77 F(ab')2 coupled to Sepharose 4B beads (lanes 3–6). Immunoprecipitates were digested or not with N-glycanase, as indicated (N-gly) and analyzed by 10% SDS-PAGE and autoradiography.

 
Fc{alpha}R on colostral neutrophils lack association with the {gamma} chain
We next investigated the physical association between Fc{alpha}R and the {gamma} subunit using digitonin-solubilized cells as described [26 ]. Fc{alpha}R were immunoprecipitated by A77 anti-Fc{alpha}R F(ab')2 fragments to avoid interaction with Fc{gamma}R. Precipitated proteins were analyzed by western blots using rabbit anti-{gamma} chain Ab. The {gamma} homodimer migrating in nonreducing gels around Mr 20,000 was detected in Fc{alpha}R precipitates of blood neutrophils but not on colostral neutrophils (Fig. 3A ). To rule out that {gamma} expression was different within these cell types, we next immunopreciptated total {gamma} chain and {gamma}-associated Fc{alpha}R using a single-cell lysate of neutrophils from blood or colostrum (Fig. 3B) . Although similar amounts of {gamma} subunits were detected in the two cell types, we confirmed that colostral neutrophils express only {gamma}-less Fc{alpha}R.



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  		Figure 3. Absence of association of {gamma} chain with human Fc{alpha}R in colostral neutrophils. Colostral and blood cells (107) were solubilized in 1% digitonin lysis buffer and immunoprecipitated using irrelevant IgG1 F(ab')2 fragments (IgG1) or A77 anti-Fc{alpha}R F(ab')2 fragments (A77) coupled to Sepharose 4B beads (A). (B) Lysates were incubated with a rabbit anti-{gamma}-chain (a-{gamma}) polyclonal Ab or A77 F(ab')2 fragments coupled to Sepharose 4B beads. Precipitated proteins were separated by 12% SDS-PAGE under nonreducing conditions and analyzed by immunoblotting using rabbit anti-{gamma}-chain polyclonal Ab and HRP-conjugated, anti-rabbit Ig Ab as described [27 ].

 
Detection of IgA on the cell surface of colostral neutrophils after endocytosis
We next examined the ability of colostral Fc{alpha}R to ingest and degrade endogenous IgA bound to Fc{alpha}R. Cells were washed and incubated at 37°C for indicated times to allow internalization. IgA detection was perfomed by flow cytometry using a PE-labeled, goat anti-IgA Ab as described [32 ]. As shown in Figure 4 , flow cytometry experiments revealed no decrease in the amounts of surface IgA even after 90 min of endocytosis. As blood neutrophils did not bear IgA, we preincubated these cells with polymeric IgA1 prior to endocytosis. As expected, blood neutrophils internalized rapidly and degraded IgA complexes as described previously [32 ]. In contrast, anti-IgA cytoplasmic stainings revealed decrease in intracellular but not surface IgA after 60–90 min of incubation of colostral neutrophils at 37°C (unpublished results).



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  		Figure 4. Detection of IgA on the cell surface after endocytosis. Colostral (open circles) neutrophils were washed and incubated at 37°C for times indicated. Cells were then surface-stained with PE-labeled, goat anti-IgA Ab and analyzed by a fluorescein-activated cell sorter (FACS). As control, blood (solid circles) neutrophils were first stained with polymeric IgA1 prior to incubation of cells at 37°C followed by anti-IgA staining as described previously [32 ]. Results were calculated as follows: 100 - [100x(x of A77 mAb incubated at 37°C-x of negative control incubated at 37°C)/(x of A77 mAb incubated at 0°C-x of negative control incubated at 0°C] in which x is the computer mean fluorescence intensity value of each FACS profile. *p < 0.001 Student’s t-test.

 
Colostral neutrophils failed to release superoxide anion in response to bacteria (EPEC)
To address the functional capacity of colostral neutrophils, we next examined superoxide anion release after phagocytosis of opsonized bacteria. As shown in Figure 5 , both cell types responded following treatment with PMA, indicating the ability of colostral neutrophils to release superoxide anion under these conditions. In contrast, no significant superoxide anion release by colostral neutrophils was observed in the presence of bacteria opsonized or not by SIgA, whereas a significant release of superoxide anion was observed by blood neutrophils in the presence of bacteria opsonized with IgA when compared with bacteria treated with medium.



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  		Figure 5. Absence superoxide release by colostral neutrophils. Bacteria were opsonized with a normal serum pool, a colostrum supernatant pool, or purified SIgA. In control assays, neutrophils were preincubated with medium 199. Maximal release was obtained after incubation of cells with PMA. PMA-induced burst release was 46.6 and 33.4 in nmol/O2- for blood and colostral neutrophils, respectively. Results are presented as % of the maximal release induced by PMA. Spontaneous release is represented by -. The results represent the mean ± SD of 8–10 experiments with cells from different individuals.

