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Department of Pathology, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is also
present) (4, 5, 6). Constitutive or regulated expression of
these and other proteins allows neutrophils to influence both innate
and adaptive immunity (1).
Although neutrophils generally have not been appreciated to have
significant function as APCs, they have been shown to present
peptide-MHC-I complexes; exogenous peptide added to neutrophils in
vitro can bind cell surface MHC-I and stimulate memory CD8 T cells
(7). In addition, neutrophils stimulated with GM-CSF and
IFN- were found to express MHC-II and mediate superantigen-induced T
cell activation, but were not found to process protein Ag for
MHC-II-restricted presentation to T cells (4, 5). Thus,
neutrophils have been shown to possess certain Ag-presenting functions,
but their ability to actively process exogenous Ag has not been
established.
The conventional MHC-I Ag-processing pathway generally involves the processing of cytosol-derived Ag. Exogenous Ag can be processed via alternate MHC-I Ag-processing mechanisms, which may involve delivery of exogenous Ag from vacuolar compartments to the cytosol for cytosolic processing (8, 9, 10) or processing wholly within vacuolar compartments without access to the cytosol (i.e., vacuolar alternate MHC-I Ag processing) (11, 12, 13, 14, 15, 16, 17). Particulate Ags, including bacteria, are efficiently processed by macrophages and dendritic cells for MHC-I presentation.
Although neutrophils phagocytose bacteria, express MHC-I, and can present exogenous synthetic peptides, previous studies have not tested whether neutrophils can process Ag expressed by bacteria for MHC-I presentation. The studies presented here establish that neutrophils process phagocytosed bacteria via an alternate MHC-I Ag-processing pathway that allows MHC-I presentation of peptides derived from the bacteria. Although this processing was inhibited by cytochalasin D, which blocks phagocytosis, it was not affected by lactacystin, a proteasome inhibitor, or brefeldin A, which blocks anterograde transport from the endoplasmic reticulum (ER) through the Golgi apparatus. These results indicate that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway. In addition, we observed that neutrophils release or "regurgitate" processed peptide into the extracellular space, and this peptide can bind MHC-I on neighboring cells (e.g., macrophages or dendritic cells) for presentation to CD8 T cells, although it remains to be determined whether the predominant mechanism for neutrophil generation of peptide-MHC-I complexes involves intravacuolar binding or regurgitation and surface binding. Our model suggests that neutrophils may directly present bacterial Ags to stimulate local effector T cell responses, including the production of cytokines, particularly when effector T cells and neutrophils are colocalized at sites of inflammation. In addition, neutrophils may regurgitate peptides to load MHC-I molecules on professional APCs, e.g., dendritic cells, which could either elicit local effector T cell responses or migrate to lymph nodes to stimulate primary T cell responses. These are the first reported studies of the ability of neutrophils to process phagocytosed bacteria for presentation to T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells were cultured at 37°C in a 5% CO2 atmosphere in standard medium composed of DMEM (Life Technologies, Grand Island, NY) with 10% FCS (HyClone Laboratories, Logan, UT), 5 x 10-5 M 2-ME, 116 mg/L L-arginine hydrochloride, 36 mg/L L-asparagine, 2 g/L NaHCO3, 1 mM sodium pyruvate, and antibiotics. Neutrophils were elicited from B6D2F1/J (H-2b x d) or CBA/J (H-2k) mice (The Jackson Laboratory, Bar Harbor, ME) by i.p. injection of 1.5 ml sterile thioglycolate (29 mg/ml) without resazurin (Difco, Detroit, MI). Four hours later, peritoneal exudate cells were removed from 3–12 mice by peritoneal lavage (using PBS with 0.02% EDTA), centrifuged for 10 min at 300 x g, and resuspended in 1–4 ml PBS (original resuspension volume). Unless otherwise noted, procedures for preparation of neutrophils were at room temperature. Cells (1 ml/tube) were layered over 9 ml of 90% Percoll (Pharmacia, Peapack, NJ) in PBS and centrifuged for 30 min at 50,000 x g (27,500 rpm using a type-40 rotor; Beckman Coulter, Fullerton, CA). Neutrophils appeared in a distinct band well below the band containing macrophages and lymphocytes. Cells were withdrawn using a Pasteur pipette and were then washed once in PBS and twice in standard medium. Cells were stained with the Hema-3 system (Fisher Scientific, Pittsburgh, PA) to confirm neutrophil purity of 93–99%. Neutrophils were further purified by complement depletion of potential residual macrophages. Cells were incubated for 20 min at 37°C in standard medium (107 cells/ml) and then for 60 min at 4°C with F4/80 Ab (Caltag Laboratories, Burlingame, CA) at 2 µg/ml in standard medium. Cells were washed three times and resuspended in PBS with 0.3% BSA to the original resuspension volume. Rabbit complement (Low-Tox-M; Accurate Chemical and Scientific, Westbury, NY) was added (1 ml/10 ml of cell suspension) for 1 h at 37°C. Cells were washed three times in standard medium and used in Ag-processing assays. Depletion of macrophages increased neutrophil purity by 1–3% (to 94–99%) by morphologic analysis and flow cytometry.
