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Originally published online as doi:10.1189/jlb.1105668 on June 12, 2006

Published online before print June 12, 2006
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(Journal of Leukocyte Biology. 2006;80:350-358.)
© 2006 by Society for Leukocyte Biology

Release of surface-expressed lactoferrin from polymorphonuclear neutrophils after contact with CD4+T cells and its modulation on Th1/Th2 cytokine production

Ko-Jen Li*, Ming-Chi Lu*, Song-Chou Hsieh{dagger}, Cheng-Han Wu{dagger}, Hsin-Su Yu{ddagger}, Chang-Youh Tsai§ and Chia-Li Yu{dagger},1

* Department of Internal Medicine, Buddhist Tzu-Chi General Hospital Taipei Branch, Taiwan; Departments of
{dagger} Internal Medicine and
{ddagger} Dermatology, National Taiwan University College of Medicine, Taipei; and
§ Section of Allergy, Immunology & Rheumatology, Veterans General Hospital-Taipei, Taiwan

1Correspondence: Department of Internal Medicine, National Taiwan University Hospital, No. 7 Chung-Shan South Road, Taipei, Taiwan 100. E-mail: clyu{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is conceivable that a membrane component(s) is transferred from antigen-presenting cells to T cells after antigenic stimulation. However, it is not clear whether a certain membrane component(s) is transferred from polymorphonuclear neturophils (PMN) to T cells for immunomodulation. In the presence study, we cocultured two of the three autologous cells—PMN, CD4+T, and red blood cells (RBC)—homotypically or heterotypically for 1 h. Spontaneous membrane exchange between autologous PMN-PMN and PMN-CD4+T but not between CD4+T-CD4+T or RBC-CD4+T was observed with a confocal microscope. Loss of membrane exchange between two paraformaldehyde-fixed cells suggests that mutual membrane exchange is via cell–cell contact. Different combinations of cellular enzyme-linked immunosorbent assay for measuring the binding between fixed cells and biotinylated cell lysates showed the same tendency. To identify the molecule(s) mediating PMN-CD4+T binding, we compared the banding of biotinylated PMN lysates and the banding of plain PMN lysate probed by biotinylated CD4+T lysate in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We found that a 75- to 80-kDa surface-expressed molecule on PMN exists constantly to mediate PMN-CD4+T binding. Peptide analysis disclosed that the molecule had 99.8% identity with lactoferrin (LF). The expression of LF on system lupus erythematosis (SLE)-PMN is less than normal PMN. PMN-CD4+T coculture increased LF expression on CD4+T. Normal PMN and human milk-derived LF suppressed interferon-{gamma} (IFN-{gamma}) but enhanced interleukin (IL)-10 production of anti-CD3+anti-CD28-activated, normal CD4+T. In contrast, coculture of SLE-PMN and autologous CD4+T suppressed IFN-{gamma} and IL-10 production. These results suggest that the surface-expressed LF released from PMN after contact with autologous CD4+T modulated its T helper cell type 1 (Th1)/Th2 cytokine production. Decreased LF expression on SLE-PMN abnormally modulates Th1/Th2 production by CD4+T cells.

Key Words: systemic lupus erythematosus • interferon-{gamma} • interleukin-10


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polymorphonuclear neutrophils (PMN) are traditionally regarded as terminally differentiated cells against bacterial pathogens. However, recent studies revealed that PMN could produce different cytokines and chemokines for interactions with other immune cells [1 , 2 ]. Activated PMN can enhance the suppressive activity of CD4 suppressor T cells [3 ] and mitogen-stimulated DNA synthesis of mononuclear cells (MNC) [4 ]. Sendo et al. [5 6 7 8 ] demonstrated that delayed-type hypersensitivity and tumor inhibitory function were suppressed, whereas humoral immune responses were enhanced in PMN-depleted rats. It is interesting that in the stimulation of interferon-{gamma} (IFN-{gamma}), interleukin (IL)-3, and granulocyte macrophage-colony stimulating factor, PMN can express major histocompatibility complex (MHC) class II and T cell costimulatory molecules CD80 and CD86 serving as antigen-presenting cells (APC) to enhance T cell proliferation [9 10 11 ]. Immunopathologically, PMN can transdifferentiate to dendritic-like cells at the sites of chronic inflammation in rheumatoid synovitis [12 ] and Wegener’s granulomatosis [13 ]. These results indicate that PMN can modulate different aspects of mononuclear immune responses. However, the molecular basis of PMN-MNC interactions remains unclear.

Intercellular membrane transfer via immune synaptic sites was found to play an important role in cell–cell interactions [14 15 16 17 ]. T cells can capture different molecules expressed on APC, such as MHC-peptide complex, CD80, and OX40L, after contact with APC [14 , 15 ]. Tabiasco et al. [16 ] reported that target cell membrane fragments captured by human natural killer (NK) cells are mediated by Src kinase, adenosine 5'-triphosphate, Ca2+, protein kinase C, and rearranged actin cytoskeletons. Poupot and Fournie [17 ] demonstrated further that spontaneous membrane transfer among certain homotypic leukemia cell lines occurred after cell–cell contact without antigen stimulation. Accordingly, it is possible that PMN can modulate T or other immune-related cell functions via membrane component transfer after cell–cell contact, in addition to the released cytokines/chemokines. In this study, we cocultured two of the three cells, PMN, CD4+T, and red blood cells (RBC), homotypically or heterotypically, in different combinations. We found that spontaneous membrane component exchange occurred between autologous PMN and CD4+T. The surface-expressed lactoferrin (LF) on PMN is one of the molecules released from PMN to modulate T helper cell type 1 (Th1)/Th2 cytokine production by CD4+T. Less LF expression on systemic lupus erythematosus (SLE)-PMN abnormally modulates Th1/Th2 cytokine production of autologous CD4+T in patients with SLE.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and controls
Twenty-three patients fulfilling 1982 revised American College of Rheumatology criteria for the classification of SLE [18 ] were enrolled. Seventeen of them were disease-active, judged by the SLE disease activity index [19 ]. The same numbers of sex- and age-matched, healthy individuals were the controls. All of the participants signed the informed consents approved by the Institutional Review Board of National Taiwan University Hospital (Taipei). All of the active SLE patients received oral prednisolone (25±7.5 mg/day) and hydroxychloroquine (200–400 mg/day), with or without azathioprine (50–100 mg/day).

