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Defining the roles of human CEACAMs during neutrophil responses to Neisseria 1
gonorrhoeae 2
3
Helen Sarantis and Scott D. Gray-Owen* 4
5
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada 6
7
Running Title: Dissecting roles of neutrophil CEA family members 8
9
*Address for Correspondence: 10
Dr. Scott D. Gray-Owen 11
University of Toronto 12
Department of Molecular Genetics 13
Toronto, Ontario 14
M5S 1A8 15
Canada 16
Tel. 416-946-5307 17
Fax. 416-978-6885 18
Email: [email protected] 19
20
21
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Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.05702-11 IAI Accepts, published online ahead of print on 7 November 2011
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ABSTRACT 23
24
Symptomatic infection of humans with Neisseria gonorrhoeae is characterized by a 25
neutrophil-rich cervical or urethral exudate, suggesting neutrophils are important both for 26
clearance of these bacteria and for the pathogenesis of gonorrhea. Neisseria interact with 27
neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion 28
molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial 29
binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by 30
human neutrophils, yet their co-expression has precluded understanding of the relative 31
contribution of each CEACAM to functional responses of neutrophils during neisserial 32
infection. In this work, we directly address the role of each CEACAM during infection 33
by introducing individual human CEACAMs into a differentiated murine MPRO cell 34
line-derived neutrophil model. Murine neutrophils cannot bind the human-restricted 35
Neisseria; yet, we show that introducing any of the Opa-binding CEACAMs of human 36
neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry of 37
Neisseria into murine neutrophils. While CEACAM1- and CEACAM6-expressing 38
neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly 39
proceeds without appreciable neutrophil activation. In stark contrast, neisserial 40
engulfment via CEACAM3 recapitulates the oxidative burst and intracellular granule 41
release seen during human neutrophil infection. Finally, by co-expressing multiple 42
CEACAMs in our model, we show that expression of CEACAM1 and CEACAM6 43
potentiate, rather than hinder, CEACAM3-dependent responses of neutrophils, exposing 44
a co-operative role for this family of proteins during neisserial infection of neutrophils. 45
These observations illustrate a divergence in function of CEACAMs in neutrophils, and 46
implicate the human-restricted CEACAM3 in the neutrophil innate response to neisserial 47
infection. 48
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INTRODUCTION 50
51
Symptomatic gonococcal infection is caused by the human-restricted bacterial 52
pathogen Neisseria gonorrhoeae, and involves a massive influx of polymorphonuclear 53
neutrophils (PMNs) into the infected urogenital tract. This results in the characteristic 54
PMN-filled urethral or cervical exudate, which is the hallmark of gonorrhea. PMNs are 55
part of a ‘first line of defense’ against bacterial infection through their prompt recruitment 56
and activation at infected sites, where they internalize and neutralize invading pathogens 57
via the production of reactive oxygen species and the release of antimicrobial agents (27). 58
Recognition of bacteria by PMNs can involve specific binding to host-derived opsonins 59
such as serum complement or immunoglobulins that coat the bacteria; however, the 60
interaction between Neisseria and PMNs can also be opsonin-independent (40). 61
Specifically, these microorganisms can bind to and activate neutrophils directly via their 62
colony opacity-associated (Opa) outer membrane proteins, the majority of which bind 63
members of the human carcinoembryonic antigen-related cellular adhesion molecule 64
(CEACAM) family (10, 44). 65
66
To date, four CEACAMs have been shown to act as receptors for the gonococcal 67
Opa proteins: CEACAM1, CEACAM3, CEACAM5 and CEACAM6 (4, 7, 10). Human 68
neutrophils express three of these (CEACAM1, CEACAM3 and CEACAM6), as well as 69
CEACAM4 and CEACAM8, which do not bind Opa (11, 32). Opa-dependent binding to 70
PMNs results in bacterial killing through the ability of neutrophils to capture, internalize, 71
and mount antimicrobial responses upon neisserial infection (28, 43). While the intimate 72
association between N. gonorrhoeae and PMNs is well-described, co-expression of 73
multiple CEACAMs in PMNs has made it difficult to specifically attribute individual 74
CEACAMs to the bactericidal response. Antibody cross-linking studies have suggested 75
that ligation of CEACAMs, individually (39) or in concert (36), results in neutrophil 76
activation. Any one of the three Opa-binding CEACAMs of PMNs can mediate bacterial 77
engulfment by transfected epithelial cell models (20). However, reflective of their 78
different cytoplasmic domains, studies of these receptors in a number of cell types, 79
including lymphocytes (5), epithelial cells (20) and endothelial cells (26), have shown 80
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that they can elicit distinct, and often opposing, cellular responses. For example, ligation 81
of CEACAM1, which contains two immunoreceptor tyrosine-based inhibition motifs 82
(ITIMs; V/L/IxYxxL/V), results in phosphatase recruitment and the suppression of 83
phosphotyrosine-based signaling cascades (8, 17). In contrast, ligation of CEACAM3, 84
which contains an immunoreceptor tyrosine-based activation motif (ITAM; YxxL/Ix6-85
8YxxL/I), results in kinase recruitment and propagation of signaling (21, 30). The effects 86
of engaging the glycosylphosphatidylinositol (GPI)-anchored CEACAM6 remain largely 87
unexplored. 88
89
In this work, we have used a genetic approach to study the individual roles of 90
CEACAM1, CEACAM3 and CEACAM6 in neutrophils. We show that all three 91
CEACAMs can bind and engulf N. gonorrhoeae. However, internalization via 92
CEACAM1 and CEACAM6 proceeds without substantial neutrophil activation, while 93
internalization via CEACAM3 results in stimulation of the PMN oxidative burst and 94
release of intracellular granules in a manner dependent on its cytoplasmic ITAM. 95
Unexpectedly, CEACAM1, which we show can be phosphorylated in PMNs, and has 96
been shown to be inhibitory in other systems (5), does not suppress CEACAM3 function 97
in the neutrophil. Instead, we show that CEACAM3 can transduce activating signals 98
upon bacterial CEACAM1 ligation, revealing a previously unrecognized co-operative 99
interaction between CEACAMs in neutrophils. Importantly, our work solidifies the 100
importance of CEACAM3 in the initiation and propagation of signaling in response to 101
neisserial infection of neutrophils. 102
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METHODS 104
105
Reagents and antibodies 106
All reagents were from Sigma (Oakville, ON) unless otherwise indicated. The 107
diisopropyl fluorophosphate (DFP) was from BioShop (Burlington, ON). Pansorbin 108
(fixed Staphylococcus aureus) was from Calbiochem (La Jolla, CA). The anti-109
gonococcal polyclonal rabbit antibody (UTR01) was described previously (21). The 110
rabbit CEACAM-specific polyclonal antiserum and normal rabbit serum were from Dako 111
(Mississauga, ON). The CEACAM pan-specific D14HD11, CEACAM1-specific 4/3/17, 112
CEACAM6-specific 9A6, and CD67 antibodies were from Genovac GmbH (Freiburg, 113
Germany). The anti-phospho Syk, anti-phospho Vav, and anti-Vav antibodies were from 114
Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Syk antibody (Syk-01) was from 115
Abcam (Cambridge, MA), the CEACAM3-specific Col-1 antibody was from Zymed (San 116
Francisco, CA), and the rat anti-mouse CD11b antibody was from BD Biosciences 117
(Mississauga, ON). Fluorescent conjugates were from Jackson ImmunoResearch 118
Laboratories (Mississauga, ON), except for Texas Red-phalloidin, which was from 119
Molecular Probes (Eugene, OR). 120
121
Primary neutrophil isolation 122
Human PMNs were isolated from citrated whole blood taken from healthy volunteers by 123
venipuncture using Ficoll-Paque Plus (Amersham Biosciences; Buckinghamshire, 124
England). Contaminating erythrocytes were removed by dextran sedimentation and 125
hypotonic shock, as described previously (21). Mouse bone marrow neutrophils were 126
taken from 8 to 10-week old FVB mice that were killed by CO2 inhalation. Specifically, 127