For control Ag-processing cells, macrophages were also obtained from peritoneal exudate cells that were harvested 4 h after injection of thioglycolate and centrifuged on Percoll gradients (as described above). Cells were withdrawn from the macrophage/lymphocyte band in the Percoll gradient and washed as for neutrophils. Of the resulting cells, 66% were macrophages (as assessed by morphology). For electroporation experiments, macrophages were generated by i.p. injection of Con A (100 µg/mouse) in B6D2F1/J mice. Five days later, cells were harvested by peritoneal lavage.
Dendritic cells were isolated from C57BL/6 bone marrow as previously described (18). Cells were flushed from femurs, passed through a 70-µm cell strainer, pelleted, and resuspended for 10 min in 0.83% NH4Cl to lyse erythrocytes. Remaining cells were incubated for 1 h at 4°C with GK1.5 (anti-CD4), 53.6-72 (anti-CD8), RA3 3A11 (anti-B220), B21-2, and 34.5.3S (anti-I-Ab or -I-Ad) (American Type Culture Collection, Manassas, VA; each Ab was present at 20 µg/108 bone marrow cells). Cells were pelleted and resuspended with complement (as described above). Remaining cells were cultured in 24-well plates (106 cells/well) with 500 U/ml GM-CSF (BD PharMingen, San Diego, CA). Nonadherent cells were removed every 2 days. Dendritic cells were harvested after 6 days by pipetting, depleted of granulocytes using magnetic beads (Dynal, Lake Success, NY), coated with anti-Gr-1 (BD PharMingen), incubated for 2 h to remove adherent macrophages, and harvested by vigorous pipetting. The resulting cells had dendritic morphology and a pattern of intracellular acid phosphatase staining typical of dendritic cells (focal perinuclear distribution for dendritic cells vs diffuse cytoplasmic distribution for macrophages) (19). Flow cytometric analysis of these cells showed that 90% had high expression of CD11c, and <1% expressed Gr-1.
Ag-processing assays
HB101.Crl-OVA and HB101.Crl-HEL are E. coli that have been transfected to constitutively express Crl-OVA or Crl-HEL, antigenic fusion proteins containing portions of OVA or hen egg lysozyme (11, 20). Crl-OVA contains the OVA257–264 epitope, which is presented by Kb. Bacteria were harvested from plates, washed in PBS with 10 mM MgCl2 and 0.04% glucose, and suspended in standard medium without antibiotics immediately before use. To produce heat-killed bacteria, bacteria were resuspended in PBS, incubated at 65°C for 60 min, and resuspended in standard medium.
In the basic Ag-processing protocol, neutrophils (0.5–2 x 105/well) or control macrophages were incubated overnight at 37°C in U-bottom 96-well plates (Falcon 3077; BD Biosciences, Franklin Lakes, NJ) with heat-killed bacteria (105 to 3 x 107/well) and CD8OVA1.3 T hybridoma cells (105/well), which detect OVA257–264-Kb complexes (11). Plates were centrifuged, and supernatants (0.1 ml) were harvested to determine IL-2 content as a measure of T cell response (see CTLL-2 bioassay for IL-2 below). A second protocol allowed the use of viable bacteria and/or pharmacologic manipulation of the Ag-processing incubation before fixation of neutrophils and subsequent incubation with CD8OVA1.3 cells. Neutrophils (2 x 105/well in 96-well plates or 106 cells in 2-ml polypropylene tubes) were incubated for 15–20 min in standard medium (without antibiotics for experiments with live bacteria) with or without brefeldin A (0.5–1 µg/ml; Sigma-Aldrich, St. Louis, MO), lactacystin (10–40 µM; purchased from E. J. Corey, Harvard University, Cambridge, MA), or cytochalasin D (10 µg/ml; Sigma-Aldrich). Bacteria were added for 90–120 min in the continued presence or absence of inhibitor. Neutrophils were fixed for 20 min in 1% paraformaldehyde in PBS, washed, incubated for 20 min with 0.2 M lysine in standard medium, and washed three times with standard medium. CD8OVA1.3 T hybridoma cells (105/well) were added, the plates were incubated 20–24 h, and supernatants were harvested for an IL-2 assay (see CTLL-2 bioassay for IL-2). A third protocol was designed to test for peptide regurgitation. Dendritic cells (104/well) or Con A-elicited macrophages from C57BL/6 or B6D2F1/J mice (105/well) were fixed with paraformaldehyde, washed, and incubated overnight with 105 viable neutrophils from CBA/J mice, various doses of heat-killed HB101.Crl-OVA, and 105 CD8OVA1.3 T hybridoma cells. Supernatants were harvested for an IL-2 assay.