Preparation of RBC, PMN, and CD4+T lymphocytes from peripheral blood of normal and SLE patients
Heparinized venous blood, obtained from healthy volunteers or SLE patients, was mixed with one-fourth volume of 2% dextran solution [molecular weight (MW) 464,000 daltons; Sigma-Aldrich, St. Louis, MO] and incubated at room temperature for 30 min. Leukocyte-enriched supernatant was collected and layered over Ficoll-Hypaque density gradient solution (specific gravity 1.77; Pharmacia Biotech, Uppsala, Sweden). RBC were collected from the bottom after dextran sedimentation and served as negative cell control, as no interaction between RBC and other immune-related cells has been reported. After centrifugation of leukocyte-enriched supernatant at 250 g for 25 min, MNC were aspirated from the interface, and PMN were obtained from the bottom. The contaminated RBC in PMN suspension were lysed by incubating in chilled 0.83% ammonium chloride solution at 4°C for 10 min. For further purification, PMN was positively selected by using monoclonal anti-Gr-1 antibody-conjugated microbeads (derived from RB6.8C5 hybridoma) and the AutoMACS (Miltenyi Biotec, Bergisch Gladback, Germany). It is well accepted that the Gr-1 molecule is a functional, important surface marker expressed on granulocytes and a memory-type CD8+T cell subset, which recognizes 21–25 kDa glycosylphosphatidylinositol-anchored protein [20 ]. The antibody has been used to purify and characterize the functions of neutrophil. The purity of PMN reached 98%, confirmed by flow cytometry after stained with anti-CD16. For purifying the CD4+T cells further, the MNC suspension was initially placed in glass Petri dishes at 37°C for 1 h to adhere the monocytes. The nonadherent cells were collected carefully and again adhered on the Petri dishes at 37°C for another 1 h. The nonadherent cells were collected and then positively selected by using anti-CD4-conjugated microbeads and the AutoMACS (Miltenyi Biotec). The purity of the CD4+T population was >98%, confirmed by flow cytometry. The cell concentration of RBC, PMN, and CD4+T was adjusted to 2 x 106/ml in 10% fetal bovine serum in RPMI 1640. The viability of PMN and CD4+T was greater than 95%, confirmed by trypan blue dye exclusion.

Detection of membrane component exchange among RBC, PMN, and CD4+T after cell–cell contact by confocal laser-scan microscope
Autologous PMN, CD4+T, and RBC were previously surface-stained with the lipophilic dyes PKH-67 (green fluorescence), PKH-26 (red fluorescence), or cytoplasmic orange-4-chloromethyl benzoyl amino tetramethyl rhodamine (CMTMR; red fluorescence) according to the manufacturer’s instruction (Sigma-Aldrich). After several washes, two of the three cells were mixed in various homotypical or heterotypical combinations at a ratio of 1:1 in conical tubes and centrifuged at 240 g for 1 min to facilitate cell–cell contact. The cell mixture was then incubated at 37ºC in 5% CO2-95% air for 1 h. In some experiments, the fluorescence-stained cells were fixed by 4% paraformaldehyde before mixing with each other in a conical tube to see the affection of cell fixation on membrane component exchange. After incubation, the cell mixture was then shaken gently and plated on a glass slide for confocal microscopic observation.

Detection of membrane exchange between PMN and CD4+T by flow cytometry
Freshly isolated PMN (2x106cells/ml) were surface-stained with PKH-67 (green fluorescence), whereas CD4+T (2x106 cells/ml) were surface-stained with PKH-26 (red fluorescence; Sigma-Aldrich), respectively, or vice versa. After several washes, autologous cells were mixed at a ratio of 1:1 in three combinations, CD4+T + CD4+T, PMN + CD4+T, and PMN + PMN, followed by centrifugation at 240 g for 1 min to facilitate cell–cell contact. The percentage of the doubly stained cells in a separated CD4+T or PMN population was analyzed by FACScan flow cytometry (Becton Dickinson, San Jose, CA) using Lysis II software after forward-scatter (FSC) and side-scatter (SSC) gating.

Preparation of biotinylated cell lysate
Freshly isolated CD4+T, PMN, or RBC (2x106cells/ml), suspended in phosphate-buffered saline (PBS), pH 7.2, were membrane-biotinylated by incubation with 1 ml biotin-labeling buffer (0.3 mg biotin/ml in PBS; Boehringer-Mannheim Biochemicals, GmbH, Mannheim, Germany) at 4°C for 1.5 h with continuous gentle shaking. After several washes with PBS, pH 7.2, the cell pellet was sonicated in an ice-bath. Following centrifugation at 11,800 g for 15 min to precipitate the cell debris, the biotinylated cell lysate was obtained from the supernatant.

Detection of PMN-CD4+T binding by cellular enzyme-linked immunosorbent assay (ELISA)
Freshly isolated, normal PMN, CD4+T, or RBC suspension (100 µL; 2x106cells/ml) were placed in U-shaped, flat-bottom microtiter wells (Linbro/Titertek®, Flow Laboratories, Inc., McLean, VA). The microtiter plate was centrifuged at 760 g for 10 min to precipitate the cells. After carefully removing the supernatant, the cells were fixed in 4% paraformaldehyde for 10 min. The microwells were then incubated with 1% bovine serum albumin (Sigma-Aldrich) for 2 h to reduce the nonspecific binding with protein molecules. Autologous, biotinylated *CD4+T, *PMN, or *RBC cell lysate (100 µL; 2x106cells/ml; please see the above paragraph for the detailed procedures) was added to the wells and incubated for 1 h at room temperature. After several washes with PBS, pH 7.2, 100 µl 10 µg/ml horseradish peroxidase (HRP)-conjugated streptavidin (BioLegend Corp., San Diego, CA) was added and incubated for another 1 h at room temperature. Color was developed by reacting with 150 µl substrate containing orthophenylenediamine (Sigma-Aldrich) for 10 min in the dark. The reaction was stopped by adding 50 µl 4N H2SO4 solution. The binding capacity between different fixed cells and biotinylated cell lysates was measured by ELISA reader (Dynex Technologist, Chantilly, VA) at optical density (OD)450 nm absorbance.