femurs and tibias were removed and bone marrow was isolated and separated on a 128
discontinuous Percoll gradient (80%/65%/55%). Neutrophils were recovered at the 129
80%/65% interface. 130
131
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MPRO cell culture and differentiation to neutrophils 133
The MPRO cells were purchased from the American Type Culture Collection, and were 134
maintained in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 1.25-135
5% conditioned HM-5 supernatant, 20% horse serum (Invitrogen; Burlington, ON) and 136
1% Glutamax supplement (Invitrogen). The HM-5 GM-CSF producing-cell line was 137
maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf 138
serum (Hyclone; Logan, UT), and was a kind gift from Dr. Steven Collins (Fred 139
Hutchinson Cancer Research Center, Seattle, WA). GM-CSF-conditioned media was 140
prepared as described in Lawson et al. (15). MPRO differentiation was induced with 10 141
μM all-trans retinoic acid (ATRA) in normal growth medium for roughly 72 hours, after 142
which cells were washed and maintained in Hank’s balanced salt solution (HBSS; 143
Invitrogen) supplemented with 10 mM HEPES, adjusted to a pH of 7.4. 144
145
Recombinant DNA constructs and establishment of human CEACAM-expressing 146
MPRO cells 147
Plasmids containing CEACAM1 and CEACAM6 in pRC/CMV were generously 148
provided by Dr. Wolfgang Zimmermann (Munich, Germany). Plasmids containing full-149
length CEACAM3, a naturally occurring isoform of CEACAM3 lacking most of the 150
cytoplasmic domain (1C1), and CEACAM3 mutant cDNAs in pCEP4, were created by 151
Dr. A. Popp and kindly provided by Prof. T. F. Meyer (Max-Planck-Institut fuer 152
Infektionsbiologie, Berlin, Germany). cDNAs were amplified via polymerase chain 153
reaction (5’ primers contained a Kozak sequence, GCC ACC ATG, for protein 154
expression), and subcloned into pMSCVpuro (all cDNAs; Clontech Laboratories, Inc.) or 155
pMSCVblast (a recombinant pMSCVpuro in which the puromycin resistance gene was 156
swapped for the blastocydin resistance gene from pCDNA6 (Invitrogen), the latter used 157
to express CEACAM1 and CEACAM6 cDNAs for creation of the MPRO-158
CEACAM1+CEACAM3 and MPRO-CEACAM3+CEACAM6 cell lines. These 159
constructs were introduced into un-differentiated MPRO cells using a pantropic retroviral 160
expression system (Clontech Laboratories, Inc.) as per the manufacturer’s instructions. 161
Briefly, the packaging cell line, GP-293, was co-transfected with the respective pMSCV 162
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vector containing CEACAM cDNA as well as pVSV-G (to allow viral incorporation of 163
the vesicular stomatitis virus envelope glycoprotein) using FuGene 6 (Roche). After 48 164
hours, virus-containing supernatants were collected and concentrated by ultra-165
centrifugation as described by Zhou et. al (46) at 120,000 x g for 2 hours at 4oC, and then 166
un-differentiated MPRO cells were infected with the VSV-G-pseudotyped virus by 167
centrifuging the cells with the concentrated virus preparation for 2 hours at 3,000 x g at 168
room temperature. The infected MPRO cells were left overnight at 37oC, and selected 169
the following day with 10 μg/ml puromycin (for pMSCVpuro-containing virus) or 10 170
μg/ml blastocydin (Invitrogen) (for pMSCVblast-containing virus). Single drug-resistant 171
cells were cloned to create monoclonal, stable cell lines, which were differentiated to 172
PMNs using ATRA for use in experiments. 173
174
175
Bacterial strains 176
All N. gonorrhoeae (‘Ngo’) strains used in this study were derived from the non-piliated 177
MS11 strain, and were kindly provided by Dr. T. F. Meyer (Max-Planck-Institut fuer 178
Infektionsbiologie, Berlin, Germany), and have been described previously (11, 14). 179
Briefly, the strains used are as follows: the non-CEACAM-binding N302 strain (referred 180
to here as Opa-), the CEACAM1/CEACAM5-binding N306 strain (expressing Opa59; 181
referred to here as OpaCCM1), and the CEACAM1/CEACAM3/CEACAM5/CEACAM6-182
binding N313 strain (expressing Opa57; referred to here as OpaCEA). Bacteria were 183
subcultured daily using a binocular microscope to monitor colony opacity phenotypes, 184
and grown on solid agar medium (GC + Isovitalex) at 37oC with 5% CO2. 185
186
Bacterial Infections for Immunofluorescence (IF) Miscroscopy and Survival Assays 187
5 x 105 MPRO PMNs were centrifuged onto washed FBS-coated coverslips at 63 g for 10 188
minutes. Where inhibitors were used, cells were incubated as described for 20 minutes at 189
37oC prior to infection. Cells were then infected at a multiplicity of infection (MOI) of 25 190
(for binding and internalization studies) or 50 (for F-actin co-localization studies and 191
survival assay) in a volume of 500 μl, re-centrifuged for 5 minutes to facilitate bacterial 192
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association with cells, and then incubated at 37oC for 5 minutes (for F-actin co-193
localization studies) or 30 minutes (for internalization and survival studies). Post-194
infection, IF samples were washed with HBSS, and fixed using 3.7% paraformaldehyde. 195
Cells were permeabilized using 0.4% Triton X-100, and stained and observed as 196
described previously (21). For assessment of intracellular versus extracellular bacteria, 197
infected cells were first stained with the rabbit polyclonal antisera against N. gonorrhoeae 198
followed by a Cy5-conjugated secondary; cells were then permeabilized and then stained 199
with the rabbit polyclonal antisera against N. gonorrhoeae followed by a FITC-200
conjugated secondary and Texas Red (TR)-conjugated phalloidin, to localize the cells. 201
Using this method, extracellular bacteria are stained with both the Cy5- and FITC-202
conjugated antibodies, whereas intracellular bacteria are only stained with the FITC-203
conjugated antibody. 204
205
Bacterial survival assay 206
Cells were infected as for IF assay (MOI of 50, 37oC for 30 minutes), and then washed 207
and incubated with 100 μg/ml gentamicin for 45 minutes to kill extracellular bacteria, 208
followed by incubation in 1 mM DFP to inhibit proteases so as to protect intracellular 209
bacteria during PMN lysis. Cells were then lysed in 0.4% saponin in HBSS for 20 210
minutes at 37oC, and then diluted and plated onto GC + Isovitalex. Colonies were 211
counted the following day. 212
213
Immunoprecipitation and western blot 214
Cells (5x106 per sample) were infected with N. gonorrhoeae at a multiplicity of infection 215
of 10 for indicated times at 37oC with 5% CO2. Infections were stopped by 216
centrifugation at 2,400 g for 3 min at 4oC, and pellets were resuspended in 50 μl of PBS-217
pervanadate buffer (1 mM EDTA, 1 mM PMSF, 1 μg/ml each aprotinin, leupeptin, and 218
pepstatin A, 1 mM NaF, 100 μM Na3V04, 10 mM H202, and 50 mg/ml soybean trypsin 219
inhibitor in PBS), and then lysed with 50 μl of radioimmunoprecipitation (RIPA) buffer 220
(1% Nonidet P-40, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1mM DFP, 1 mM 221
PMSF, 1 mg/ml each aprotinin, leupeptin, and pepstatin A, 1 mM NaF, 100 mM Na3V04, 222
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10 mM H202, and 50 μg/ml soybean trypsin inhibitor) containing 2% SDS (except for co-223
immunoprecipitation), and left on ice for 15 minutes. Lysates were then topped up with 224
900 μl of RIPA, rotated for 30 minutes at 4oC, immunoprecipitated with anti-225
phosphotyrosine antibody (4G10) (for phosphorylation studies) or anti-CEACAM 226
antibody (Dako) (for co-immunoprecipitation studies) for 2 h, incubated with Protein A-227
sepharose for 1 h, washed two times with RIPA, and boiled for 5 minutes. 228
Immunoprecipitated samples were separated by SDS-PAGE and transferred to a 229
nitrocellulose membrane (Amersham Biosciences). The membrane was blocked with 5% 230
milk in TBS with 0.05% Tween for 30 minutes at room temperature, incubated with 231
appropriate primary and HRP-conjugated secondary antibodies, and chemiluminescent 232
detection was performed using ECL+ (Amersham Biosciences). 233
234
235
Syk and Vav phosphorylation assays 236
106 MPRO PMNs were infected with N. gonorrhoeae at a multiplicity of infection of 10, 237
in 250 μl of HBSS at 37oC with 5% CO2 for the times indicated. Infections were stopped 238
by centrifugation at 2,400 g for 3 min at 4oC, lysed in boiling SDS sample buffer, and 239
boiled for a further 5 minutes. Samples were resolved and immunoblotted as described 240
above. 241
242
Oxidative burst assays 243