Peritoneal macrophages were used in control experiments to confirm the activity of inhibitors for blockade of the conventional MHC-I Ag-processing pathway, because these cells were more amenable to electroporation than neutrophils. Cells were resuspended at 8 x 106/ml in DMEM with or without brefeldin A (0.5–1 µg/ml) or lactacystin (10–40 µg/ml), incubated for 10 min at 37°C in polypropylene tubes with mixing, and cooled to 4°C. OVA protein (Sigma-Aldrich, catalog no. A-5503) was added (0.6 mg/ml final concentration), and macrophages were electroporated at 4°C with 200V, 800 µF, and low resistance settings using a Cell-Porator and 4-mm gap cuvettes (Life Technologies) (21). Macrophages were washed, plated (105/well), incubated for 2 h at 37°C in standard medium with the continued presence or absence of inhibitor, fixed, and washed. CD8OVA1.3 T hybridoma cells were added for 24 h, and supernatants were harvested as described above.
CTLL-2 bioassay for IL-2
Supernatants from Ag-processing experiments were frozen, thawed, and assessed for IL-2 using a colorimetric CTLL-2 bioassay (18, 22). IL-2-dependent CTLL-2 cells were washed three times and incubated (5 x 103/well) in the supernatants for 20–24 h at 37°C. Alamar blue (Accumed, Chicago, IL) was added (15 µl/well) to assess metabolic activity. The plates were incubated for 20–24 h, and the extent of Alamar blue reduction was determined by the difference in optical density at 550 and 595 nm using a Bio-Rad (Hercules, CA) model 550 microplate spectrophotometer (18).
Flow cytometry
To assess neutrophil purity, 5 x 105 cells were stained for 30 min at 4°C in FACS buffer (PBS with 1% BSA) containing 10 µg/ml RB6-8C5 anti-Gr-1 Ab (23) or rat IgG2b isotype control (Caltag Laboratories), washed three times with FACS buffer, incubated with FITC-conjugated goat F(ab')2 anti-rat IgG H and L (Caltag Laboratories) for 1 h, washed three times, and analyzed with a flow cytometer (FACScan, BD Biosciennes; or Epics elite ESP, Beckman Coulter, Miami, FL). To assess MHC-I expression, neutrophils were stained with biotinylated 28-8-6 anti-H-2Kb/H-2Db (BD PharMingen) or biotinylated mouse IgG2a isotype control Ab (BD PharMingen) and streptavidin-CyChrome (BD PharMingen). Fluorescein- labeled bacteria were used to assess phagocytosis. Heat-killed HB101. Crl-OVA (1 ml at 109 bacteria/ml in PBS (pH 9.1)) were declumped by aspiration 10–20 times through a 27-gauge needle, centrifuged for 2 min at 16,000 x g, resuspended in 1 ml PBS (pH 9.1), and declumped again. Bacteria were combined in the dark with 100 µl of Fluos (Boehringer Mannheim, Indianapolis, IN; 10 mg/ml in DMSO), incubated for 70 min at room temperature with rocking and declumping every 15 min, washed four times in PBS, declumped, and resuspended in standard medium. Neutrophils (5 x 105/ml) were incubated with bacteria (at titers used in Ag-processing experiments) for 60 min at 37°C in 1 ml standard medium in round-bottom polypropylene tubes with rocking. Neutrophils were washed, fixed for 20 min in 1% paraformaldehyde in PBS, and evaluated by flow cytometry. Events were gated to exclude those with insufficient forward and side scatter to qualify as mammalian cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils phagocytose and kill bacteria but have not been demonstrated to process bacterial Ags for presentation to T cells. Because neutrophils, with some exceptions (4, 5, 6), do not generally express MHC-II molecules, their potential ability to function as APCs for CD4 T cell responses appears limited. However, neutrophils do express MHC-I (1, 2, 3) and have been shown to present MHC-I-restricted peptides (7). Thus, we propose that alternate MHC-I Ag-processing mechanisms could allow the processing of bacteria that are phagocytosed by neutrophils.
To establish a system for analysis of MHC-I expression and Ag
processing, murine neutrophils were isolated from thioglycolate-induced
acute peritoneal exudates. Exudate cells consisted primarily of
neutrophils with small numbers of macrophages and lymphocytes (66% of
exudate cells were neutrophils by microscopic analysis of stained
cells, and 64% had high expression of Gr-1, a neutrophil marker, by
flow cytometry; Fig. 1
A).