Identification of the membrane protein(s) on PMN binding with CD4+T by Western blot analysis
Freshly isolated PMN were membrane-biotinylated as mentioned in the previous paragraph. Sample buffer [100 µL; 1x=125 nM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 5% glycerol, 0.03% bromophenol blue, and 1% ß-mercaptoethanol] was added to lyse 1 x 107-biotinylated PMN in pellet followed by boiling for 7 min with frequent, vigorous vortexing. The precipitates were removed by centrifugation at 11,800 g at 4°C for 15 min. The clear PMN lysates (15 µl/well) were electrophoresed in 10% SDS-polyacrylamide gel electrophoresis (PAGE). The dispersed proteins in gel were transferred to a nitrocellulose paper by a semidry transfer system. The membrane was immersed in the blocking buffer (5% nonfat milk in wash buffer containing 10 nM Tris, pH 7.5, 100 nM NaCl, and 0.1% Tween-20) for 1 h at room temperature and then probed by HRP-conjugated streptavidin (1 µg/ml, BioLegend Corp.). The antigen–antibody reaction was detected by the enhanced chemiluminescence protein detection system (Amersham International, Amersham, UK) to show the profile of PMN surface membrane proteins.

The plain PMN lysate (1x107cells/ml) was electrophoresed in 10% SDS-PAGE for Western blot analysis. The following procedures were the same as described above, except that the dispersed molecules were probed by biotinylated CD4+T cell lysate.

The overlapped band(s) described in the above paragraphs was considered the surface-expressed molecule(s) on PMN, which can react with CD4+T.

The plain CD4+T cell lysates (1x107cells/ml) were electrophoresed in 10% SDS-PAGE. After reacting with plain PMN cell lysates (1x107cells/ml) in a nitrocellulose membrane, the complexes were probed by 1:100x diluted monoclonal HRP-conjugated anti-human LF antibody (Biodesign, Beverly, MA) for detecting whether LF is present in PMN-CD4+T-binding sites or not.

Peptide mapping of surface-expressed molecule(s) on PMN binding with CD4+T surface membrane
The surface-expressed molecules on PMN, which reacted with CD4+T cell membrane in Western blot as mentioned above were excised and eluted. After Lys C protease cleavage and alkylation to prevent the linkage of multiple peptides through disulfide bonds, the digested peptides were separated further by reverse-phase high-pressure liquid chromatography on an octadecyl silica gel. The peptide sequence was analyzed using the ProFound computer program at the Beckman Center, Stanford University Medical Center (CA).

Detection of LF expression on PMN and CD4+T of normal and SLE patients by flow cytometry
Freshly isolated PMN or CD4+T was incubated with mouse monoclonal anti-human LF antibody (anti-LF, 1:100x dilution, Biodesign) in an ice-bath (0°C) for 1 h. After several washes with PBS, pH 7.2, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (IgG) with 1:2000x dilution as secondary antibodies for 1 h. The PMN or CD4+T cells only stained with secondary antibodies were the negative control. The percent and mean fluorescence intensity (MFI) of LF-expressed cells were detected by FACScan flow cytometry (Becton Dickinson) using Lysis II software after gating by FSC and SSC to identify a PMN or CD4+T population. As we stained the viable cells (viability >95%), only surface-expressed LF but not cytoplasmic granular LF was detected by flow cytometry.

Comparing the MW of PMN-derived and human milk-derived LF by Western blot
Commercially available human milk-derived LF (Sigma-Aldrich) and PMN cell lysate were electrophoresed in 10% SDS-PAGE and probed by mouse monoclonal anti-human LF antibody (Biodesign) for comparing the MW of PMN- and human milk-derived LF.

Detection of the binding capacity of human milk-derived LF to CD4+T
Different amounts of purified human milk LF (1.25–10 µg/ml) were incubated with normal, viable CD4+T cells for 1 h at 37°C. After three washes, the CD4+T was lysed in lysis buffer with sonication. The CD4+T lysates were electrophoresed in 10% SDS-PAGE probed by HRP-conjugated monoclonal anti-LF antibody.

Detection of LF expression on CD4+T after coculture with autologous PMN by flow cytometry
Freshly isolated, normal CD4+T was mixed with/without autologous PMN at a ratio of 1:1 in a conical tube for 1 h followed by staining with monoclonal anti-LF in an ice-bath (0°C) for 30 min. The percent and MFI of LF on the CD4+T population were detected by FACScan flow cytometry after FSC and SSC gating to identify the CD4+T population.

The effects of autologous PMN and different doses of human milk-derived LF on normal and SLE CD4+T cell Th1 (IFN-{gamma}) and Th2 (IL-10) cytokine production by ELISA
Anti-CD3- and anti-CD28 (CD3+CD28)-activated CD4+T (2x106cells/ml) was incubated with autologous PMN (2x106/ml) at a 1:1 ratio from normal or active SLE patients or different doses of human milk-derived LF (1–200 µg/ml) at 37ºC in 5% CO2-95% air for 24 h. The concentration of IFN-{gamma} and IL-10 in cultured supernatants was measured by commercially available, human ELISA kits (ABcam, Cambridge, MA). The detailed procedures were described in the respective manufacturer’s instruction booklet. The minimal detectable concentration was 2 pg/ml for IFN-{gamma} and 3 pg/ml for IL-10.