For DHR assay, 106 MPRO PMNs were treated with 2 μM of dihydrorhodamine-123 244
(DHR) for 20 minutes at 37 oC at a concentration of 107 cells/ml prior to agonist 245
treatment. Samples were then treated with agonists (N. gonorrhoeae at a multiplicity of 246
infection of 10, unless otherwise indicated, or PMA at 1 μg/ml) in 500 μl of HBSS for 60 247
minutes at 37oC. Infections were stopped by centrifugation at 2,400 g for 3 min at room 248
temperature, and cell pellets were fixed in 1% PFA prior to analysis by flow cytometry 249
using a FACSCalibur with CellQuest software (Becton Dickinson, San Diego, CA), 250
gathering the FL-1 signal from a gated sample of 10,000 cells. For chemiluminescence 251
assay, 5x105 cells were incubated with 25 μg/ml 5-amino-2,3-dihydro-1,4-252
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phthalazinedione (Luminol) in a volume of 100 μl, and then treated with agonists in a 253
total volume of 200 μl, in triplicate. Infections were allowed to proceed for 60 minutes at 254
37oC after which luminescence was read using a Tecan plate reader with i-control 255
software. Plotted are the data from the 60 minute time point, which was consistently the 256
time at which peak fluorescence occurred in the infected samples. 257
258
Degranulation assays 259
For flow-based assays of secondary granule degranulation (CD11b release for murine 260
neutrophils and CD67 release for human neutrophils), 106 PMNs were treated with 261
agonists in 500 μl medium for 30 minutes at 37oC. Infections were stopped by 262
centrifugation at 2,400 g for 3 min at room temperature. Cell pellets were fixed in 1% 263
PFA, and stained with 1.25 μg of PE-conjugated rat anti-mouse CD11b (or 1 μg of anti-264
CD67 antibody, followed by a PE-conjugated secondary for human neutrophil studies) in 265
a total volume of 50 μl. Flow cytometry was conducted as above, gathering the FL-2 266
signal from a gated sample of 10,000 cells. Myeloperoxidase, elastase, and lactoferrin 267
release assays were performed essentially as described in (1). Briefly, for all three 268
assays, 106 PMNs were infected with agonists in a total volume of 500 μl and infections 269
proceeded for 30 minutes at 37oC. Cells were then pelleted and supernatants were 270
collected. For myeloperoxidase assay, 50 μl of supernatant was mixed with 150 μl 271
SureBlue tetremethylbenzidine peroxidase substrate (KPL; Gaithersberg, MD), and plates 272
were read spectrophotometrically at 650 nm. For elastase assay, 50 μl of supernatant was 273
diluted 2-fold in PBS and incubated with 100 μl DQ elastin substrate conjugated to 274
BODIPY FL (from the EnzCheck Elastase kit; Molecular Probes), and incubated for 24 275
hours at room temperature before reading fluorescence at 488 for excitation and 515 for 276
emission. For both the MPO and elastase assay, a % release is shown and was calculated 277
as the amount of the protein in the supernatant divided by the total amount in 106 278
CHAPS-lysed cells. Lactoferrin (Lf) release was assayed by ELISA as described in (23). 279
Because HBSS would often cause spontaneous specific granule release in cells, CD11b 280
and lactoferrin degranulation assays were conducted in Medium 199 (Invitrogen). 281
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Statistical Analyses 283
Immunofluorescence experiments and plating assays were conducted with triplicate 284
samples, and standard deviations (represented as error bars) were calculated from 285
averages between three individual coverslips (25 cells counted per coverslip) from a 286
single representative experiment. Chemiluminescence-based oxidative burst assays, 287
bacterial survival assays, and MPO, elastase, and lactoferrin release assays were always 288
conducted with triplicate samples, with means and standard deviations presented as error 289
bars. All results depicted in this study are representative of those obtained from at least 3 290
independent experiments, each conducted on separate days. Where indicated, samples 291
were analyzed by 1) a two-tailed Student’s t-test assuming equal variance, taking into 292
account all cells from each condition of a particular experiment, with p values of < 0.05 293
being deemed significant; or 2) 1-way ANOVA with Tukey’s post-test when comparing 294
means between different cell lines treated with the same agonist. 295
296
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RESULTS 297
Introduction of human CEACAMs into murine neutrophils is sufficient for 298
neisserial capture and engulfment 299
To delineate the individual functions of CEACAMs 1, 3 and 6 in the neutrophil, we used 300
a ‘knock-in’ approach to introduce human CEACAMs into mouse promyelocytic cells 301
(MPRO)(41). Rather than being immortalized by an oncogenic event, the normal 302
differentiation of MPRO cells is arrested by a dominant-negative retinoic acid receptor 303
alpha (RARα), a defect that can be overcome with supraphysiological doses of retinoic 304
acid. The MPRO model allowed us to circumvent two issues: one, since the cells are 305
murine, they contain no Opa-binding CEACAMs, and Opa-dependent effects could be 306
specifically attributed to introduced human CEACAMs (Fig. 1a, top; compare with 307
human neutrophils, Fig. 1a, bottom); and two, since the cells are promyelocytes, we 308
could create stable cell lines for propagation in culture, which could be differentiated in 309
vitro to mature, functional neutrophils (15, 18). 310
311
We introduced cDNAs encoding human CEACAM1 (“1”), CEACAM3 (“3”) and 312
CEACAM6 (“6”), as well as the empty vector (“puro”) into MPRO cells using retroviral 313
transduction. In both the promyelocytes, and the neutrophils differentiated from the 314
promyelocytes, all three human CEACAMs were expressed in much the same manner as 315
in human neutrophils, with CEACAM1 and CEACAM6 in larger quantities than 316
CEACAM3 (Fig. 1b,c). In all the experiments performed in this work, we used 317
neutrophils differentiated from the CEACAM-expressing promyelocytes. As with 318
normal mouse bone marrow neutrophils (Fig. 1a, top), MPRO-puro cells were unable to 319
substantially bind or engulf N. gonorrhoeae, while introduction of CEACAM1, 320
CEACAM3 or CEACAM6 permitted the binding and engulfment of N. gonorrhoeae by 321
the mouse neutrophils (Fig. 1d). More bacteria consistently adhered to cells expressing 322
CEACAM1 or CEACAM6, relative to CEACAM3. Strikingly, bacterial association with 323
MPRO-CEACAM3 cells frequently resulted in the appearance of F-actin rich structures 324
surrounding the bound bacteria (Fig. 1e,f). Such structures were infrequently apparent in 325
infected MPRO-CEACAM6 cells, and were never observed during neisserial infection of 326
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MPRO-CEACAM1. These observations suggested that these receptors were signaling 327
differently in response to engagement by N. gonorrhoeae, arguing for different functional 328
outcomes upon bacterial infection. 329
330
CEACAM3 is distinct in its ability to elicit neutrophil oxidative burst and 331
degranulation responses to N. gonorrhoeae 332
Once we had established that human CEACAMs were functionally expressed in the 333
murine background, we sought to study the contribution of the different CEACAMs to 334
other neutrophil functions. Human neutrophils respond to Opa-expressing N. 335
gonorrhoeae by triggering an increased consumption of oxygen, resulting in the 336
production of oxygen radicals in the cell (the ‘oxidative burst’ response), as well as by 337
releasing granule components to the cell surface or into the newly formed phagosome 338
(‘degranulation’). Using both a single cell-based assay (oxidation of dihydro-rhodamine, 339
DHR) (Fig. 2a), as well a chemiluminescence-based protocol measuring the oxidation of 340
luminol (Fig. 2b), we observed that MPRO-CEACAM3 cells could stimulate the 341
oxidative burst upon neisserial infection, whereas the CEACAM1- and CEACAM6-342
expressing cell lines did not lead to appreciable oxidative burst activation in the cells. 343
This did not result from a difference in bacterial association, as all three CEACAM-344
expressing MPRO lines were able to bind and internalize Neisseria (Fig. 1d). The 345
magnitude of the response measured with DHR reflected that observed in human 346
neutrophils (Fig. 2c). Using fixed Staphylococcus aureus particles (‘Pansorbin’) coated 347
with anti-CEACAM monoclonal antibodies to infect human neutrophils, we observed 348