Purification of neutrophils on Percoll density gradients resulted in
neutrophil purity of 93–99% (range in different experiments) by
morphology and a virtually identical percentage of cells with high
expression of Gr-1 by flow cytometry (Fig. 1A). In contrast,
resident peritoneal macrophages from naive mice or bone marrow-derived
macrophages had no significant expression of Gr-1, and macrophages from
acute thioglycolate-elicited peritoneal exudates had at most only low
expression of Gr-1 (data not shown, corresponding to gate M2 in Fig. 1A, below the high expression gate containing neutrophils).
In some experiments, neutrophils isolated on Percoll gradients were
further purified by treatment with F4/80 and complement to increase
neutrophil purity by an additional 1–3% (as assessed by morphology).
Flow cytometry also revealed that 99% of neutrophils expressed MHC-I
(Fig. 1B). These approaches provided highly purified
neutrophil preparations for analysis of Ag-processing function.
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To assess bacterial uptake, neutrophils were incubated for 60 min
with varying numbers of fluorescein-labeled heat-killed HB101.Crl-OVA
(E. coli bearing a fusion protein that contains the
OVA257–264 epitope) and evaluated by flow
cytometry (Fig. 1
C). To calculate the number of bacteria per
neutrophil, events were first selected by gating to include
fluorescein-positive events (gate M1, Fig. 1C) with
scatter properties of intact neutrophils. The mean fluorescent value
(MFV) of these events was then divided by the MFV of isolated
fluorescein-labeled HB101.Crl-OVA to obtain mean bacterial uptake per
neutrophil (Table I). Of neutrophils that
internalized bacteria (i.e., fluorescein-positive neutrophils), the
mean bacterial uptake per neutrophil ranged from 0.9 to 5.9 at
different bacterium:neutrophil ratios (Table I). Theoretically, these
numbers may slightly underestimate bacterial uptake, because bacterial
degradation may decrease cell-associated fluorescence. However, at
lower limiting bacterial dilutions (bacterium:neutrophil ratio from 1:1
to 10:1), this approach indicated that fluorescein-positive neutrophils
(M1 gate of Fig. 1C) contained an average of close to 1
bacterium per neutrophil (Table I). Most of the variation in
fluorescence of these cells was explained by variation in the level of
labeling of individual bacterium (Fig. 1C). If bacterial
degradation or other artifacts substantially affected this calculation,
the number of bacteria per fluorescein-positive neutrophil would not
approach 1 (e.g., degradation of bacteria would reduce the MFV of
neutrophils at limiting bacterial dilutions). Therefore, bacterial
uptake can be approximated by dividing the MFV of neutrophils by the
MFV of isolated bacteria.
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C and Table I). In conclusion,
conditions that produced detectable Ag presentation (see
Alternate MHC-I processing of bacterial Ag by
neutrophils) involved phagocytosis of as little as 1–2
bacteria/neutrophil.
The calculations of total bacterial uptake do not reveal whether
neutrophil-associated bacteria were intracellular or on the cell
surface. To address this question, neutrophils were incubated with
fluorescein-labeled bacteria for 60 min, transferred to 4°C without
fixation, and directly analyzed by flow cytometry with and without
addition of ethidium bromide (100 µg/ml) to quench the fluorescence
of extracellular or surface-bound bacteria as previously described
(24, 25, 26). The fluorescence of isolated bacteria (without
exposure to neutrophils) was decreased by 88% when quenched by 100
µg/ml ethidium bromide (data not shown). When neutrophils were
exposed to bacteria at 4°C, the fluorescence of neutrophil-associated
bacteria was decreased by 92% with ethidium bromide (Fig. 2), indicating that essentially all of
these bacteria were surface bound. Uptake of bacteria was greater at
37°C than at 4°C, and bacterial fluorescence was decreased by only
44% with ethidium bromide (Fig. 2). These results indicate that
56% of neutrophil-associated bacteria were intracellular after a
60-min incubation at 37°C (the presence of some bacteria at the cell
surface is consistent with the lack of a chase period).
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To examine processing of phagocytosed bacteria,
B6D2F1/J neutrophils were purified on Percoll
gradients and incubated overnight with heat-killed HB101.Crl-OVA and
CD8OVA1.3 T cell hybridoma cells (which recognize
OVA257–264-Kb complexes).
