Published online before print April 7, 2006
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Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, Canada
1Correspondence: Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Rd., Saskatoon, SK, S7N 5E3, Canada. E-mail: philip.griebel{at}usask.ca
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Key Words: neutrophils • MHC II • APC • antigen presentation • microparticles • CD3
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-), interleukin (IL)-1, IL-3, IL-8, and tumor necrosis factor [2
]. Thus, PMNs are transcriptionally active and have the potential to vary in their responses to a broad range of innate immune stimuli.
Until recently, there was no evidence to suggest that neutrophils might play a role in the induction of an adaptive immune response [2
]; however, there are several reports that PMNs from a variety of species can express major histocompatibility complex (MHC) class II and costimulatory molecules (CD80 and CD86) [3
4
5
6
]. Coexpression of MHC class II and costimulatory molecules would potentially endow PMNs with the capacity to influence the adaptive immune system through antigen presentation. Previous investigations with mouse, human, and goat PMNs defined conditions whereby MHC class II expression was observed following stimulation with IFN-
, granulocyte macrophage-colony stimulating factor (GM-CSF), or IL-3 [7
8
9
10
] or in human patients, with Wegener’s granulomatosis [11
, 12
]. In some cases, PMN expression of MHC II was reported as constitutive [4
, 13
], and human PMNs were also shown to function as accessory cells for primed T cell activation with protein antigens and superantigens [6
, 11
, 14
, 15
]. One of our earlier observations that recombinant bovine adenovirus vaccine vectors had a tropism for PMNs triggered an interest in determining the role of PMNs in the induction of a specific immune response. As bovine adenoviral vectors can stimulate strong immune reactions [16
], we tested the hypothesis that bovine PMNs might function as antigen-presenting cells (APCs), thereby providing a link between the innate and adaptive branches of the immune system. As there was a growing body of literature to suggest that PMNs from other species could assume such a role, we began by investigating the expression of MHC class II, a key protein involved in antigen presentation, on bovine PMNs. We observed that bovine PMNs expressed detectable levels of MHC class II on their surface, only when cocultured with peripheral blood mononuclear cells (PBMC). This observation suggested that PMNs may up-regulate endogenous MHC class II expression or passively acquire MHC class II protein from other leukocytes.
Recent observations suggest that cell membrane fragments are readily exchanged between certain immune cells [17 18 19 20 21 ]. Although the consequences of this event are not fully understood, several interesting theories are emerging to explain its significance. "Cross-presentation", for example, is a term coined for the circumstance where a DC can acquire antigen from another cell and present this antigen on endogenous MHC class I. What is especially unique about this process is that DC are able to sample extracellular membranes and intracellular proteins from live cells [20 ]. Other cell types, such as macrophages, can only acquire cytoplasmic membranes from dead or dying cells [20 ]. T cells have also been shown to acquire membrane proteins from APCs in an activation-dependent manner [17 , 19 ]. In particular, T cells can acquire MHC class II proteins and costimulatory molecules, which support antigen presentation to other primed T cells [17 , 19 ]. Furthermore, some B cell lines are capable of a continuous uptake of autologous cell membrane and proteins through an immunological synapse [22 ].
The purpose of this study was to investigate if MHC class II detected on the surface of bovine PMNs was the result of passive acquisition or endogenous synthesis of MHC class II protein. The data presented demonstrate that bovine PMNs have a remarkable capacity to passively acquire plasma membrane lipids and a variety of integral membrane proteins from several types of dead or dying cells.
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was from Novartis (Basel, Switzerland).
Purification of cells
Blood was collected from the jugular vein of male or female Holstein cattle (age >12 months) using 0.3% EDTA (Sigma-Aldrich). Whole blood was centrifuged at 1400 g without braking for 20 min to separate the buffy coat layer, which was removed at the interface between the red blood cell (RBC) pellet and the plasma, diluted 1:1 with calcium and magnesium-free phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3), layered onto Ficoll-Hypaque (Pharmacia LKB Biotechnology, Uppsala, Sweden), and centrifuged at 1400 g (without braking) for 20 min to isolate the PBMC fraction. After removal of plasma and buffy coat, the top half of the remaining RBC/PMN fraction was also discarded. RBCs were lysed by adding 5–10 ml RBC/PMN fraction to 40 ml lysis solution (distilled water with 0.17 M NH4Cl, 10 mM KHCO3, and 0.11 mM EDTA, pH 7.3), followed by three washes with PBS. Cells prepared by this method were consistently >98% viable, as determined by trypan blue dye exclusion, and contained less than 4% mononuclear cells. No attempt was made to differentiate between PMN and eosinophilic granulocytes, but the latter were usually present in low numbers.