only those Pansorbin particles coated with CEACAM3-specific antibody were able to 349
stimulate an oxidative burst response compared to Pansorbin particles coated with the 350
isotype control or CEACAM1- or CEACAM6-specific antibodies (Fig. 2d). These 351
experiments clearly implicate CEACAM3, and not CEACAM1 or CEACAM6, as being 352
sufficient to signal the oxidative burst upon neisserial infection. 353
354
To monitor neutrophil degranulation in response to neisserial infection, we first 355
used a single cell-based flow cytometric assay measuring degranulation of the specific 356
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granule protein CD11b. As with the oxidative burst experiments, we observed that 357
MPRO-CEACAM3 cells initiated this response upon neisserial infection, while the 358
mouse neutrophils lacking human CEACAMs, or those expressing CEACAM1 or 359
CEACAM6, did not (Fig. 3a). We performed a similar assay in human neutrophils, 360
measuring degranulation of the specific granule protein CD67, and observed that only 361
CEACAM3-specific Pansorbin particles, and not those with isotype or antibodies to 362
CEACAM1 or CEACAM6, could stimulate degranulation of CD67 (Fig. 3b). The 363
magnitude of the degranulation response to CEACAM3-binding Pansorbin particles 364
reflected that seen with pan-CEACAM-binding bacteria (OpaCEA; Fig. 3c). We further 365
confirmed the CEACAM3-dependent degranulation responses in MPRO PMNs using an 366
ELISA-based assessment of degranulation in the population: first, by measuring 367
degranulation of two azurophilic granule proteins, myeloperoxidase (MPO; Fig. 3d) and 368
elastase (Fig. 3e), as well as by measuring the release of a specific granule-containing 369
protein, lactoferrin (Fig. 3f). 370
371
Because Neisseria gonorrhoeae are particularly susceptible to the azurophilic 372
granule protein cathepsin G (34), we reasoned that degranulation responses to 373
CEACAM3 ligation should also result in neisserial killing. To test this, we performed 374
gentamicin-protection assays with MPRO PMNs. We observed that the amount of 375
gentamicin-protected bacteria recovered from MPRO-CEACAM3 PMNs reflected that of 376
MPRO-puro (i.e. background levels). In contrast to this, MPRO-CEACAM1 and MPRO-377
CEACAM6 PMNs contained an increased number of viable internalized bacteria (Fig. 378
3g). Because MPRO-CEACAM3 PMNs are fully capable of internalizing bacteria (Fig. 379
1d), this result suggests that those bacteria that are internalized via CEACAM3 do not 380
survive the infection. These results, as well as those in the previous section, suggest that 381
ligation of CEACAM3 elicits signals that lead to both the neisserial-induced PMN 382
activation and may also result in subsequent bacterial killing. 383
384
CEACAM3 is phosphorylated and recruits murine Syk upon N. gonorrhoeae 385
infection 386
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Because CEACAM3 encodes a cytoplasmic ITAM that is phosphorylated upon ligation 387
(21, 29), we speculated that this motif was responsible for the functional responses we 388
were observing. To assess CEACAM phosphorylation in response to infection, we 389
infected the various MPRO neutrophils with Opa-expressing N. gonorrhoeae and 390
immunoprecipitated either total CEACAM or phosphotyrosine-containing proteins, 391
which we then probed with a pan-specific antibody that recognizes all the CEACAMs 392
used in this study. Consistent with past work using human-derived cell lines (17, 21, 29), 393
the CEACAMs containing tyrosines in their cytoplasmic tails (CEACAM1 and 394
CEACAM3) were phosphorylated upon neisserial infection (Fig. 4a). CEACAM6 395
phosphorylation was not observed, which was expected since it is a GPI-anchored protein 396
lacking a cytoplasmic domain. We considered whether the phosphorylated CEACAMs 397
could activate mouse Syk, a tyrosine kinase known to be centrally involved in signaling 398
for oxidative burst and degranulation by neutrophils (42). To test for this, we monitored 399
the phosphorylation state of Syk and a downstream effector, Vav, in infected whole cell 400
lysates using phospho-specific antibodies. Upon neisserial infection, CEACAM3-401
expressing cells showed an increase in Syk and Vav phosphorylation; this did not occur 402
in CEACAM1 or CEACAM6-expressing cells or in CEACAM3-expressing cells that 403
were uninfected or infected with Opa- bacteria (Fig. 4b,c). Furthermore, treatment of 404
MPRO-CEACAM3 PMNs with either the Src family kinase inhibitor PP2 or the Syk 405
inhibitor piceatannol inhibited Vav phosphorylation upon neisserial infection (Fig. 4d), 406
confirming that the majority of the Vav phosphorylation seen was dependent on both the 407
phosphorylation of the CEACAM3 ITAM its the subsequent recruitment and activation 408
of Syk, as observed previously in human neutrophils (29). Finally, co-409
immunoprecipitation of Syk with CEACAM3 confirmed that these two proteins became 410
transiently associated upon infection with Opa-expressing N. gonorrhoeae (Fig. 4e). 411
Thus, human CEACAM3 ligation is connected to Syk downstream signaling responses in 412
this murine neutrophil system. 413
414
CEACAM3-specific activation of neutrophils is ITAM-dependent 415
Using an epithelial-based model, we previously observed that Syk recruitment to 416
CEACAM3 is critically dependent on the presence of both phosphorylatable tyrosines in 417
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the cytoplasmic ITAM (26). We therefore reasoned that if CEACAM3-dependent 418
recruitment of Syk was required for PMN activation in response to neisserial infection, 419
then the intact ITAM would be required for this as well. To directly test for the 420
requirement of the ITAM in the response to neisserial infection by neutrophils, we 421
constructed MPRO lines expressing CEACAM3 proteins with either one (MPRO-422
CEACAM3-Y230F or MPRO-CEACAM3-Y241F) or both (MPRO-CEACAM3-423
Y230F/Y241F) ITAM tyrosines mutated to phenylalanine (and therefore unable to be 424
phosphorylated) as well as a natural splice variant of CEACAM3 lacking the ITAM and 425
most of the cytoplasmic domain (MPRO-CEACAM3-1C1). The various mutant alleles 426
were expressed at similar levels as the wild type CEACAM3 (WT) protein in MPRO 427
cells (Fig. 5a). Each mutant CEACAM3 could bind and internalize OpaCEA-expressing N. 428
gonorrhoeae when expressed by the neutrophils (Fig. 5b), highlighting that the 429
cytoplasmic domain was not essential for bacterial uptake. The CEACAM3 mutant 430
proteins were not, however, able to induce oxidative burst (Fig. 5c) or degranulation (Fig. 431
5d) responses upon neisserial infection. Thus, an intact ITAM is not required for 432
gonococcal uptake by PMNs. The ITAM is, however, required for activation of PMNs in 433
response to neisserial infection. 434
435
We confirmed that infection of cells expressing mutant CEACAM3 proteins did not lead 436
to Syk or Vav activation (Fig. 6a). Consistent with the role of Syk activation in the 437
CEACAM3-specific PMN responses, inhibition of Syk function using the 438
pharmacological inhibitor piceatannol effectively abolished the ability of MPRO-439
CEACAM3 cells to mount an appreciable oxidative burst (Fig. 6b) or degranulation 440
response (Fig. 6c,d) upon neisserial infection, with the response becoming 441
indistinguishable to that by cells expressing the ITAM-less CEACAM3-1C1. Taken 442
together, these data demonstrate that CEACAM3-dependent neutrophil responses require 443
an intact ITAM to allow the recruitment and activation of Syk and its effectors. 444
Importantly, these data also re-iterate that these responses are not simply the result of 445
CEACAM-dependent binding or bacterial uptake, but rather require the initiation of 446
ITAM-dependent signaling as a result of CEACAM3 ligation by Opa. 447
448
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CEACAM3 transmits CEACAM1-dependent signals for neutrophil activation 449
Our results to this point show that MPRO-CEACAM3 neutrophils function remarkably 450
similarly to human neutrophils. CEACAM1 encodes a cytoplasmic immunoreceptor 451
tyrosine-based inhibitory motif (ITIM) with the potential to oppose ITAM activation 452
signals through the recruitment of inhibitory phosphatases in some cell types (5, 17). 453
Because CEACAM3 is always co-expressed with CEACAMs 1 and 6 in the human 454
neutrophil, we sought to determine whether either of these CEACAMs could modulate 455