 
Decreased IgA-mediated bacterial (EPEC)-killing by colostral neutrophils despite of normal phagocytosis levels
First, we showed that colostral neutrophils were able to internalize bacteria with or without SIgA as opsonins at similar levels and as blood neutrophils (Fig. 6 ). To examine the ability of phagocytes to mediate bacterial-killing spontaneously, we first performed the assay using bacteria alone. As shown in Figure 7 , both cell types were able to kill EPEC at a rate of about 20% and without significant differences. Significant increases in killing rates were observed when blood and colostral neutrophils were incubated with serum-opsonized bacteria compared with bacteria with medium alone or opsonized with serum from an agammaglobulinemia patient. In contrast, no significant killing was obtained when colostral neutrophils were incubated with colostrum-opsonized bacteria, whereas killing was observed with blood neutrophils. SIgA-mediated bacterial-killing was observed with blood neutrophils but not with colostral neutrophils (Fig. 7) . To verify the effects of superoxide release on EPEC-killing, we analyzed bactericidal activity by colostral neutrophils in the presence or absence of SOD, an enzyme that metabolizes anion superoxide. As shown in Figure 8 , when colostral neutrophils were preincubated in the presence of SOD, decreased bactericidal activity (bacteria opsonized or not with SIgA) was seen in these cells. No effects of SOD were observed in EPEC-killing using serum-opsonized bacteria.



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  		Figure 6. Bacterial phagocytosis by colostral neutrophils. Cells were incubated with bacteria opsonized (A) or not (B) with SIgA on a shaker for 30 min at 37°C. After washing at 4°C, cells were stained with acridine orange and wet-mounted as described [38 ]. (C) Comparative phagocytosis performed with colostral and blood PMN cells. % of phagocytosis was calculated after counting 100 cells containing bacteria or not._art>

 


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  		Figure 7. Decreased SIgA-mediated EPEC-killing by colostral neutrophils. Phagocytosis of bacteria was performed, extracellular bacteria were removed, and cells were lysed to release the internalized bacteria followed by the microbiological plate technique. Results represent the mean ± SD of 10 experiments. Opsonin sources were used as indicated. Agamma represents serum from an agammaglobulinemia patient. *P < 0.05.

 


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  		Figure 8. Action of SOD on EPEC-killing by colostral neutrophils. Bacteria were opsonized with a normal serum pool and purified SIgA. In control assays, colostral PMN cells were incubated with medium 199. Bacterial-killing by phagocytes from colostrum was determined using a microbiological plate technique in the presence or not of SOD (140 units). The SOD was incubated with the bacteria and PMN cell suspensions during 2 h at 37°C under continuous shaking. Results represent the mean ± SD of four experiments with cells from different individuals. *P < 0.05 comparing the SOD-treated group with the untreated group for the same opsonin source.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified colostral neutrophils as cells expressing Fc{alpha}R (a.1 isoform) without {gamma} subunit association. In these cells, we did not detect the conventional Fc{alpha}R-{gamma}2 molecular complex as described for blood myeloid cells [23 24 25 26 ]. We demonstrated that colostral neutrophils did not present significant bacterial-killing and could not release superoxide anion efficiently when incubated with IgA-coated bacteria, whereas blood neutrophils were able to perform these functions as expected [40 ]. Indeed, IgA immune complexes (IC) or cross-linked, anti-Fc{alpha}R mAb have been shown to trigger Fc{alpha}R+ blood cells, resulting in various immune-effector functions such as phagocytosis, oxidative burst, and cytokine release [40 41 42 ]. These functions require the common immunoreceptor tyrosine-based activation motif (ITAM) in the {gamma} cytoplasmic tail [43 ]. The decrease in bactericidal activity in colostral neutrophils cannot be explained by differences in expression, because blood and colostrum PMN display similar levels of CD89 (see Table 1 ). Moreover, experiments using SOD indicated that killing by colostral neutrophils of SIgA-opsonized bacteria was specific and dependent on superoxide anions. However, killing of serum-opsonized bacteria was SOD-independent, indicating involvement of other mechanisms via IgG and C3 as described previously [12 ]. Other studies addressing the microbicidal mechanism of colostral PMN phagocytes for serum-opsonized bacteria oxygen independently should also be evaluated.

The identification of Fc{alpha}R as the a.1 isoform indicates that colostral neutrophils could originate from the blood pool. However, colostral neutrophils differ from blood cells by the lack of {gamma} chain association. Although inhibitory effects of milk on blood neutrophils have been described earlier [44 ], our preliminary studies failed to demonstrate that soluble factors present in colostrum could down-regulate Fc{alpha}R-{gamma}2 association on blood neutrophils in vitro, because no significant alterations in the amounts of FcR{gamma} chain associated with Fc{alpha}R were detected (unpublished results).