T cell stimulation was determined by IL-2 secretion as assessed with a
colorimetric CTLL-2 bioassay. Ag expressed by bacteria was processed
and presented by neutrophils to CD8OVA1.3 T hybridoma cells (Fig. 3A). Ag presentation was
detected with as few as 1–3 x 106
bacteria/well (bacterium:neutrophil ratio from 10:1 to 30:1),
corresponding to phagocytosis of only 1–2 bacteria by 25.7–44% of
neutrophils (see above and Table I). No significant presentation was
observed when T hybridoma cells alone were incubated with bacteria
(i.e., in the absence of neutrophils; data not shown) or when T
hybridoma cells were incubated with bacteria and MHC-disparate
(H-2k) neutrophils (data not shown and section on
peptide regurgitation). Significant Ag presentation was observed
with as few as 2 x 104 neutrophils/well
(97–98% pure neutrophils; data not shown). Heat-killed bacteria were
used to avoid bacterial contamination in the previous experiments
(which used a simple, nonfixation protocol), but similar results were
obtained in experiments with live bacteria and a fixation protocol
(Fig. 3B). In addition, alternate MHC-I processing of
HB101.Crl-OVA was inhibited by cytochalasin D, which inhibits
actin-dependent functions such as phagocytosis (Fig. 4), indicating that phagocytic
internalization of bacteria by neutrophils was necessary for alternate
MHC-I processing and presentation of bacterial Ag.
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). In addition, macrophages were
isolated from acute, thioglycolate-induced peritoneal exudates using
Percoll gradients. The Ag processing and presentation activity of
neutrophils was then compared with that of macrophages isolated from
the same exudate (Fig. 6). Neutrophils
and macrophages were both capable of alternate MHC-I Ag processing and
presentation, but neutrophils were more efficient than macrophages at
the same titer (105 cells/well). Furthermore,
numbers of macrophages that could potentially contaminate 93–99% pure
neutrophil preparations (1–7% of the population, i.e., 1–7 x
103 cells in wells containing
105 cells) generated small or undetectable levels
of presentation, significantly (p < 0.01, Fig. 6) below the level produced by 105 neutrophils.
These observations indicate that alternate MHC-I Ag-processing activity
of purified neutrophil preparations was truly attributable to
neutrophils.
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Alternate MHC-I Ag processing may involve cytosolic Ag processing, following escape of Ag from the phagosome into the cytosol, or vacuolar Ag processing within the phagosome, followed by peptide binding to MHC-I molecules within the vacuole. A third possibility is that vacuolar processing is followed by secretion or regurgitation of peptide and binding of peptide to MHC-I at the cell surface. More than one of these mechanisms may contribute to alternate MHC-I Ag processing, and their relative contributions may vary under different conditions.
To determine the mechanism(s) of neutrophil Ag processing, we used
inhibitors with differential effects on cytosolic and vacuolar
processing mechanisms. Proteasome inhibitors and brefeldin A (an
inhibitor of anterograde ER-Golgi transport) inhibit cytosolic
processing and the conventional MHC-I pathway (21, 27, 28, 29, 30)
but do not block vacuolar alternate MHC-I Ag processing (11, 12, 13, 14, 15, 16, 21). Neutrophils were incubated with brefeldin A, lactacystin (a
proteasome inhibitor), or control medium for 15 min and then incubated
for 90 min with HB101.Crl-OVA in the continued presence or absence of
the inhibitors. The neutrophils were then fixed, washed, and incubated
overnight with CD8OVA1.3 T hybridoma cells to determine the extent of
Ag processing. Alternate MHC-I Ag-processing activity of neutrophils
was completely resistant to both lactacystin and brefeldin A (Fig. 7A). To provide a positive
control for the pharmacologic activities of lactacystin and brefeldin
A, we confirmed that both inhibitors were able to block the processing
of OVA protein that was electroporated into the cytosol of macrophages
(Fig. 7B; neutrophils were not used because extensive cell
death occurred with these cells upon electroporation). The continued
processing of bacterial Ag by neutrophils under conditions that block
cytosolic processing indicates that alternate MHC-I Ag processing by
neutrophils occurs by vacuolar mechanisms.