CEACAM3 function. Importantly, we observed that co-expression of CEACAMs 1 or 6 456
with CEACAM3 did not affect CEACAM3 function in neutrophils. Specifically, MPRO-457
CEACAM1 + CEACAM3 (“1 + 3”) and MPRO-CEACAM3 + CEACAM6 (“3 + 6”) 458
neutrophils mounted oxidative burst and degranulation responses to N. gonorrhoeae 459
indistinguishable from those seen in MPRO-CEACAM3 neutrophils (Fig. 7a,b). 460
Similarly, we confirmed that CEACAM3, as well as Syk and Vav, could be 461
phosphorylated in the presence of CEACAM1 (Fig. 7c-e). Our results imply that the 462
mechanisms of inhibition by CEACAM1 are receptor- and/or cell type-specific, and the 463
lack of inhibition of CEACAM3 by CEACAM1 ligation may reflect the special ‘wiring’ 464
of biochemical pathways within the neutrophil. 465
466
It is known that of the hundreds of Opa variants potentially encoded by N. gonorrhoeae, 467
the majority bind CEACAM1 (44) while only a small proportion bind to CEACAM3. 468
Based upon work such as described herein, this specificity may be attributed to selective 469
pressures against CEACAM3 binding in vivo. Importantly, Opa proteins that bind 470
CEACAM3 always bind CEACAM1, while numerous Opa proteins exist that bind 471
CEACAM1 and not CEACAM3 (4, 7, 11). As such, we deemed it important to 472
determine the outcome of a CEACAM1-binding strain on cells expressing CEACAM1 in 473
the presence or absence of CEACAM3, the former being the normal situation in human 474
neutrophils. To address this, we infected MPRO cells expressing CEACAM1 or 475
CEACAM3, or both together, with a N. gonorrhoeae MS11 strain that binds to 476
CEACAM1 but not CEACAM3 (hereafter referred to as OpaCCM1 to distinguish it from 477
our regular strain, OpaCEA which binds all CEACAMs in this study). At a low 478
multiplicity of infection, we observed that degranulation of MPRO-CEACAM1 + 479
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CEACAM3 neutrophils were not stimulated by N. gonorrhoeae expressing the 480
CEACAM1-specific Opa variant, in contrast to the response apparent when these cells 481
were infected with the isogenic strain that instead expressed the CEACAM1- and 482
CEACAM3-bispecific OpaCEA (Fig. 8a, top). Therefore, it seemed the CEACAM1-483
binding strain was evading the CEACAM3 response. Unexpectedly, however, when a 484
higher multiplicity of infection was used, the MPRO-CEACAM1+CEACAM3 cells were 485
observed to degranulate in response to specific CEACAM1 binding (Fig. 8a, bottom). 486
This required CEACAM3, as MPRO-CEACAM1 neutrophils did not degranulate in 487
response to either gonococcal strain. When the same two strains were used to infect 488
human neutrophils, a similar phenomenon was observed; at low MOIs, the CEACAM1-489
binding strain (OpaCCM1) elicited a response indistinguishable from that to the non-490
CEACAM binding (Opa-) strain, while at a higher MOI it behaved like the pan-491
CEACAM binding strain (OpaCEA) (Fig. 8b). Furthermore, the OpaCCM1 strain, as well 492
two other non-CEACAM3-binding strains (expressing Opa50, “50” which binds 493
proteoglycan receptors on cells, leading to cross-linking of β1 integrins known to activate 494
Syk (22); or Opa54, “54” which also binds CEACAM1) were able to stimulate Syk and 495
Vav phosphorylation in infected human neutrophils (Fig. 8c). These data suggest that 496
CEACAM3 can transduce signals from other CEACAMs, albeit less effectively than 497
through direct CEACAM3 engagement. This previously unappreciated interaction lends 498
credence to a previous suggestion that CEACAMs may exist as a complex on the 499
activated PMN surface (36). 500
501
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DISCUSSION 502
503
In this work, we have used a murine neutrophil model to describe the contribution of 504
individual human CEACAMs on neutrophil responses to neisserial infection. We show 505
that N. gonorrhoeae, which normally cannot bind murine cells, can be captured and 506
internalized by any one of the Opa-binding CEACAMs expressed by human PMNs 507
(CEACAM1, CEACAM3 or CEACAM6). Strikingly, however, only CEACAM3-508
expressing mouse PMNs elicited an oxidative burst and degranulation response upon 509
neisserial infection, is unique in its ability to signal for these responses in the cell. 510
Accordingly, we observed that the Src family kinase-dependent phosphorylation of 511
tyrosine residues within the CEACAM3 cytoplasmic ITAM was required for these 512
effects, apparently due its ability to recruit and activate the protein tyrosine kinase, Syk. 513
Indeed, when Syk function was inhibited in CEACAM3-expressing PMNs, oxidative 514
burst and degranulation responses were lost, mirroring the effects of expressing an 515
ITAM-less CEACAM3 in PMNs. These results reveal that CEACAM-dependent 516
neisserial uptake in PMNs is ITAM-independent, while activation of the neutrophil 517
oxidative burst and degranulation are ITAM-dependent. Finally, we show that co-518
expression of CEACAM1 and CEACAM6 with CEACAM3 in mouse PMNs potentiates 519
the neutrophil response to neisserial infection; this unexpected finding contrasts that 520
which is observed in lymphocytes, in which CEACAM1 engagement has been shown to 521
inhibit T cell receptor-mediated ITAM-based signaling. While CEACAM1 is the only 522
CEACAM expressed by lymphocytes, our data suggests that CEACAMs may act 523
cooperatively in the neutrophil, allowing high-density bacterial CEACAM1 or 524
CEACAM6 binding to elicit activating signals via CEACAM3. 525
526
The central importance of neutrophils in the pathogenesis of gonorrhea has prompted 527
focused research seeking to characterize molecular mechanisms of neutrophil-expressed 528
CEACAM functions in response to N. gonorrhoeae infection (3, 20, 31). This work has 529
largely been conducted in transfected epithelial cell lines in an effort to study individual 530
CEACAMs in the absence of co-expressed CEACAMs and/or other potential receptors 531
on the neutrophil (3, 20, 32). While helpful, this approach does not permit the study of 532
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specialized PMN functions, including oxidative burst, degranulation and cytokine 533
responses, which are centrally important to the biology of these potently bactericidal 534
cells. Furthermore, epithelial cells lack signaling proteins, such as the tyrosine kinase 535
Syk, that are critical for the function of immunoreceptors (29). As such, we felt that 536
studying these receptors in the natural context of the neutrophil was critical to understand 537
their authentic function. 538
539
In order to express human CEACAMs in mouse neutrophils to demonstrate 540
distinct functions for neutrophil-expressed CEACAMs that bind neisserial Opa proteins, 541
we exploited a developmentally arrested murine promyelocyte cell line (MPRO) that has 542
been shown to be differentiated into functional neutrophils in vitro. MPRO cells were 543
created by transducing bone marrow cells ex vivo with a dominant negative form of the 544
retinoic acid receptor alpha (RARα), a neutrophil differentiation factor, which resulted in 545
cells arrested at the promyelocyte stage of neutrophil differentiation (41). Addition of 546
supraphysiological concentrations of retinoic acid is sufficient to overcome the arrest, 547
likely stimulating endogenous RARα proteins that are not associated with the dominant 548
negative form. These cells are, therefore, completely normal PMNs once differentiation 549
is complete, not transformed or defective as are leukemic cell lines. Indeed, it has been 550
shown that HL-60 cells (6) lack the ability to undergo chemotaxis in response to f-Met-551
Leu-Phe (37), and mis-sort secondary granule proteins such as CD11b/CD18 (12, 16), 552
while these and other functions are normal in MPRO-differentiated PMNs (9, 15, 18). 553
554
There are some technical issues that must be kept in mind when discussing results 555
from the MPRO cells. We find that these cells are less robust in their stimulation, 556
particularly with respect to oxidative burst. This reflects what is seen with other cultured 557
phagocytes, including the widely used RAW mouse macrophage-like cells, which display 558
a considerably less robust oxidative burst when compared with primary mouse 559
macrophages (19). MPRO cells may also have a lower capacity to degranulate in 560
response to stimulation, particularly in the case of the primary granules. Whether this is a 561
generalized property of the cells themselves or may depend upon the nature of the 562