Anti-inflammatory properties of human colostrum have been described for more than three decades [29 , 45 , 46 ]. They have been imputed to the paucity of soluble initiators and mediators as well as to the presence of some anti-inflammatory agents such as catalase, histaminase, arylsulfatase, alpha tocopherol, alpha 1-antichymotrypsin, and alpha 1-antitrypsin [11 , 45 , 46 ]. Conversely, the anti-inflammatory role of colostral phagocytes was unknown. Here, we provide evidence that colostral neutrophils mediate noninflammatory functions. The absence of IgA-mediated, downstream responses on colostral neutrophils may be, at least in part, explained by the absence of association of {gamma} with Fc{alpha}R ({gamma}-less Fc{alpha}R). Indeed, Morton et al. [25 ] and our recent studies [26 ] have demonstrated that cells expressing {gamma}-less Fc{alpha}R alone cannot deliver downstream signals such as interleukin (IL)-2 release, ß-hexosaminidase release, and calcium influx. However, although these observations were obtained with transfectants overexpressing a mutated Fc{alpha}R (Arg to Leu at position 209), the present results provide for the first time a formal demonstration that this type of cell exists in humans and may function as a noninflammatory mediator in colostrum.

It is noteworthy that Ig-mediated phagocytosis is an ITAM-dependent function [43 ]. Mice deficient in the {gamma} subunit are unable to perform IgG-mediated phagocytosis [47 ]. It is interesting that in our study, colostral neutrophils could internalize bacteria and IgA-coated bacteria at similar levels as blood cells but could not mediate killing of IgA-coated bacteria likely because of the absence of {gamma} association with Fc{alpha}R. Low bacterial-killing mediated by colostral neutrophils in the absence of opsonins might be explained by other opsonic receptors different than CD89. Whether total {gamma} chains detected in the cell lysates (see Fig. 3 ) are associated with other receptors remains to be demonstrated.

One of the roles of {gamma}-less Fc{alpha}R is to mediate endocytosis and recycling of IgA [26 ]. It is noteworthy that the two types of Fc{alpha}R ({gamma}-associated and {gamma}-less) showed similar kinetics of Fc{alpha}R-mediated endocytosis; however, the endocytosis pathways of the two types of receptors differed. Although {gamma}-less Fc{alpha}R was localized in early endosomes mainly, Fc{alpha}R-{gamma}2 was found in endolysosomal compartments. {gamma}-less Fc{alpha}R recycled the internalized IgA toward the cell surface and protected against IgA degradation [26 ]. In this study, the detection of IgA on the surface of colostral neutrophils after incubation at 37°C for 90 min, associated with decrease of cytoplasmic IgA, suggests that {gamma}-less Fc{alpha}R binds SIgA and uses the previously proposed [26 ] recycling pathway continuously to avoid IgA degradation. Thus, {gamma}-less Fc{alpha}R might play a powerful role in the transport of SIgA. Indeed, one of the major roles of SIgA in innate and adaptative immunity is to block adherence and entry of pathogenic microorganisms such as bacteria and viruses [4 ]. This intracellular storage of SIgA would allow the maternal pre-armed immune response protecting the neonate from pathogens. Neonate-digestive functions are immature, even in term infants [48 ]. The pH of the gastric contents is increased because of decreased basal and stimulated hydrochloride secretion. Pepsin is inactivated, and there is no intragastric digestion of proteins in infants 5 to 8 days of age. Chymotrypsin and trypsin activities are present in duodenal fluids but at decreased levels at birth. Fat and carbohydrate digestion are also immature during the perinatal period [48 ]. Considering these data as a whole, it is plausible to propose that colostral leukocytes cross the stomach and reach the small instestine.

Taken together, our findings point to the existence of myeloid cells in colostrum expressing only Fc{alpha}R without {gamma} chains that carry IgA constitutively. As these receptors are involved in IgA recycling, one of their functions could be the intracellular transport of IgA for protection from the action of bacterial IgA proteases. The protective role of {gamma}-less Fc{alpha}R may be important in view of maintaining SIgA antibody concentrations in distal parts of the newborn gut to allow local IgA-mediated protection.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from INSERM, the Association pour la Recherche sur le Cancer (grant n° 5349), FRM-Sidaction, and by FAPESP grants n° 95/9843-0, 98/06432-8, 95/4466-4, and 95/6754-7. A. C. H-F. and P. L. contributed equally to this work. We thank Drs. U. Blank for providing anti-{gamma} Abs, M. Netter for preparing prints, and D. Broneer for critical reading of this manuscript. We also thank the medical and nursing staffs of the Obstetric Clinics of Hospital Universitário da USP and l’Hôpital Notre Dame du Bon Secours, Paris.

Received October 14, 1999; revised September 24, 2000; accepted September 25, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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