Statistical analysis
The results were represented by mean ± SD. Continuous variables were analyzed using the nonparametric Wilcoxon rank-sum test using a commercially available software package: Stata/SE 8.0 for Windows. A P value <0.05 was considered statistically significance. Spearman correlation coefficient by rank and multiple linear regression were applied to test the correlation and significance between two parameters.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spontaneous membrane component(s) exchange between autologous PMN and CD4+T and between PMN and PMN after cell–cell contact
To understand whether a certain surface membrane component(s) is exchanged between PMN and autologous CD4+T, PKH-67 (green fluorescence) surface-stained PMN were mixed with autologous cytoplasmic orange-CMTMR (red fluorescence)-stained CD4+T for 1 h. As demonstrated in Figure 1A , yellowish-green fluorescence was observed on the CD4+T surface by confocal microscope. It indicates that a certain surface component(s) was transferred from PMN to an autologous CD4+T surface. In contrast, a cytoplasmic component of CD4+T was not transferred to an autologous PMN surface. No membrane component exchanged between PKH-26 (red fluorescence) surface-stained CD4+T and PKH-67 (green fluorescence) surface-stained RBC, which were used as negative cell control, as no cell–cell interaction has been reported between these two cells (Fig. 1B) . Conversely, for investigating whether membrane components transfer from CD4+T to autologous PMN or not, PKH-67 (green fluorescence) surface-stained CD4+T cocultured with cytoplasmic orange-CMTMR (red fluorescence)-stained PMN for 0 min (Fig. 1C-1) and 60 min. (Fig. 1C-2) . We noted that yellowish fluorescence also appeared on the surface of PMN at 60 min but not at 0 min. Putting these results together, it is conceivable that mutual surface membrane component(s) exchange occurs between PMN and CD4+T. In addition, the coculture of paraformaldehyde-fixed surface fluorescence-stained, autologous PMN and CD4+T failed to exchange surface components with each other (data not shown). For measuring the percent of membrane exchange in an individual cell population after PMN-CD4+T contact, the mixed cells were gated by FSC and SSC. As shown in Figures 2 and 3 , PMN (R1 in Figs. 2E and 3E ) and CD4+T (R2 in Figs. 2E and 3E ) populations were identified clearly after gating. We found membrane component(s) exchange between autologous CD4+T-CD4+T was minimum (3.96% in Fig. 2D ). However, the exchange between autologous PMN and CD4+T increased to 9.98% (Fig. 2G) . It is interesting that the membrane exchange between autologous PMN and PMN was high, up to 24.3% (Fig. 3D) . It is possible that mutual membrane exchange between homotypical, adherent cells such as PMN proceed continuously. It is surprising that the membrane transfer from CD4+T to autologous PMN was higher (18.2% in Fig. 2F and 14.6% in Fig. 3F ) than that from PMN to autologous CD4+T (9.98% in Fig. 2G and 8.64% in Fig. 3G ). These results suggest that spontaneous membrane component(s) exchange occurs after cell–cell contact in PMN-CD4+T and PMN-PMN but not CD4+T-CD4+T coculture.


Figure 1
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  		Figure 1. Exchange of membrane component(s) between autologous PMN and CD4+T observed by confocal laser-scan microscope. (A) PKH-67 (green fluorescence) surface-stained PMN cocultured with cytoplasmic orange-CMTMR (red fluorescence)-stained CD4+T cells for 1 h. The yellowish fluorescence appears on the CD4+T surface. (B) PKH-26 (red) surface-stained CD4+T cocultured with PKH-67 (green) surface-stained RBC for 1 h. No membrane component was exchanged between RBC and CD4+T. (C) PKH-67 (green) surface-stained CD4+T cocultured with cytoplasmic orange-CMTMR (red)-stained PMN at Time 0 min (C-1) and at Time 60 min (C-2). Yellowish fluorescence appears on the PMN surface.

 

Figure 2
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  		Figure 2. A representative case of membrane component exchange between autologous CD4+T-CD4+T and PMN-CD4+T cells detected by flow cytometry. (A) CD4+T cells without staining. (B) CD4+T cells were surface-stained with PKH-26 (red). (C) CD4+T cells were surface-stained with PKH-67 (green). (D) PKH-26 (red) surface-stained CD4+T cocultured with PKH-67 (green)-stained, autologous CD4+T for 1 h. The doubly stained cell in the CD4+T population was 3.96%. (E) PKH-67 surface-stained CD4+T cocultured with PKH-26 surface-stained PMN for 1 h. The mixed cells were then gated by FSC and SSC. R1 denotes PMN population, and R2 denotes CD4+T population. (F) The doubly stained cell in the PMN population was 18.2%. (G) The doubly stained cell in CD4+T was 9.98%. The same experiment was conducted three times using different samples with a similar tendency.

 

Figure 3
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  		Figure 3. A representative case of membrane component exchange between autologous PMN-PMN and PMN-CD4+T cells detected by flow cytometry. (A) PMN without staining. (B) PMN was stained with PKH-26 (red). (C) PMN was stained with PKH-67 (green). (D) PKH-26 (red) surface-stained PMN cocultured with autologous PKH-67 (green) surface-stained PMN for 1 h. The doubly stained cell in the PMN population was 24.3%. (E) PKH-26 (red) surface-stained CD4+T cocultured with autologous PKH-67 (green) surface-stained PMN for 1 h. The mixed cells were then gated by FSC and SSC. R1 denotes PMN population, and R2 denotes CD4+T population. (F) The doubly stained cell in the PMN population was 14.6%. (G) The doubly stained cell in the CD4+T population was 8.64%. The same experiment was conducted three times using different samples with a similar tendency.

 
Detection of autologous PMN-CD4+T and RBC-CD4+T cell-surface binding by cellular ELISA and Western blot
The first step to detect surface component exchange between two cells is to confirm the binding between two cells. The binding of biotinylated *PMN, *CD4+T, or *RBC lysate with 4% paraformaldehyde-fixed CD4+T or PMN was conducted by cellular ELISA. We found that the binding of biotinylated *CD4+T membrane to fixed PMN cells was higher than biotinylated *PMN membrane to fixed CD4+T cells (Fig. 4A ), whereas the binding of biotinylated *RBC membrane to fixed CD4+T was negligible. These results are consistent with those of flow cytometric determination (Figs. 2 and 3F and 3G ). To identify the nature of surface component(s) mediating PMN-CD4+T binding, we compared the overlapped, electrophoretic band(s) appearing in biotinylated *PMN lysates and plain PMN lysate probed by biotinylated *CD4+T lysate (Fig. 4B , lanes 1 and 2). We found at least three proteins with MW of 75–80, 65–70, and 20–25 kDa were the candidates for membrane exchange (Fig. 4B) . However, only the 75- to 80-kDa molecule was detected constantly in different experiments. We excised and eluted this 75- to 80-kDa band for peptide analysis. The result disclosed that the molecule is 99.8% identity with human LF (MW=77 kDa). For investigating whether LF is present in PMN-CD4+T-binding sites or not, the CD4+T cell lysate was electrophoresed in 10% SDS-PAGE followed by reacting with PMN lysate. The complexes were then probed by anti-human LF antibody. We confirmed that the 75- to 80-kDa molecule was the cognate antigen of anti-LF antibody (Fig. 4B , lane 3).