stimulus remains to be determined. 563
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564
One impetus for studying the individual CEACAMs in neutrophils was to 565
consider the relative effect of CEACAM1 and CEACAM3 phosphotyrosine-based signals 566
in response to extracellular ligation (38). That CEACAM3 is responsible for the 567
neutrophil responses that we observed makes biological sense given the presence of the 568
activating ITAM. Considering the potent response of neutrophils to bacterial-derived 569
products, it was unexpected that neutrophils that bind and engulf N. gonorrhoeae by 570
CEACAM1 and CEACAM6 do not initiate a bactericidal degranulation and oxidative 571
burst response. The absence of response upon CEACAM1 does not seem to emanate 572
from its co-inhibitory function, which can oppose activating signals when bound by 573
neisserial Opa proteins in other (non-neutrophil) cell types via the phosphorylation of its 574
ITIM tyrosines and subsequent recruitment of phosphatases (17). Our finding that co-575
expression of CEACAM1 and CEACAM3 does not suppress the activating effect of the 576
latter suggests that this co-inhibitory activity does not occur in neutrophils. We suspect 577
this may be attributed to the special characteristics of the PMN, since these cells are 578
short-lived and their activation is reigned in through apoptosis and clearance via surface 579
phosphatidylserine recognition by macrophages (33), they may not require intricate co-580
inhibitory signaling seen in other cell types. 581
582
While engulfment by CEACAM3 appears to occur via a process that reflects that 583
by phagocytic immunoglobulin-specific Fc receptors (13, 29, 3, 30), the mechanism by 584
which N. gonorrhoeae are engulfed upon CEACAM1 and CEACAM6 binding remains 585
uncharacterized. In this regard, it is of interest that we observed F-actin rich regions 586
surrounding some bacteria being internalized via CEACAM6, something that has not 587
been observed in epithelial cell models (3); this may suggest a neutrophil-specific binding 588
partner for CEACAM6. Alternatively, because CEACAM6 is a GPI-anchored protein 589
that has been shown to reside in cholesterol-rich membrane microdomains (24), its 590
inclusion in such regions of the plasma membrane may promote association with 591
signaling factors that themselves reside in these regions (35). Further studies 592
characterizing proteins involved in CEACAM-mediated uptake processes in neutrophils 593
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will be useful to determine how these processes differ from those observed in epithelial 594
cells. 595
596
Another unexpected result emanating from this work was the capacity for a 597
CEACAM1-binding Opa protein to elicit CEACAM3-dependent PMN activation. This 598
effect was not apparent at low bacterial densities, suggesting that it is less efficient than 599
direct CEACAM3 engagement. Whether it reflects a CEACAM-specific co-operative 600
response (as suggested by (36)) or the result of non-specific clustering of receptors on 601
PMNs, such as through presence in similar membrane micro-environments, remains to be 602
determined. In considering the lack of activating signals upon exposure to CEACAM1-603
specific bacteria at low multiplicity of infection (Fig. 8a,b), it is interesting to consider 604
that there is variability in PMN responses to gonococci bearing different Opa variants (2, 605
11). The ability of different Opa variants to activate PMNs had been speculated to be a 606
product of the Opa proteins themselves, as a result of variable sequence conferring 607
different affinities for a single receptor (2). Our data support this proposal, since the 608
variability may reflect the specific outcome of binding to CEACAM3 directly versus 609
indirect CEACAM3-engagement via Opa protein binding to CEACAM1 and/or 610
CEACAM6. 611
612
While the number of physiological functions attributed to the CEA family is vast, 613
ranging from cell adhesion, insulin signaling, and angiogenesis, the only known role for 614
CEACAM3 in human tissues is as a receptor for the Gram-negative, human-restricted 615
pathogens Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, and 616
Haemophilus influenzae (30). This peculiar observation has been postulated by ourselves 617
and others as a function of co-evolution between pathogen and host. While CEACAM1, 618
CEACAM5 and CEACAM6 are useful for colonization (25, 45) and immune evasion (5), 619
CEACAM3 is an evolutionarily 'new' receptor that is clearly deleterious for bacteria. As 620
such, CEACAM3 represents a 'molecular mimic' that allows neutrophil-dependent 621
clearance of pathogens that target other CEACAM family receptors. The striking 622
parallels between the response of CEACAM3-expressing murine neutrophils and primary 623
neutrophils from humans reflects the ability of human CEACAM3 to engage downstream 624
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mouse effectors, as seen in this work. By directly engaging Syk kinase, the CEACAM3-625
dependent signals converge with those from immunoreceptors including the phagocytic 626
immunoglobulin Fc domain-specific receptors and Dectin-1, eliciting an evolutionarily 627
conserved and highly effective bactericidal response to these highly adapted human 628
pathogens. 629
630
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ACKNOWLEDGEMENTS 631
632
The authors thank Dr. Wendy Dobson-Belaire for help in establishing the retrovirus 633
transduction protocols, as well as Shannon McCaw and Dr. Nancy So for technical 634
assistance during the human neutrophil experiments. We also thank Drs. Sergio 635
Grinstein and Michael Glogauer for helpful comments during the course of the work. 636
This work was supported by funding from the Canadian Institutes for Health Research 637
grant no. MOP-15499. H.S. was supported by the Ontario Graduate Scholarship Program. 638
639
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37. Sirak, A. A., J. D. Laskin, F. M. Robertson, and D. L. Laskin. 1990. Failure of 767 F-Met-Leu-Phe to induce chemotaxis in differentiated promyelocytic (HL-60) 768 leukemia cells. J Leukoc Biol 48:333-342. 769
38. Skubitz, K. M., K. D. Campbell, K. Ahmed, and A. P. Skubitz. 1995. CD66 770 family members are associated with tyrosine kinase activity in human neutrophils. 771 J Immunol 155:5382-5390. 772
39. Skubitz, K. M., K. D. Campbell, and A. P. Skubitz. 1996. CD66a, CD66b, 773 CD66c, and CD66d each independently stimulate neutrophils. J Leukoc Biol 774 60:106-117. 775
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40. Swanson, J., E. Sparks, B. Zeligs, M. A. Siam, and C. Parrott. 1974. Studies 776 on gonococcus infection. V. Observations on in vitro interactions of gonococci 777 and human neutrophils. Infect Immun 10:633-644. 778
41. Tsai, S., and S. J. Collins. 1993. A dominant negative retinoic acid receptor 779 blocks neutrophil differentiation at the promyelocyte stage. Proc Natl Acad Sci U 780 S A 90:7153-7157. 781
42. Van Ziffle, J. A., and C. A. Lowell. 2009. Neutrophil-specific deletion of Syk 782 kinase results in reduced host defense to bacterial infection. Blood 114:4871-783 4882. 784
43. Virji, M., and J. E. Heckels. 1986. The effect of protein II and pili on the 785 interaction of Neisseria gonorrhoeae with human polymorphonuclear leucocytes. J 786 Gen Microbiol 132:503-512. 787
44. Virji, M., S. M. Watt, S. Barker, K. Makepeace, and R. Doyonnas. 1996. The 788 N-domain of the human CD66a adhesion molecule is a target for Opa proteins of 789 Neisseria meningitidis and Neisseria gonorrhoeae. Mol Microbiol 22:929-939. 790
45. Wang, J., S. D. Gray-Owen, A. Knorre, T. F. Meyer, and C. Dehio. 1998. Opa 791 binding to cellular CD66 receptors mediates the transcellular traversal of 792 Neisseria gonorrhoeae across polarized T84 epithelial cell monolayers. Mol 793 Microbiol 30:657-671. 794
46. Zhou, Y., H. Zhang, J. D. Siliciano, and R. F. Siliciano. 2005. Kinetics of 795 human immunodeficiency virus type 1 decay following entry into resting CD4+ T 796 cells. J Virol 79:2199-2210. 797
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800
FIGURE LEGENDS 801
802
Figure 1. Human CEACAM expression in murine neutrophils permits the binding 803
and engulfment of N. gonorrhoeae 804
(a) Murine neutrophils do not bind N. gonorrhoeae, and human neutrophils bind N. 805
gonorrhoeae in an OpaCEA-dependent manner. Neutrophils from mouse bone marrow 806