To obtain an enriched population of B cells, PBMC were collected, resuspended in PBS at 4 x 107 cells/ml, and incubated with 1 ml MM1A (anti-CD3; VMRD, Pullman, WA) and DH59B (anti-CD172a; VMRD) sodium azide-free monoclonal antibodies (mAb) at 5 µg/ml for 30 min on ice. Cells were then incubated with goat anti-mouse immunoglobulin G (IgG)-coated magnetic beads (Dynabeads® M-450, Dynal, Great Neck, NY) at a bead-to-target cell ratio of 5:1. Bead-bound cells were removed with a magnet as described by the manufacturer, and the remaining cells were washed in PBS and resuspended in AIM-V medium.
Clone 2 B cells (a sheep B cell line) were cultured as described previously [23
]. An enriched T cell (CD3 cells) population was obtained by culturing 5 x 106 PBMC in 5 ml AIM-V medium with 5% v/v FBS, 4 µg/ml Concanavalin A (Con A; Sigma-Aldrich), and 4 ng/ml human IL-2. Cells were passaged, and the media were replaced every 2–3 days. This culture system produced an activated T cell population (>99% CD3+), which expressed MHC class II (
50% MHC II+).
PMNs were collected from the mammary gland of heifers following injection into each teat canal of 5 ml minimal essential medium (MEM) containing 1 µg/ml LPS (Sigma-Aldrich). PMNs were collected 16 h after LPS injection by infusing each teat with 10 ml MEM, massaging the mammary gland, and then expressing the liquid from the teat canal into a sterile bottle. This procedure was repeated with 5 ml MEM. Cytospins stained with DifQuick (Baxter Scientific, Miami, FL) were examined with a microscope, and PMN purity was consistently 95–98% [24
].
Transwell-separated cocultures were performed using a 0.4-µm transwell plate (Corning Costar, Cambridge, MA). All cell cultures were incubated at 37°C in 5% CO2, and all cocultures were carried out at a 1:3 ratio (PMN:other cell) unless otherwise specified.
PMN coculture design
Cocultures were carried out in six-well culture dishes (Corning Costar) containing 6 ml complete AIM-V medium, 2–3 x 106 PMNs, and three times this number of PBMC. Cultures were incubated for 20–24 h in a 37°C humidified chamber, or cell populations were cooled to 4°C prior to mixing and culturing at 4°C in a refrigerator. Cells were collected for analysis by gently pipetting cocultures to resuspend the cells, which were then washed once with PBS prior to cell labeling and analysis.
Biotin and fluorescence labeling
Cells were labeled with PKH-26 red fluorescent cell linker kit (Sigma-Aldrich), according to the manufacturer’s instructions. Similarly, according to the manufacturer’s instructions, viable cells were labeled with EZ-Link Sulfo-NHS-Biotin (Pierce, Rockford, IL). Biotinylation of surface proteins was confirmed by flow cytometric analysis of streptavidin-fluorescein isothiocyanate (FITC; Southern Biotechnology Birmingham, AL) binding.
Induction of apoptosis or necrosis
Cell lysis was performed by three cycles of snap-freezing in liquid nitrogen and thawing in a 37°C water bath (freeze/thaw, 1x107cells/ml). Cells were then sonicated for 20 s using a microtip vibrating at 20 kHz and amplitude of 40% (VibraCellTM sonicator, Betatek Inc., ON, Canada). This process resulted in plasma membrane disruption and lysis of all cells and is subsequently described as a form of cell "necrosis". Cells were -irradiated with 11,000 rads to induce apoptosis, and the induction of apoptosis was confirmed by using the deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Flow TACSTM in situ apoptosis detection kit, R&D Systems) to detect DNA fragmentation.
Membrane extraction
Clone 2 B cells or bovine B cells were washed two times in PBS, and 8 x 107 cells were resuspended in 2 ml homogenization buffer (20 mM Tris HCl, pH 7.5, 10 mM NaCl, 0.1 mM MgCl2, 10 mM phenylmethylsulfonyl fluoride). Cells were sonicated for 20 s, underlaid with 5 ml 40% sucrose (in homogenization buffer), and centrifuged at 75,000 g for 1 h at 4°C. The white floccular material at the interface between the buffer and sucrose was collected, diluted in PBS, and pelleted at 210,000 g for 1 h at 4°C. The pellet was resuspended in PBS and stored at –70°C.