Figure 4
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  		Figure 4. Detection of cell membrane binding between PMN and CD4+T by (A) cellular ELISA and (B) Western blot. (A) PMN or CD4+T at a concentration of 2 x 106/ml were fixed by 4% paraformaldehyde in U-shaped, flat-bottom microwells. The lysates of biotinylated *PMN, *RBC, or *CD4+T were then added to microwells. The binding capacity of different combinations was measured at OD450 nm absorbance after color development. Statistical significance was assessed by nonparametric Wilcox rank-sum test. (B) A representative case showing PMN membrane protein(s) bound to CD4+T by Western blot analysis. Lane 1: Biotinylated *PMN lysate was electrophoresed in 10% SDS-PAGE and probed by HRP-conjugated streptavidin. Lane 2: Plain PMN lysate was electrophoresed in 10% SDS-PAGE and probed by biotinylated and HRP-conjugated streptaviden. Lane 3: Plain CD4+T cell lysate was electrophoresed in 10% SDS-PAGE and then incubated with PMN-lysate for 2 h. The complexes were probed by HRP-conjugated anti-human LF antibody. The same experiment was conducted five times using different samples with a similar tendency. Only a 75- to 80-kDa molecule (indicated by the arrow) was detected constantly.

 
The expression of LF on PMN and CD4+T from normal and SLE patients
PMN from patients with active SLE exhibit defective expression of many surface molecules including Fc receptor for IgG I–III, complement receptors types I and III, CXC chemokine receptor 1, and adhesion molecules. As LF is present in secondary cytoplasmic granules and the surface membrane of PMN, it would be interesting to compare the expression of LF on PMN and CD4+T from normal and SLE patients. As illustrated in Figure 5A , normal PMN expressed much more LF on the cell surface than normal CD4+T. The MW of LF in PMN was identical to human milk-derived LF by Western blot analysis (Fig. 5B) . Human milk-derived LF dose-responsively bound to the cell surface of CD4+T as shown in Figure 5C . It suggests that exogenous LF can adhere to the CD4+T surface. Coculture of autologous PMN and CD4+T for 1 h increased LF expression on CD4+T detected by flow cytometry (Fig. 5D) . These results suggest that LF really expresses on PMN surface and can be transferred to the surface of CD4+T cell after coculture. The paraformaldehyde-fixed PMN failed to increase LF expression on CD4+T after coculture (data not shown). These observations further confirm surface LF exchange between PMN and CD4+T after cell–cell contact. As PMN can modulate immune functions of MNC [3 , 4 ], and LF can be transferred from PMN to CD4+T, it is worthwhile to compare the surface expression of LF on normal and SLE-PMN. As demonstrated in Figure 5E , we found SLE-PMN expresses less LF on the cell surface than normal PMN. As all of our SLE patients received prednisolone (25±7.5 mg/ml), it is possible that the decreased LF expression on SLE-PMN was a result of the suppressive effect by corticosteroids. Accordingly, the correlation of LF expression on SLE-PMN and the daily prednisolone dose was tested. As shown in Figure 5F , no correlation was found between prednisolone dose and LF expression on SLE-PMN (r=–0.14, P=0.68). In addition, hydrocortisone at a concentration 5x higher than the physiologic dose did not affect phagocytosis and IL-8 production of PMN in the in vitro study (data not shown).


Figure 5
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  		Figure 5. Detection of LF expression on PMN and CD4+T cells from normal and active SLE. (A) LF expression on the surface of normal PMN and CD4+T detected by flow cytometry. Negative control denotes that the cells only stained with secondary antibodies. (B) Normal PMN lysate and human milk-derived LF were compared in Western blot probed by antihuman LF antibody. (C) Human milk-derived LF (indicated by arrow) dose-dependently bound to CD4+T cell surface probed by antihuman LF antibody in Western blot. (D) A representative case of LF expression on CD4+T incubating with medium or autologous PMN detected by flow cytometry. The same experiment was conducted for three times using different samplers with a similar tendency. (E) Comparison of LF expression on PMN from normal and SLE patients detected by flow cytometry. Statistical significance was assessed by nonparametric Wilcoxon rank-sum test. (F) Correlation of LF expression on SLE-PMN and the prednisolone dose (mg/day) in patients with SLE. Statistical assessment was calculated by linear regression test.

 
The effects of autologous PMN and exogenous LF on Th1/Th2 cytokine production by the anti-CD3+CD28-activated CD4+T cell from normal and SLE patients
In our previous study, we found that peritoneal exudate PMN from an autoimmune MRL-lpr/lpr mouse suppressed autologous, peritoneal, mononuclear Th1/Th2 cytokine expression, different from a normal BALB/c mouse [21 ]. It would be interesting to know any difference in Th1/Th2 cytokine production by CD4+T lymphocytes after coculture with autologous PMN or different amounts of exogenous LF in normal and SLE patients. Autologous PMN or different amounts of human milk-derived LF from 1 to 200 µg/ml were added to anti-CD3+anti-CD28-actviated CD4+T cells from normal and SLE patients. Their effects on Th1 (represented by IFN-{gamma}) and Th2 (represented by IL-10) production were compared. As demonstrated in Figure 6A and 6B , normal PMN and exogenous LF significantly suppressed IFN-{gamma} and a tendency to enhance IL-10 production of normal-activated CD4+T. In contrast, autologous SLE-PMN significantly suppressed IFN-{gamma} and IL-10 production by SLE-CD4+T (Fig. 6C and 6D) .