(top) or human blood (bottom) were infected with N. gonorrhoeae either lacking (Opa-) 807
or expressing an Opa adhesin that binds all the CEACAMs used in this study (OpaCEA). 808
Cells were visualized by staining with phalloidin (red), and bacteria are shown in green. 809
(b) Human CEACAMs are expressed in MPRO neutrophils. (i) Human neutrophils were 810
fixed and stained with antibodies specific for CEACAM1 (4/3/17), CEACAM3 (Col-1), 811
or CEACAM6 (9A6), and analyzed by flow cytometry. Tinted histograms correspond to 812
the isotype control. (ii) MPRO PMNs were fixed and stained with the same antibodies 813
used in (i). 814
(c) Western blot analysis of cell lines shown in (b). 815
(d) All human neutrophil-expressed CEACAMs are capable of binding and internalizing 816
Opa-expressing N. gonorrhoeae. Differentiated MPRO cells were infected on glass 817
coverslips with N. gonorrhoeae expressing OpaCEA for 60 minutes at 37oC at a 818
multiplicity of infection (MOI) of 25, followed by fixation with paraformaldehyde. Intra- 819
and extra-cellular bacteria were differentially stained as described in Methods, and 820
quantified via immunofluorescence microscopy. Total bacteria bound or internalized per 821
cell, for a total of 25 cells counted, were averaged and plotted. 822
(e) F-actin dynamics during bacterial internalization by CEACAM1, CEACAM3 and 823
CEACAM6. Cells were infected as in (d), except for a shorter duration (5 minutes at 824
37oC) and with an MOI of 50. “F-actin associated” refers to the number of bacteria co-825
localizing with F-actin, divided by the total number of bound bacteria per cell. 25 cells 826
were counted, and the average is shown. In (d) and (e), two-tailed t-tests were conducted 827
for relevant samples; an asterisk denotes p values less than 0.05. 828
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(f) MPRO-CEACAM3 (and to a lesser extent, MPRO-CEACAM6) neutrophils 829
internalize N. gonorrhoeae with re-organization of F-actin at sites of bacterial attachment. 830
Images from (e) are shown. 831
832
Figure 2. CEACAM3 expression in mouse neutrophils recapitulates the oxidative 833
burst response of human PMNs to N. gonorrhoeae 834
(a) CEACAM3-dependent oxidative burst response to N. gonorrhoeae infection in 835
murine neutrophils. MPRO neutrophils were treated with the oxidative burst reagent 836
DHR-123 (which fluoresces upon oxidation), followed by infection with Opa- N. 837
gonorrhoeae (dashed line), OpaCEA-expressing N. gonorrhoeae (black line), PMA (gray 838
line), or left uninfected (tinted). Bacterial multiplicity of infection was 10. After 60 839
minutes at 37oC, cells were fixed and the FL-1 signal was collected from a gated sample 840
of 10,000 cells by flow cytometry to monitor the oxidative burst response. The geometric 841
mean of the FL-1 signal is shown for uninfected cells (“unstim”) or for OpaCEA-infected 842
samples (“CEA”). In all panels, an arrow denotes a CEACAM-dependent (c) or 843
CEACAM3-dependent (a and d) event. 844
(b) CEACAM3-dependent chemilumiscent response to luminol. MPRO neutrophils were 845
pre-incubated with luminol, and then infected with the indicated strains of N. 846
gonorrhoeae at an MOI of 10 or 50, or left uninfected. Luminescence was read after 60 847
minutes at 37oC. Relevant samples were analyzed by 1-way ANOVA with Tukey’s post-848
test. An asterisk denotes that sample is significantly different than the corresponding 849
samples from the other cell lines. 850
(c) Human neutrophils undergo oxidative burst when infected with CEACAM-binding N. 851
gonorrhoeae. Human neutrophils were treated with DHR-123, infected, and analyzed as 852
in (a). The geometric mean of the FL-1 signal is shown for cells infected with Opa- 853
bacteria (“none”) or OpaCEA-expressing bacteria (“CEA”). 854
(d) Human neutrophils exhibit oxidative burst when infected with CEACAM-binding 855
Pansorbin particles. Human neutrophils were treated with DHR-123, and infected as in 856
(a) with fixed S. aureus particles (‘Pansorbin’) coated with anti-CEACAM1 (4/3/17; gray 857
line), anti-CEACAM3 (Col-1; black line), anti-CEACAM6 (9A6; dotted line), or isotype 858
(tinted), or PMA (dashed line). 859
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860
Figure 3. CEACAM3 expression by mouse neutrophils allows primary and 861
secondary granule release in response to N. gonorrhoeae infection 862
(a) CEACAM3-dependent degranulation of the specific granule protein CD11b in 863
response to N. gonorrhoeae infection of murine neutrophils. Cells were infected with 864
Opa- N. gonorrhoeae (dashed line), OpaCEA-expressing N. gonorrhoeae (black line), 865
PMA (gray line), or left uninfected (tinted). Bacterial multiplicity of infection was 10. 866
After 30 minutes at 37oC, cells were fixed and stained with a PE-conjugated rat anti-867
mouse CD11b antibody. The FL-2 signal was collected from a gated sample of 10,000 868
cells by flow cytometry to monitor the oxidative burst response. The geometric mean of 869
the FL-2 signal is shown for OpaCEA-infected cells. In all panels, an arrow denotes a 870
CEACAM3-dependent (a and b) or CEACAM-dependent (c) event. 871
(b) Human neutrophils degranulate the secondary granule protein CD67 when infected 872
with CEACAM-binding Pansorbin particles. Human neutrophils were infected with 873
Pansorbin particles coated with anti-CEACAM1 (4/3/17; gray line), anti-CEACAM3 874
(Col-1; black line), anti-CEACAM6 (9A6; dashed line) or isotype antibody (tinted), and 875
analyzed as in (a), except using an antibody to CD67 followed by a PE-conjugated 876
secondary. 877
(c) Human neutrophils degranulate CD67 when infected with CEACAM-binding N. 878
gonorrhoeae. Human neutrophils were infected and analyzed as in (b). Histograms 879
labeled as follows: Opa- N. gonorrhoeae-tinted, OpaCEA-expressing N. gonorrhoeae- 880
black line, PMA-gray line. The geometric mean of the FL-2 signal is shown for cells 881
infected with Opa- bacteria (“none”) or OpaCEA-expressing bacteria (“CEA”). 882
(d,e,f) CEACAM3-specific degranulation in response to neisserial infection. Cells were 883
pre-incubated with cytochalasin B and infected as shown with N. gonorrhoeae at an MOI 884
of 50 (d and e) or 1 (f), for 30 minutes at 37oC. Post-infection, supernatants were 885
collected and analyzed for myeloperoxidase (d), elastase (e), or lactoferrin (f) release as 886
described in Methods. 887
(g) N. gonorrhoeae engulfed by CEACAM3 are less able to survive the infection when 888
compared to those engulfed by CEACAM1 and CEACAM6. MPRO PMNs were infected 889
with OpaCEA-expressing N. gonorrhoeae at an MOI of 50 in 24-well tissue culture plates 890
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(5 x 105 cells per well). Bacteria were allowed to interact with the neutrophils for 30 891
minutes, and then treated with gentamicin to kill extracellular bacteria for 45 minutes 892
37oC. Cells were then treated with the cell-permeable protease inhibitor DFP (1 mM) for 893
15 minutes to prevent killing of viable intracellular bacteria during lysis, and then lysed 894
with 0.4% saponin in HBSS to release viable bacteria for dilution plating. Colony 895
forming units (cfu) were counted the following day. Plotted are the results of 4 896
experiments, with puro set at “1” to allow normalization of samples. Samples in (d)-(g) 897
were analyzed by 1-way ANOVA with Tukey’s post-test. An asterisk denotes that 898
sample is significantly different than the corresponding samples from the other cell lines. 899
900
Figure 4. CEACAM3 ligation by N. gonorrhoeae results in phosphotyrosine-based 901
signaling in mouse neutrophils 902
(a) CEACAM1 and CEACAM3 are phosphorylated on tyrosine upon N. gonorrhoeae 903
infection. MPRO-puro (‘puro’), MPRO-CEACAM1 (‘1’), MPRO-CEACAM3 (‘3’) and 904
MPRO-CEACAM6 (‘6’) neutrophils were infected with OpaCEA-expressing N. 905
gonorrhoeae for indicated times and then lysed, followed by immunoprecipitation of 906
phosphotyrosine-containing proteins (top panels), or total CEACAM (bottom panels). 907
Immunoprecipitates were resolved by SDS-PAGE, transferred, and all membranes were 908
probed for CEACAM. 909
(b) Syk phosphorylation upon N. gonorrhoeae infection of MPRO-CEACAM3 910
neutrophils. Cells were infected as shown (MPRO-puro, MPRO-CEACAM1, and 911
MPRO-CEACAM6 with OpaCEA-expressing bacteria, and MPRO-CEACAM3 PMNs 912
with Opa- and OpaCEA bacteria, or uninfected), followed by lysis of the cell pellet, 913
resolution of lysates by SDS-PAGE, and probing for a phosphorylated form of Syk 914