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Peptides generated by vacuolar alternate MHC-I Ag processing may
bind to MHC-I molecules within vacuolar compartments or may be
regurgitated into the surrounding milieu and bind to MHC-I molecules on
the surface of the same cell or a neighboring cell (11, 31). To evaluate peptide regurgitation, we incubated viable
MHC-mismatched (CBA/J) neutrophils with HB101.Crl-OVA and fixed
B6D2F1/J macrophages. Viable CBA/J neutrophils
were capable of phagocytic processing of bacteria but lacked expression
of Kb to present OVA peptide to CD8OVA1.3 T
cells, whereas the fixed B6D2F1/J macrophages
were unable to phagocytose or process bacteria but were capable of
presenting exogenous peptide to CD8OVA1.3 T cells. Processing and
presentation of bacterial Ag occurred under these conditions, although
neither viable CBA/J neutrophils nor fixed
B6D2F1/J macrophages alone elicited a response
after overnight incubation with HB101.Crl-OVA and CD8OVA1.3 cells (Fig. 8A). Thus, neutrophils can
process bacterial Ags and regurgitate peptides that bind to MHC-I
molecules on macrophages for subsequent presentation to T cells. Fixed
dendritic cells were also shown to bind and present peptides
regurgitated by CBA/J neutrophils (Fig. 8B). In addition,
chloroquine and ammonium chloride inhibited the ability of neutrophils
to process HB101.Crl-OVA for regurgitation of peptides onto fixed
macrophages for presentation to T cells (Fig. 9). This observation indicates that
processing for regurgitation was dependent on intracellular vacuolar
processing mechanisms that are inhibited by these lysosomotropic amines
(which disrupt vacuolar pH gradients). We conclude that neutrophils
phagocytose and process bacterial Ags and that presentation to T cells
is mediated both directly by neutrophils and indirectly by professional
APCs, e.g., macrophages and dendritic cells, after regurgitation of
peptide from neutrophils.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils are most likely limited to the presentation of MHC-I-restricted Ag, because they generally lack MHC-II molecules (with some specific exceptions; Ref. 4, 5, 6). Processing of Ags expressed by phagocytosed bacteria must occur by an alternate MHC-I Ag-processing mechanism, but several different alternate MHC-I Ag-processing mechanisms have been proposed and may exist in different situations. Cytosolic processing mechanisms involve exit of Ag from vacuoles into the cytosol, degradation of Ag by proteasomes, transport of peptides by TAP into the ER, and transport of peptide-MHC-I complexes through the ER-Golgi to the cell surface. In contrast, vacuolar processing mechanisms involve vacuolar Ag proteolysis and binding of peptides to MHC-I molecules within vacuolar compartments or on the cell surface after peptide regurgitation. Thus, vacuolar processing is independent of proteasome-mediated proteolysis and ER-Golgi transport, and it is resistant to inhibitors (e.g., lactacystin and brefeldin A, respectively) that block these functions and block cytosolic processing. These inhibitors were used to distinguish between potential cytosolic and vacuolar processing mechanisms in neutrophils. Processing of phagocytosed bacteria by neutrophils was not blocked by inhibitors of cytosolic Ag processing, indicating that alternate MHC-I Ag processing by neutrophils can involve vacuolar mechanisms. It is possible that alternate MHC-I processing of other types of Ag could involve cytosolic mechanisms, but there are currently no data to assess this hypothesis. We conclude that neutrophils mediate alternate MHC-I Ag processing, including contributions by vacuolar processing mechanisms.
Previous studies have suggested that vacuolar alternate MHC-I Ag processing may involve binding of peptides to MHC-I molecules either within vacuolar compartments (e.g., phagosomes) or on the cell surface (following peptide regurgitation), and it is possible that both of these mechanisms occur. We observed that peptides were regurgitated by MHC-mismatched neutrophils and bound to MHC-I molecules on fixed macrophages or dendritic cells for presentation to T cells. This observation suggests two interesting conclusions. First, peptide regurgitation may contribute, at least in part, to generation of peptide-MHC-I complexes on neutrophils and direct presentation of Ag by neutrophils to T cells ("autoregurgitation"). This does not exclude additional generation of peptide-MHC-I complexes within intracellular vacuolar compartments. Second, peptide regurgitation may allow neutrophils to deliver peptides to other APCs, e.g., macrophages and dendritic cells ("trans-regurgitation"). In this case, neutrophils would indirectly contribute to Ag presentation by performing the initial proteolytic steps of Ag processing, but the final steps of direct presentation to T cells would be mediated by professional APCs. Professional APCs have properties (life span, costimulator expression, and the ability to migrate to lymph nodes) that promote their ability to induce T cell responses. Thus, neutrophils may provide both direct and indirect contributions to presentation of Ag to T cells.
The indirect and direct contributions of neutrophils to Ag presentation may affect different stages of T cell responses. Neutrophils primarily encounter and phagocytose bacteria at peripheral (nonlymphoid) sites of infection and inflammation, and this tissue localization provides potential interaction with effector or memory T cells but little chance of interaction with naive T cells, which are primarily localized within lymphoid tissues. Furthermore, in agreement with other studies, we found that costimulatory molecules (CD80 or CD86) were not expressed or expressed at very low levels by neutrophils (data not shown), which limits their potential to activate naive T cells effectively. Therefore, direct Ag presentation by neutrophils is unlikely to impact on priming of T cell responses. Effector T cells may respond to direct Ag presentation by neutrophils to activate various effector mechanisms, including cytokine production, which may influence immune responses and host defense. Direct presentation of Ag by neutrophils may also contribute to the amplification of CD8 responses or the longevity of CD8 T cell memory.