Flow cytometry
mAb specific for bovine CD3 (Clone MM1A), CD14 (Clone MM61A), CD21 (Clone Baq15A), CD172a (Clone DH59B), and the DR region of bovine MHC II (Clone TH14B) were purchased from VMRD. FITC- and phycoerythrin-conjugated, isotype-specific goat anti-mouse Ig antibodies were purchased from Southern Biotechnology. The level of specific mAb binding was quantified by subtracting cells that reacted with an equivalent concentration of an isotype-matched, irrelevant mAb (Cedar Lane Laboratories Ltd., ON, Canada). Cells were fixed in 2% paraformaldehyde in PBS (pH 6.7) and stored in the dark at 4°C until data acquisition and analysis were performed with a FACScan flow cytometer using the CellQuest program (Becton Dickinson, Mountain View, CA). When PBMC or other cells were cocultured with PMNs, it was possible to specifically analyze PMN phenotype by collecting events within a region (R1) defined by the relatively high 90° light side-scatter (SSC) generated by the cytoplasmic granules of PMNs (see Fig. 1C
, R1).
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Figure 1. Cell isolation methodology alters MHC class II expression on bovine PMNs. (A) Three isolation techniques were compared: Purified PMNs were isolated from blood (n=10) by using centrifugation to separate mononuclear cells from PMNs (Centrifugation); lysis buffer was used to remove erythrocytes from blood (n=10) and leave a combined PMN and mononuclear cell population (Lysis); collection of fluid was from the mammary gland (n=6) following LPS injection, which specifically recruited PMNs (Mammary Gland). Data presented are values for individual animals (bar=group median value), and matched blood samples were used to compare isolation methods. The centrifugation and lysis techniques were also compared at 0 h and 24 h after blood collection. Centrifugation versus lysis at 0 h and 24 h (P<0.001 and 0.004, respectively); centrifugation at 0 h versus 24 h (P=0.002) (B) A representative fluorescein-activated cell sorter histogram (i) and dot plots (ii, iii) of anti-MHC class II mAb-stained bovine PMNs (FL1 vs. forward angle light scatter (FALS)) following coculture for 24 h with irradiated PBMC. PMN labeled with an isotype control mAb are represented as dotted line (i) or scatter plot (ii), and PMN labeled with anti-MHC class II mAb are represented as a solid line (i) or scatter plot (iii). (C) Dot scatter profile of lysed bovine blood with cell size [x-axis: forward-scatter (FSC), height] and cell granularity (y-axis: SSC, height) is presented. The region used for PMN phenotypic analyses in B is defined (R1), and PBMC are visible as the ungated SSCLo population.
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Induction of MHC II expression
Previous investigators reported that MHC class II expression could be induced when purified human PMNs were cultured with GM-CSF or IFN- [7
8
9
10
]. Purified bovine PMNs were incubated with recombinant bovine GM-CSF (1 ng/ml), IFN- (10 or 100 ng/ml), LPS (0.5 ug/ml), or a combination of all three stimuli, but no detectable change in MHC class II expression was observed (Fig. 2A
). It has also been reported that a soluble T cell factor induced MHC class II expression on human PMNs [6
]. To determine if a soluble T cell factor might induce MHC II expression, purified PMNs were cocultured for 24 h with a 0.4-µm transwell membrane separating PMNs from PBMC or Con A/IL-2-activated T cells. The transwell membrane should facilitate an exchange of secreted, soluble factors but prevent direct cell-cell contact. PMNs cocultured with PBMC or activated T cells by this method showed no detectable MHC II expression (Fig. 2B)
. Only when PMNs were cocultured in contact with PBMC was detectable MHC class II expression detected on PMNs, and this apparent expression occurred within 40 min of coculture and with equal efficiency at 4°C and 37°C (Fig. 2B)
. These observations supported the conclusion that a form of cell-cell contact might be required for PMNs to express MHC class II molecules on their surface.