Figure 6
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  		Figure 6. Effects of autologous PMN and different doses of LF (1–200 µg/ml) on IFN-{gamma} and IL-10 production by anti-CD3+anti-CD28-activated CD4+T cells (1x106/ml) of normal (A and B) and SLE patients (C and D). Statistical significance was assessed by the nonparametric Wilcoxon rank-sum test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Many authors have reported [4 5 6 7 8 , 10 11 12 13 ] interactions between PMN and MNC in immune responses. However, the molecular basis for the interactions remains unexplored. Immune synapse was found recently to involve the membrane transfer among different cells including homotypical leukemia cell lines [17 ], T cell-APC [22 23 24 ], cytotoxic T cell-target cell [25 ], and NK cell-target cell [16 , 26 ]. The intercellular membrane transfer requires direct cell–cell contact but does not involve cytosolic molecules or exosomal release. Savina et al. [27 ] proved no significant membrane transfer among K562 erythroleukemia cell lines, although these cells were potent exosome producers. In the present study, several original observations are found: Spontaneous membrane component(s) exchange occurs between autologous PMN and CD4+T, and the transfer from CD4+T to PMN was greater than from PMN to CD4+T; the membrane exchange between autologous CD4+T and CD4+T is minimal; LF is one of the membrane components transferred from PMN to CD4+T after cell–cell contact; surface-expressed LF on SLE-PMN was less than normal PMN; exogenous LF attached to the CD4+T surface and suppressed IFN-{gamma} but enhanced IL-10 production in normal and SLE; normal PMN suppressed IFN-{gamma} but enhanced IL-10 production of autologous CD4+T cells; and SLE-PMN suppressed IFN-{gamma} and IL-10 production of autologous CD4+T cells. These observations have not been reported in the literature.

LF is an iron-containing protein usually found in the secondary granules of PMN and can be released after activation [28 ]. The molecule may translocate spontaneously to the surface membrane of resting PMN [29 ]. In contrast, many other PMN granular proteins including cathepsin G, elastase, proteinase 3, myeloperoxidase, and inhibitory tumor necrosis factor {alpha} (TNF-{alpha}) translocate to the cell surface only after activation [30 31 32 33 34 ]. These secondary granules of PMN were regarded previously as reservoirs of enzymes or proteins. However, recent evidence disclosed that the secondary granular membrane may fuse with the PMN surface membrane after activation and serve as a new receptor or ligand for interacting with the environment [34 35 36 ]. The expression of LF on the PMN surface can modulate diverse, immunological/inflammatory functions including inhibition of PMN recruitment [37 ], enhancement of Th1 response to Staphylococcus aureus infection [38 ], suppression on feline mononuclear IFN-{gamma} secretion [39 ], and trinitrobenzene sulfonic acid-induced bowel inflammation [40 ]. Recently, Moed et al. [41 ] demonstrated that LF inhibited nonselective T cell proliferation, chemokine receptor expression, and cytokine production. Furthermore, Dhennin-Duthille et al. [42 ] reported that LF up-regulated the expression of CD4 antigen through mitogen-activated protein kinase isoforms Lck and extracellular signal-regulated kinase 2 signaling pathways to enhance the immune responses. It is conceivable that membrane-bound LF on PMN reduced its surface charge, which in turn, promoted PMN attachment to another cell surface [43 ]. In the present study, we noted that LF can be transferred from PMN to CD4+T after cell–cell attachment, and the transfer would modulate autologous CD4+T Th1/Th2 cytokine production. We found that autologous, normal PMN and exogenous LF suppressed Th1 cytokine (IFN-{gamma}) but exhibited a tendency to enhance Th2 cytokine (IL-10) production, which is consistent with our previous study in mice [21 ]. These results suggest that PMN-CD4+T interactions may prevent overwhelming, inflammatory reactions in normal immune responses. The effect of LF on normal CD4+T Th1/Th2 cytokine production reached a plateau at the concentration of 100 µg/ml, which is compatible with the maximal physiological concentration as reported by Adeyemi et al. [44 ].

Conversely, the immunological/inflammatory roles of LF in autoimmune disease have not been reported. The serum concentration of LF in rheumatoid arthritis patients is statistically higher than SLE or the normal group and correlated with rheumatoid disease activity [44 ]. A study about the Th1/Th2 cytokine modulation after PMN-CD4+T interactions in patients with systemic autoimmunity was also quite rare in the literature. Matsuyama et al. [45 ] reported that TNF-related apoptosis-inducing ligand (TRAIL) is involved in SLE neutropenia, as serum TRAIL and TRAIL expressed on T cells are higher, which would kill autologous PMN in patients with SLE. In our previous study, we demonstrated that the peritoneal exudate PMN suppressed peritoneal exudate mononuclear Th1 and Th2 cytokine expression in MRL-lpr/lpr mice [21 ]. In the present study, we originally found that the surface-expressed LF on SLE-PMN was significantly less than normal PMN. As the Th1/Th2 cytokine production profile in SLE-CD4+T is different from normal CD4+T, it is worthwhile to compare Th1/Th2 cytokine production after autologous PMN-CD4+T coculture and exogenous LF supplement in normal and SLE. We disclosed that SLE-PMN suppressed IFN-{gamma} and IL-10 cytokine production of autologous-activated CD4+T cells, different from normal PMN. This might reflect a compensatory mechanism counteracting the overproduction of Th2 cytokines, especially IL-10 in SLE. We believe that LF is one of the membrane components transferred from PMN to autologous CD4+T cells for immunomodulation on Th1/Th2 cytokine expression. However, whether LF is transferred directly from PMN to autologous CD4+T, or LF is released from PMN and then attached to autologous CD4+T is not settled in the present study. Less membrane expression of LF on SLE-PMN seems irrelevant to the daily prednisolone use as shown in Figure 5F . The in vitro study also revealed that hydrocortisone at a concentration higher than the physiological dose did not affect PMN phagocytosis (data not shown). However, the real mechanism of the decreased LF expression on SLE-PMN needs further investigation.

Poupot and Fournie [17 ] demonstrated that nonantigenic-induced, homotypic membrane transfer strictly required cell–cell contact and was confined to a few leukemia cell lines but not among homotypic {alpha}ß-T, {gamma}{delta}-T, B, or NK cells. In this study, we demonstrated that the spontaneous membrane exchange may occur between normal, autologous PMN-PMN and PMN-CD4+T. It is reported that the exchange of surface component(s) in many homotypic transformed cells in the sites of immunological synapse [42 ]. Whether immunological synapse forms between PMN and CD4+T, their signaling pathways, and the nature of the other exchangeable membrane components between PMN and CD4+T is now under investigation.

In conclusion, we demonstrate that spontaneous membrane exchange between autologous PMN and CD4+T occurs after cell–cell contact without antigen stimulation. LF is one of the exchangeable molecules for modulating Th1/Th2 cytokine production by CD4+T after PMN-CD4+T contact. This novel finding becomes one of the molecular bases of PMN-CD4+T interactions in modulating immune responses.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from National Science Council (NSC93-2314-B002-085) and Tzu-Chi General Hospital (TCRD-TPE-95-01), Taiwan.