In contrast, neutrophils may contribute indirectly to presentation of Ags for priming of T cell responses. Professional APCs such as macrophages and dendritic cells are often resident in tissues and may also migrate to sites of inflammation. Thus, neutrophils present at a site of acute infection or inflammation are likely to encounter both bacterial Ag and professional APCs. Peptide regurgitation by neutrophils, which are highly phagocytic and may be present in large numbers, may contribute substantially to formation of peptide-MHC-I complexes on nearby professional APCs. Dendritic cells in receipt of neutrophil-derived peptides may migrate to lymph nodes and present Ag to activate naive T cells, providing a mechanism for neutrophils to indirectly contribute to priming of T cell responses.
Neutrophils directly present bacterial Ags on MHC-I molecules but not MHC-II molecules, but one can consider the possibility that neutrophils contribute indirectly to MHC-II-restricted Ag presentation via regurgitation of peptides onto professional APCs. Stern and colleagues (32, 33) have recently suggested that peptide-MHC-II complexes may be formed on the surface of dendritic cells, although their system did not involve any contribution of neutrophils. Although previous studies have demonstrated that at least some MHC-I Ag presentation can result from peptide regurgitation (11, 31), peptide regurgitation has not been demonstrated to contribute substantially to MHC-II Ag presentation (34). Thus, it remains unclear whether indirect contributions of neutrophils to bacterial Ag presentation via trans-regurgitation of peptides apply only to presentation by MHC-I molecules or to presentation by both MHC-I and MHC-II molecules.
Neutrophils may make unique contributions to processing of bacterial Ags. The high phagocytic capacity of neutrophils makes them a repository of bacterial Ags for processing. Direct or indirect contributions by neutrophils could allow enhanced presentation and generation of T cell responses to these Ags, which would otherwise be lost to the immune system. In addition, the extensive capacities of neutrophils for phagocytosis, microbicidal functions, and catabolism of phagocytosed material may make them uniquely capable of processing certain bacteria, especially pathogenic bacteria that are resistant to host defense mechanisms. These bacteria may be more efficiently killed and processed by neutrophils than by other cells (e.g., dendritic cells), which may have less developed microbicidal functions.
In summary, the results presented here provide the first demonstration that neutrophils can mediate alternate MHC-I Ag processing of bacterial Ags and their direct presentation to T cells. Neutrophils can also contribute to the formation of peptide-MHC-I complexes on other APCs, providing an indirect contribution to presentation of bacterial Ags. Although neutrophils were once thought to provide only microbicidal function to innate immune defense, they are now appreciated to influence adaptive immunity, including T cell responses, via a number of mechanisms, including cytokine secretion, synthesis and expression of MHC molecules, and formation of peptide-MHC complexes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Clifford V. Harding, Department of Pathology, Case Western Reserve University, Biomedical Research Building 925, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail address: cvh3{at}po.cwru.edu
3 Abbreviations used in this paper: MHC-I, class I MHC; MHC-II, class II MHC; ER, endoplasmic reticulum; MFV, mean fluorescence value.
Received for publication January 22, 2001. Accepted for publication June 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway. J. Immunol. 156:4182.[Abstract]
TCR+ cells by Mycobacterium tuberculosis via an alternate class I MHC antigen-processing pathway. J. Immunol. 162:372.-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation. J. Biol. Chem. 272:13437.This article has been cited by other articles:
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  M. Pletneva, H. Fan, J.-J. Park, V. Radojcic, C. Jie, Y. Yu, C. Chan, A. Redwood, D. Pardoll, and F. Housseau IFN-Producing Killer Dendritic Cells Are Antigen-Presenting Cells Endowed with T-Cell Cross-Priming Capacity Cancer Res., August 15, 2009; 69(16): 6607 - 6614. [Abstract] [Full Text] [PDF]
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 E. F. Lind, C. L. Ahonen, A. Wasiuk, Y. Kosaka, B. Becher, K. A. Bennett, and R. J. Noelle Dendritic Cells Require the NF-{kappa}B2 Pathway for Cross-Presentation of Soluble Antigens J. Immunol., July 1, 2008; 181(1): 354 - 363. [Abstract] [Full Text] [PDF]
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 C. Beauvillain, Y. Delneste, M. Scotet, A. Peres, H. Gascan, P. Guermonprez, V. Barnaba, and P. Jeannin Neutrophils efficiently cross-prime naive T cells in vivo Blood, October 15, 2007; 110(8): 2965 - 2973. [Abstract] [Full Text] [PDF]