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Figure 2. PMN stimulation does not alter expression of MHC class II. (A) Purified PMNs were cultured for 24 h with medium alone, recombinant bovine IFN- (100 ng/ml), recombinant bovine GM-CSF (1 ng/ml), Escherichia coli LPS (0.5 µg/ml), or a combination of all three stimuli (I+G+L). Data presented are values for individual animals (bar=median value for each group), and PMNs from each animal (n=8) were used to compare all culture conditions. (B) Purified PMNs were cultured alone (Medium) or cocultured for 40 min at 4°C and 37°C or 24 h at 37°C in direct contact with PBMC (PBMC 40 min 4°C; PBMC 40 min 37°C; PBMC 24 h). All cocultures were significantly different from the medium control (P<0.008). PMNs were also cocultured for 24 h with PBMC (PBMC+ transwell) or activated T cells (T-cell + transwell) with a 0.4-µm transwell membrane separating each population. Transwell cultures were not significantly different from medium control. Data presented are values for individual animals (bar=median value for each group), and PMNs from each animal (n=8) were used to compare all culture condition.
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Figure 3. Passive transfer of murine MHC class II (Mur MHC II) to bovine PMNs (Bov MHC II). Purified bovine PMNs were cocultured for 24 h with murine splenocytes. The mAb used to detect species-specific MHC class II and the coculture conditions are indicated beneath the x-axis. Comparison of mAb labeling within each coculture group revealed a significant difference (P<0.004). Data presented are values for individual animals (n=9), and the median value (bar) is indicated for each group.
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Figure 4. Identification of MHC class II donor cell phenotype. (A) PBMC were labeled with anti-MHC II, anti-CD3 (T cells), anti-CD14 (monocytes), and anti-CD21 (B cells) mAb to determine viable cell number before and after a 24-h coculture with PMNs. A significant reduction in MHC II+ (P<0.005) and CD21+ (P<0.006) cells was observed. (B) Purfied PMNs were cultured in medium alone or cocultured for 24 h with bovine PBMC, purified CD3+ T cells (T-cells), Con A-stimulated T cells (Activated T-cells), CD14+ monocytes (Monocytes), or an enriched [PBMC–(CD3+ and C14+ cells)] B cell population (B-cells). Viable Clone 2 sheep B cells (Clone 2), isolated Clone 2 cell membranes (Cl2-memb), and freeze-thaw and lysed Clone 2 B cells (Cl2-F/T) were also cocultured with purified PMNs for 24 h. Data presented are values for PMNs isolated from six animals, and the median value (bar) is indicated for each group. When compared with the medium control, significant differences were observed following PMN coculture with PBMC (P<0.003), activated T cells (P<0.03), B cells (P<0.01), Clone 2 membranes (P<0.03), and lysed Clone 2 cells (P<0.003).
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17.0% of PMNs, and induction of T cell apoptosis with -irradiation (confirmed by TUNEL assay) significantly increased CD3 transfer (Fig. 5
). Thus, the passive acquisition of MHC class II and CD3 molecules suggested that PMN acquisition of integral membrane proteins might reflect a general process. Two different labeling techniques were then used to determine if PMNs passively acquired membrane lipids and a broad range of integral membrane proteins. Bovine PBMC were labeled with the lipophilic PKH-26 dye, or PBMC surface proteins were biotinylated before PBMC coculture with PMNs. PKH was transferred from PBMC to all PMNs within 24 h (Fig. 6
), and this transfer was also observed within 40 min of coculture (data not shown). A similar pattern of transfer was also observed for biotinylated surface molecules (Fig. 6)
, which supported the conclusion that PMNs passively acquired plasma membrane lipids and integral membrane proteins from donor cells.
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Figure 5. PMNs passively acquire CD3 and MHC class II from bovine T cells. Purified PMNs were cultured for 24 h in medium alone or cocultured with PBMC, activated T cells (T-cells), or -irradiated PBMC and T cells (PBMCs+irr; T-cells+irr), and then, PMN were labeled with anti-MHC class II or anti-CD3 mAb, as indicated. Data presented are values for PMN isolated from individual animals (n=9), and group medians (bar) are indicated. All cocultures were significantly different from the medium control (P=0.004), and anti-CD3 labeling increased significantly (P<0.008) when PMNs were cocultured with -irradiated T cells.