Received November 16, 2005; revised April 3, 2006; accepted April 20, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cassatella, M. A. (1995) The production of cytokines by polymorphonuclear neutrophils Immunol. Today 16,21-26[CrossRef][Medline]
  2. Lloyd, A. R., Oppenheim, J. J. (1992) Poly’s lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response Immunol. Today 13,169-172[CrossRef][Medline]
  3. Hirohata, S., Yanagida, T., Yoshino, Y., Miyashita, H. (1995) Polymorphonuclear neutrophils enhance suppressive activities of anti-CD3-induced CD4+ suppressor T cells Cell. Immunol. 160,270-277[CrossRef][Medline]
  4. El Hag, A., Clark, R. A. (1987) Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system J. Immunol. 139,2406-2413[Abstract]
  5. Kudo, C., Yamashita, T., Araki, A., Terashita, M., Watanabe, T., Atsumi, M., Tamura, M., Sendo, F. (1993) Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. I. Inhibition by RP-3 treatment of the priming and effector phases of delayed type hypersensitivity to sheep red blood cells in rats J. Immunol. 150,3728-3738[Abstract]
  6. Kudo, C., Yamashita, T., Terashita, M., Sendo, F. (1993) Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. II. Inhibition by RP-3 treatment of mononuclear leukocyte recruitment in delayed-type hypersensitivity to sheep red blood cells in rats J. Immunol. 150,3739-3746[Abstract]
  7. Tamura, M., Sekiya, S., Terashita, M., Sendo, F. (1994) Modulation of the in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. III. Enhancement by RP-3 treatment of the anti-sheep red blood cell plaque-forming cell response in rats J. Immunol. 153,1301-1308[Abstract]
  8. Tanaka, E., Sendo, F. (1993) Abrogation of tumor-inhibitory MRC-OX8+ (CD8+) effector T-cell generation in rats by selective depletion of neutrophils in vivo using a monoclonal antibody Int. J. Cancer 54,131-136[Medline]
  9. Gosselin, E. J., Wardwell, K., Rigby, W. F., Guyre, P. M. (1993) Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-{gamma}, and IL-3 J. Immunol. 151,1482-1490[Abstract]
  10. Fanger, N. A., Liu, C., Guyre, P. M., Wardwell, K., O’Neil, J., Guo, T. L., Christian, T. P., Mudzinski, S. P., Gosselin, E. J. (1997) Activation of human T cells by major histocompatibility complex class II expressing neutrophils: proliferation in the presence of superantigen, but not tetanus toxoid Blood 89,4128-4135[Abstract/Free Full Text]
  11. Radsak, M., Iking-Konert, C., Stegmaier, S., Andrassay, K., Hansch, G. M. (2000) Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation Immunology 101,521-530[CrossRef][Medline]
  12. Iking-Konert, C., Ostendorf, B., Sander, O., Jost, M., Wagner, C. (2005) Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells Ann. Rheum. Dis. 64,1436-1442[Abstract/Free Full Text]
  13. Iking-Konert, C., Vogt, S., Radsak, M., Hansch, G. M. (2001) Polymorphonuclear neutrophils in Wegener’s granulomatosis acquire characteristics of antigen presenting cell Kidney Int. 60,2247-2262[CrossRef][Medline]
  14. Wetzel, S. A., McKeithan, T. W., Parker, D. C. (2005) Peptide-specific intercellular transfer of MHC class II to CD4+ T cells directly from the immunological synapse upon cellular dissociation J. Immunol. 174,80-89[Abstract/Free Full Text]
  15. Baba, E., Takahashi, Y., Lichtenfeld, J., Tanaka, R., Yoshida, A., Sugamura, K., Yamamoto, N., Tanaka, Y. (2001) Functional CD4 T cells after intercellular molecular transfer of OX40 ligand J. Immunol. 167,875-883[Abstract/Free Full Text]
  16. Tabiasco, J., Espinosa, E., Hudrisier, D., Joly, E., Fournie, J. J., Vercellone, A. (2002) Active trans-synaptic capture of membrane fragments by natural killer cells Eur. J. Immunol. 32,1502-1508[CrossRef][Medline]
  17. Poupot, M., Fournie, J. J. (2003) Spontaneous membrane transfer through homotypic synapses between lymphoma cells J. Immunol. 171,2517-2523[Abstract/Free Full Text]
  18. Tan, E. M., Cohen, A. S., Fries, J. F., Masi, A. T., McShane, D. J., Rothfield, N. F., Schaller, J. G., Talal, N., Winchester, R. J. (1982) The 1982 revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum. 25,1271-1277[Medline]
  19. Bombardier, C., Gladman, D. D., Urowitz, M. B., Caron, D., Chang, C. H. (1992) Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE Arthritis Rheum. 35,630-640[Medline]
  20. Matsuzaki, J., Tsuji, T., Chamoto, K., Takeshima, T., Sendo, F., Nishimura, T. (2003) Successful elimination of memory-type CD8 T cell subset by the administration of anti-Gr-1 monoclonal antibody in vivo Cell. Immunol. 224,98-105[CrossRef][Medline]
  21. Yu, C. L., Sun, K. H., Tsai, C. Y., Tsai, Y. Y., Tsai, S. T., Huang, D. F., Yu, H. S. (1998) Expression of Th1/Th2 cytokine mRNA in peritoneal exudative polymorphonuclear neutrophils and their effects on mononuclear cell Th1/Th2 cytokine production in MRL-lpr/lpr mice Immunology 95,480-487[CrossRef][Medline]
  22. Huang, J. F., Yang, Y., Sepulveda, H., Shi, W., Hwang, I., Peterson, P. A., Jackson, M. R., Sprent, J., Cai, I. (1999) TCR-mediated internalization of peptide-MHC complexes acquired by T cells Science 286,952-954[Abstract/Free Full Text]
  23. Hudrisier, D., Bongrand, P. (2002) Intercellular transfer of antigen-presenting cell determinants onto T cells: molecular mechanisms and biological significance FASEB J. 16,477-486[Abstract/Free Full Text]