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S. B. Lovitch, T. J. Esparza, G. Schweitzer, J. Herzog, and E. R. Unanue Activation of Type B T Cells after Protein Immunization Reveals Novel Pathways of In Vivo Presentation of Peptides J. Immunol., January 1, 2007; 178(1): 122 - 133. [Abstract] [Full Text] [PDF]
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L. B. Arruda, D. Sim, P. R. Chikhlikar, M. Maciel Jr, K. Akasaki, J. T. August, and E. T. A. Marques Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes J. Immunol., August 15, 2006; 177(4): 2265 - 2275. [Abstract] [Full Text] [PDF]
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S. B. Lovitch, Z. Pu, and E. R. Unanue Amino-Terminal Flanking Residues Determine the Conformation of a Peptide-Class II MHC Complex. J. Immunol., March 1, 2006; 176(5): 2958 - 2968. [Abstract] [Full Text] [PDF]
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D. Martinez, M. Vermeulen, A. Trevani, A. Ceballos, J. Sabatte, R. Gamberale, M. E. Alvarez, G. Salamone, T. Tanos, O. A. Coso, et al. Extracellular Acidosis Induces Neutrophil Activation by a Mechanism Dependent on Activation of Phosphatidylinositol 3-Kinase/Akt and ERK Pathways J. Immunol., January 15, 2006; 176(2): 1163 - 1171. [Abstract] [Full Text] [PDF]
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 R. Rosales-Reyes, C. Alpuche-Aranda, M. d. l. L. Ramirez-Aguilar, A. D. Castro-Eguiluz, and V. Ortiz-Navarrete Survival of Salmonella enterica Serovar Typhimurium within Late Endosomal-Lysosomal Compartments of B Lymphocytes Is Associated with the Inability To Use the Vacuolar Alternative Major Histocompatibility Complex Class I Antigen-Processing Pathway Infect. Immun., July 1, 2005; 73(7): 3937 - 3944. [Abstract] [Full Text] [PDF]
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 K. P.J.M. van Gisbergen, M. Sanchez-Hernandez, T. B.H. Geijtenbeek, and Y. van Kooyk Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN J. Exp. Med., April 18, 2005; 201(8): 1281 - 1292. [Abstract] [Full Text] [PDF]
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E. J. Cheadle, D. O'Donnell, P. J. Selby, and A. M. Jackson Closely Related Mycobacterial Strains Demonstrate Contrasting Levels of Efficacy as Antitumor Vaccines and Are Processed for Major Histocompatibility Complex Class I Presentation by Multiple Routes in Dendritic Cells Infect. Immun., February 1, 2005; 73(2): 784 - 794. [Abstract] [Full Text] [PDF]
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 T. Engeman, A. V. Gorbachev, D. D. Kish, and R. L. Fairchild The intensity of neutrophil infiltration controls the number of antigen-primed CD8 T cells recruited into cutaneous antigen challenge sites J. Leukoc. Biol., November 1, 2004; 76(5): 941 - 949. [Abstract] [Full Text] [PDF]
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A. R. Tvinnereim, S. E. Hamilton, and J. T. Harty Neutrophil Involvement in Cross-Priming CD8+ T Cell Responses to Bacterial Antigens J. Immunol., August 1, 2004; 173(3): 1994 - 2002. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, N. S. Potter, L. Ramachandra, R. K. Pai, M. Convery, W. H. Boom, and C. V. Harding Alternate Class I MHC Antigen Processing Is Inhibited by Toll-Like Receptor Signaling Pathogen-Associated Molecular Patterns: Mycobacterium tuberculosis 19-kDa Lipoprotein, CpG DNA, and Lipopolysaccharide J. Immunol., August 1, 2003; 171(3): 1413 - 1422. [Abstract] [Full Text] [PDF]
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P. J. Chefalo, A. G. Grandea III, L. Van Kaer, and C. V. Harding Tapasin-/- and TAP1-/- Macrophages Are Deficient in Vacuolar Alternate Class I MHC (MHC-I) Processing due to Decreased MHC-I Stability at Phagolysosomal pH J. Immunol., June 15, 2003; 170(12): 5825 - 5833. [Abstract] [Full Text] [PDF]
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S. B. Lovitch, J. J. Walters, M. L. Gross, and E. R. Unanue APCs Present A{beta}k-Derived Peptides That Are Autoantigenic to Type B T Cells J. Immunol., April 15, 2003; 170(8): 4155 - 4160. [Abstract] [Full Text] [PDF]
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S. A. Fulton, S. M. Reba, T. D. Martin, and W. H. Boom Neutrophil-Mediated Mycobacteriocidal Immunity in the Lung during Mycobacterium bovis BCG Infection in C57BL/6 Mice Infect. Immun., September 1, 2002; 70(9): 5322 - 5327. [Abstract] [Full Text] [PDF]
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A.-M. Lennon-Dumenil, A. H. Bakker, R. Maehr, E. Fiebiger, H. S. Overkleeft, M. Rosemblatt, H. L. Ploegh, and C. Lagaudriere-Gesbert Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation J. Exp. Med., August 19, 2002; 196(4): 529 - 540. [Abstract] [Full Text] [PDF]
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S. H. Gregory and E. J. Wing Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections J. Leukoc. Biol., August 1, 2002; 72(2): 239 - 248. [Abstract] [Full Text] [PDF]
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