(pSyk). Total Syk is shown as a loading control. 915
(c) Vav phosphorylation upon N. gonorrhoeae infection of MPRO-CEACAM3 916
neutrophils. Experiment was conducted in as (b), except lysates were probed for a 917
phosphorylated form of Vav (pVav) with total Vav shown as a loading control. 918
(d) Inhibition of Src and Syk kinase activity abolishes Vav phosphorylation upon 919
neisserial infection of MPRO-CEACAM3 PMNs. Cells were treated with the Src- and 920
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Syk-specific inhibitors PP2 and piceatannol (both at 10 μM), or carrier (DMSO) for 30 921
minutes at 37oC. Cells were then infected and probed for pVav as in (c). 922
(e) Syk co-immunoprecipitates with CEACAM3 upon infection of MPRO-CEACAM3 923
PMNs with OpaCEA-expressing bacteria. MPRO-puro and MPRO-CEACAM3 PMNs 924
were infected as shown for indicated times and then lysed, followed by 925
immunoprecipitation of CEACAMs. Immunoprecipitates were resolved by SDS-PAGE, 926
transferred, and membranes were probed for Syk (top) or total CEACAM (bottom). 927
928
Figure 5. Mutation of critical tyrosines in CEACAM3 ITAM does not impede 929
bacterial internalization, but affects oxidative burst and degranulation 930
(a) Expression of CEACAM3 mutants in MPRO cells. ‘WT’ denotes wild-type 931
CEACAM3, ‘Y230F’ is CEACAM3 with the first ITAM tyrosine mutated to 932
phenylalanine, ‘Y241F’ is the second tyrosine to a phenylalanine, ‘Y230F/Y241F’ 933
contains both mutations, and ‘1C1’ is a natural splice isoform of CEACAM3 lacking the 934
ITAM. Cells were fixed and stained for CEACAM3 expression using the Col-1 antibody, 935
followed by a PE-conjugated secondary. FL-2 signal was acquired by flow cytometry as 936
described in Methods. 937
(b) CEACAM3 mutants are able to bind and internalize OpaCEA-expressing N. 938
gonorrhoeae. Cells were plated on glass coverslips followed by neisserial infection for 939
30 minutes at 37oC, fixation, and processing for immunoflorescence microscopy as 940
described in Methods. F-actin (to see cell shape) is stained with phalloidin (red), total 941
bacteria are in green, and extracellular bacteria appear blue. 942
(c) Mutation of either tyrosine in the CEACAM3 ITAM is sufficient to lose oxidative 943
burst upon CEACAM3 binding. Shown are CLU counts from cells infected for 60 944
minutes. Relevant samples were analyzed by 1-way ANOVA with Tukey’s post-test. An 945
asterisk denotes that sample is significantly different than the corresponding samples 946
from the other cell lines. 947
(d) CD11b degranulation to the cell surface does not occur when CEACAM3 ITAM is 948
mutated. MOI was 10. Post-infection, cells were fixed and stained with a PE-conjugated 949
rat anti-mouse CD11b antibody. The FL-2 signal was collected from a gated sample of 950
10,000 cells by flow cytometry. The geometric mean of the FL-2 signal is indicated. 951
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Arrow denotes a CEACAM3-dependent event. Histograms are labeled as follows: 952
OpaCEA-expressing N. gonorrhoeae (black line), PMA (grey line), or uninfected (filled). 953
954
Figure 6. CEACAM3-dependent Syk activation is responsible for the PMN 955
oxidative burst and degranulation responses to N. gonorrhoeae infection 956
(a) Mutation of either tyrosine in the CEACAM3 ITAM ablates Syk and Vav 957
phosphorylation upon N. gonorrhoeae binding. Cells were infected as shown, and then 958
lysed, followed by resolution of whole cell lysates by SDS-PAGE and probing for 959
activated forms of Syk (pSyk) and its downstream effector, Vav (pVav). Bottom panels 960
show total Syk and Vav, confirming equal loading of samples. 961
(b,c,d) Disruption of Syk function using the pharmacological inhibitor piceatannol ('Pic'), 962
or use of an ITAM-deficient CEACAM3 ('1C1'), abolishes CEACAM3-mediated 963
oxidative burst and degranulation in response to N. gonorrhoeae infection. Cells were 964
infected as shown, and then analyzed via luminol-dependent chemiluminescence for 965
oxidative burst (b), or supernatants were assayed for elastase (c) or lactoferrin (d) release 966
as described in Methods. Relevant samples were analyzed by 1-way ANOVA with 967
Tukey’s post-test. An asterisk denotes that sample is significantly different than the 968
corresponding samples from the other cell lines. 969
970
Figure 7. Co-expression of CEACAM1 or CEACAM6 with CEACAM3 does not 971
affect CEACAM3-mediated signaling or functional effects of PMNs 972
(a) CEACAM1 or CEACAM6 co-expression with CEACAM3 does not hinder 973
CEACAM3 oxidative burst activation. CEACAM1 or CEACAM6 were introduced into 974
MPRO-CEACAM3 cells to create two double-CEACAM-expressing cell lines, MPRO-975
CEACAM1+CEACAM3 (“1 + 3”) and MPRO-CEACAM3+CEACAM6 (“3 + 6”). Cells 976
were differentiated for 72 hours and then treated with DHR-123 and infected as described 977
in Fig. 2a. Histograms: uninfected (tinted), OpaCEA-expressing N. gonorrhoeae (black 978
line), PMA-treated (gray line). 979
(b) Introduction of CEACAM1 or CEACAM6 into MPRO-CEACAM3 does not inhibit 980
degranulation of CD11b in response to N. gonorrhoeae infection. Cells were infected as 981
in Fig. 3a. Histograms are labeled as in (a). 982
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(c) CEACAM3 is phosphorylated in the presence of CEACAM1. MPRO-CEACAM3 983
(‘3’) and MPRO-CEACAM1+CEACAM3 (“1 + 3”) neutrophils were infected with 984
OpaCEA-expressing N. gonorrhoeae for indicated times and then lysed, followed by 985
immunoprecipitation of phosphotyrosine-containing proteins. Immunoprecipitates were 986
resolved by SDS-PAGE, transferred, and probed for CEACAM. 987
(d) Syk phosphorylation upon N. gonorrhoeae infection of MPRO-CEACAM3, MPRO-988
CEACAM1+3, and MPRO-CEACAM3+6 neutrophils. Cells were infected with OpaCEA-989
expressing bacteria, followed by lysis of the cell pellet, resolution of lysates by SDS-990
PAGE, and probing for a phosphorylated form of Syk (pSyk). Tubulin is shown as a 991
loading control. 992
(e) Vav phosphorylation upon N. gonorrhoeae infection MPRO-CEACAM3, MPRO-993
CEACAM1+3, and MPRO-CEACAM3+6 neutrophils. Experiment was conducted in as 994
(d), except lysates were probed for a phosphorylated form of Vav (pVav) with tubulin 995
shown as a loading control. 996
997
Figure 8. Co-operative CEACAM signaling in response to Neisserial infection 998
(a) Activation of CEACAM3-dependent signaling with a CEACAM1-binding Neisseria 999
strain. We used a N. gonorrhoeae strain expressing a CEACAM1-specific Opa variant 1000
(OpaCCM1) to determine if bacteria could evade the CEACAM3-dependent neutrophil 1001
response. Differentiated MPRO-puro, MPRO-CEACAM1, MPRO-CEACAM3 and 1002
MPRO-CEACAM1+CEACAM3 cells were infected with OpaCCM1-expressing N. 1003
gonorrhoeae (gray line) or OpaCEA-expressing N. gonorrhoeae (black line) as shown with 1004
specific MOIs, or left untreated (tinted). Cells were then fixed, stained for surface 1005
appearance of the granule marker CD11b, and then analyzed by flow cytometry for FL-2 1006
signal. 1007
(b) Human neutrophils undergo oxidative burst in response to infection by a CEACAM1-1008
binding Neisseria strain, at a high MOI. Human PMNs were treated with DHR-123 and 1009
then infected with Opa- N. gonorrhoeae (left panels; black histogram), OpaCCM1-1010
expressing N. gonorrhoeae (middle panels; black histogram), or OpaCEA-expressing N. 1011
gonorrhoeae (right panels; black histogram) as shown with specific MOIs. Untreated 1012
(tinted; FL-1: 6.16) and PMA-treated (gray histogram; FL-1: 19.3) are superimposed on 1013
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all histograms for comparison. Geometric means of the FL-1 signal are shown for the N. 1014
gonorrhoeae strains. 1015
(c) CEACAM3 effectors are activated when human PMNs are infected with Neisseria 1016
that bind CEACAM1 and not CEACAM3. 106 human PMNs were infected with N. 1017
gonorrhoeae strains expressing the following Opa proteins: “-“ (no Opa); “50” (binds 1018
heparan sulfate proteoglycans and crosslinks integrins); “57” (OpaCEA); “54” (binds 1019
CEACAM1 but not CEACAM3); and “59” (OpaCCM1). MOIs were 10; cells were 1020
infected at 37oC as shown, followed by lysis of the cell pellet, resolution of lysates by 1021
SDS-PAGE, and probing for a phosphorylated form of Syk (pSyk) and Vav (pVav). 1022
Tubulin is shown as a loading control. An asterisk denotes non-specific bands. 1023
1024
1025
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