  24. Hwang, I., Sprent, J. (2001) Role of the actin cytoskeleton in T cell absorption and internalization of ligands from APC J. Immunol. 166,5099-5107[Abstract/Free Full Text]
  25. Hudrisier, D., Riond, J., Mazarguil, H., Gairin, J. E., Joly, E. (2001) CTLs rapidly capture membrane fragments from target cells in a TCR signaling-dependent manner J. Immunol. 166,3645-3649[Abstract/Free Full Text]
  26. Sjostrom, A., Eriksson, M., Cerboni, C., Johansson, M. H., Sentman, C. L., Karre, K., Hoglund, P. (2001) Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors J. Exp. Med. 194,1519-1530[Abstract/Free Full Text]
  27. Savina, A., Vidal, M., Colombo, M. I. (2002) The exosome pathway in K562 cells is regulated by Rab11 J. Cell Sci. 115,2505-2515[Abstract/Free Full Text]
  28. Afeltra, A., Caccavo, D., Ferri, G. M., Addessi, M. A., De Rosa, F. G., Amoroso, A., Bonomo, L. (1997) Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies Clin. Exp. Immunol. 109,279-285[CrossRef][Medline]
  29. Deriy, L. V., Chor, J., Thomas, L. L. (2000) Surface expression of lactoferrin by resting neutrophils Biochem. Biophys. Res. Commun. 275,241-246[CrossRef][Medline]
  30. Attucci, S., Korkmaz, B., Juliano, L., Hazouard, E., Girardin, C., Brillard-Bourdet, M., Anthonioz, R. P., Gauthier, F. (2002) Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates Biochem. J. 366,965-970[Medline]
  31. Bangalore, N., Travis, J. (1994) Comparison of properties of membrane-bound versus soluble forms of human leukocytic elastase and cathepsin G Biol. Chem. Hoppe Seyler 375,659-666[Medline]
  32. Mulder, A. H., Heeringa, P., Brouwer, E., Limburg, P. C., Kallenberg, C. G. (1994) Activation of granulocytes by anti-neutrophil cytoplasmic antibodies (ANCA): a Fc{gamma}RII-dependent process Clin. Exp. Immunol. 98,270-278[Medline]
  33. Daley, J. M., Reichner, J. S., Mahoney, E. J., Manfield, L., Henry, W. L., Jr, Mastrofrancesco, B., Albina, J. E. (2005) Modulation of macrophage phenotype by soluble product(s) released from neutrophils J. Immunol. 174,2265-2272[Abstract/Free Full Text]
  34. Borregaard, N., Cowland, J. B. (1997) Granules of the human neutrophilic polymorphonuclear leukocyte Blood 89,3503-3521[Free Full Text]
  35. Balazovich, K. J., Fernandez, R., Hinkovska-Galcheva, V., Suchard, S. J., Boxer, L. A. (1996) Transforming growth factor-ß 1 stimulated degranulation and oxidant release by adherent human neutrophils J. Leukoc. Biol. 60,772-777[Abstract]
  36. Elbim, C., Reglier, H., Fay, M., Delarche, C., Andrieu, V., El Benna, J., Gougerot-Pocidalo, M. A. (2001) Intracellular pool of IL-10 receptors in specific granules of human neutrophils: differential mobilization by proinflammatory mediators J. Immunol. 166,5201-5207[Abstract/Free Full Text]
  37. Na, Y. J., Han, S. B., Kang, J. S., Yoon, Y. D., Park, S. K., Kim, H. M., Yang, K. H., Joe, C. O. (2004) Lactoferrin works as a new LPS-binding protein in inflammatory activation of macrophages Int. Immunopharmacol. 4,1187-1199[CrossRef][Medline]
  38. Guillen, C., McInnes, I. B., Vaughan, D. M., Kommajosyula, S., Van Berkel, P. H., Leung, B. P., Aguila, A., Brock, J. H. (2002) Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice J. Immunol. 168,3950-3957[Abstract/Free Full Text]
  39. Kobayashi, S., Sato, R., Inanami, O., Yamamori, T., Yamato, O., Maede, Y., Sato, J., Kuwabara, M., Naito, Y. (2005) Reduction of concanavalin A-induced expression of interferon-{gamma} by bovine lactoferrin in feline peripheral blood mononuclear cells Vet. Immunol. Immunopathol. 105,75-84[Medline]
  40. Togawa, J., Nagase, H., Tanaka, K., Inamori, M., Umezawa, T., Nakajima, A., Naito, M., Sato, S., Saito, T., Sekihara, H. (2002) Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance Am. J. Physiol. Gastrointest. Liver Physiol. 283,G187-G195[Abstract/Free Full Text]
  41. Moed, H., Stoof, J., Boorsma, D. H., von Blomberg, M. E., Gibbs, S., Bruynzeel, D. P., Sckper, R. J., Rustemeyer, T. (2004) Identification of anti-inflammatory drugs according to their capacity to suppress type-1 and type-2 T cell profiles Clin. Exp. Allergy 34,1868-1875[CrossRef][Medline]
  42. Dhennin-Duthille, I., Masson, M., Damiens, E., Fillebeen, C., Spik, G., Mazurier, J. (2000) Lactoferrin upregulates the expression of CD4 antigen through the stimulation of motogen-activated protein-kinase in the human lymphoblastic T Jurkat cell line J. Cell. Biochem. 79,583-593[CrossRef][Medline]
  43. Boxer, L. A., Haak, R. A., Yang, H. H., Wolach, J. B., Whitcomb, J. A., Butterick, C. J., Baehner, R. L. (1982) Membrane-bound lactoferrin alters the surface properties of polymorphonuclear leukocytes J. Clin. Invest. 70,1049-1057[Medline]
  44. Adeyemi, E. O., Campos, L. B., Loizou, S., Walport, M. J., Hodgson, H. J. (1990) Plasma lactoferrin and neutrophil elastase in rheumatoid arthritis and systemic lupus erythematosus Br. J. Rheumatol. 29,15-20[Abstract/Free Full Text]
  45. Matsuyama, W., Yamamoto, M., Hagashimoto, I., Oonakahara, K., Watanabe, M., Machida, K., Yoshimura, Y., Eiraku, N., Kawabata, M., Osame, M., Arimura, K. (2004) TNF-related apoptosis-inducing ligand is involved in neutropenia of systemic lupus erythematosus Blood 104,184-191[Abstract/Free Full Text]



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