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Figure 6. Plasma membrane proteins and lipids are transferred from PBMC to PMNs. Purified PMNs were cultured for 24 h in medium alone or cocultured with unlabeled PBMC (MHC class II; CD3), biotin-labeled PBMC (Biotin), or PKH-26 labeled PBMC (PKH). Flow cytometry was used to detect MHC class II, CD3, biotin, or PKH transfer to PMNs from PBMC. Data presented are values for PMNs isolated from individual animals (n=7), and the median value (bar) is indicated for each group. When compared with medium control, there was significant transfer to PMNs of MHC class II (P<0.001) and CD3 (P<0.001) from unlabeled PBMC, biotinylated proteins from biotinylated PBMC (P<0.001), and PKH lipophilic label (P<0.03) from PKH-labeled PBMC.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A previous report [8
] that human PMNs expressed MHC class II did not provide sufficient technical detail to determine if passive transfer of MHC class II was a possibility during cell isolation or culture. However, culture conditions used in this study may have supported the increased expression of MHC class II on contaminating mononuclear cells. Thus, there was no assurance that MHC class II detected on PMNs wasn’t passively acquired from other contaminating cells. Radsak et al. [6
] used reverse transcriptase-polymerase chain reaction (PCR) to confirm endogenous MHC class II expression in PMNs, but the presence of even low numbers of monocytes or DC could have affected PCR results. This group also suggested that a soluble T cell factor was responsible for the induction of MHC class II expression on human PMNs. In contrast, bovine PMNs cocultured in the presence of activated T cells or PBMC separated by a 0.4-um transwell membrane did not show detectable MHC class II expression (Fig. 2)
. Furthermore, studies with recombinant cytokines were consistent with a previous report that IFN- could not induce MHC class II expression on purified, caprine PMNs, another ruminant species [4
]. Thus, the present observations consistently supported the conclusion that bovine PMNs passively acquired MHC class II.
Of particular importance was the rapid acquisition of MHC class II by bovine PMNs within 40 min of coculture with PBMC and the observation that this acquisition occurred with equal efficiency at 4°C and 37°C (Fig. 2B) . Mammalian cell transcription and translation are markedly decreased at 4°C [25 ], and this limits metabolically active processes. Furthermore, reduced membrane fluidity at 4°C restricts the potential for membrane fusion and phagocytosis. Thus, the rapid acquisition of MHC class II on the surface of PMNs at 4°C is not consistent with phagocytosis but is more consistent with a direct attachment of membrane fragments to the PMN surface. A proposed model for the observed transfer of membrane lipids and integral membrane protein (Fig. 6) from dead or dying cells (Figs. 4 and 5) is presented in Figure 7 . This model proposes that membrane fragments, such as microvesicles, which can contain integral membrane proteins [26 ], are shed from dying cells. These microvesicles can exhibit increased adherence [27 ], and we hypothesize that they attach to the PMN surface before being incorporated into the PMN membrane. This model would also be consistent with our present observations (Fig. 1) and previous observations with caprine PMNs [4 ] that PMN isolation by centrifugation can decrease the apparent expression of MHC class II. Centrifugation may be able to remove loosely attached microvesicles, and the observation that centrifugation no longer reduced MHC class II on PMNs following incubation of blood at 37°C for 24 h (Fig. 1) may be consistent with the phagocytic uptake of donor cell fragments or fusion of microparticles with the PMN plasma membrane. However, if phagocytosis is involved in this membrane transfer, then a mechanism must exist by which there is an efficient recycling of membrane lipids and a broad range of integral membrane proteins from the phagolysosome to the cell surface. The observation that PKH, a lipophilic fluorescent dye, was transferred from PBMC to PMNs (Fig. 6) indicates that donor cell membranes were transferred with integral membrane proteins. This observation is consistent with a transfer of membrane proteins through direct PMN-donor cell contact or the release of membrane fragments or microparticles from dead or dying cells. Furthermore, there appeared to be a relationship between the level of protein expression on a donor cell and the efficiency with which the protein was transferred to PMNs. For example, all T cells expressed CD3, but only a subpopulation expressed a relatively lower level of MHC class II protein. We observed a much greater transfer of CD3 (Fig. 5) than MHC class II (Fig. 4B) from bovine T cells to PMN. This apparent lack of specificity in the transfer of membrane proteins implicates a mechanism that does not rely on specific recognition signals for protein uptake or insertion into the PMN plasma membrane. The binding of microparticles to the PMN surface and the subsequent fusion of these microparticles with the plasma membrane could explain this nonspecific process.
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Figure 7. Model for the passive acquisition of plasma membrane lipids and proteins by bovine PMNs. The induction of apoptosis or necrosis induces donor cells (D) to release microparticles (i–iii), which can contain biotin-labeled (B) integral membrane proteins (IMP) and lipophilic PKH dye (PKH) inserted into the plasma membrane. These microparticles then attach to the PMN plasma membrane (iv and v) and with time, integral membrane proteins fuse with the plasma membrane (vi and vii), such that acquired proteins and lipids can be detected on the cell surface.
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Received May 28, 2005; accepted February 27, 2006.
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