Interferon-gamma coordinates CCL3-mediated neutrophil recruitment in vivo
© Bonville et al; licensee BioMed Central Ltd. 2009
Received: 12 October 2008
Accepted: 19 March 2009
Published: 19 March 2009
We have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice.
In this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNγ). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNγ receptor gene-deleted mice, although neutrophils from IFNγR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNγ. Interestingly, although supplemental IFNγ restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNγ -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-γ together are sufficient to promote neutrophil recruitment but not pathologic activation.
Our findings reveal a heretofore unrecognized hierarchical interaction between the IFNγ and CCL3, which demonstrate that IFNγ is crucial for CCL3-mediated neutrophil recruitment in vivo.
Most respiratory virus infections are relatively benign and self-limited events. However, infection with highly pathogenic viruses can result in more severe sequelae, in which disease progresses to respiratory failure due to uncontrolled inflammation, pulmonary edema, and damage to lung tissue [1–5].
As part of an ongoing effort to understand inflammatory responses during severe respiratory virus infection, we have developed an inhalation model using the natural rodent pathogen, pneumonia virus of mice (PVM). Originally identified by Horsfall and colleagues [6, 7], PVM is a pneumovirus (family Paramyxoviridae) that is closely related to respiratory syncytial virus (RSV), and is among the few characterized mouse models of virus-induced acute respiratory distress syndrome (ARDS) [7–9]. Among the prominent features of this infection, a minimal intranasal inoculum (30 – 100 pfu) results in robust virus replication within bronchial epithelial cells that is accompanied by profound granulocyte recruitment. In the absence of pharmacologic intervention, PVM infection progresses to pulmonary edema and respiratory compromise, similar to the more severe forms of RSV infection experienced by human infants [10, 11]. In our earlier studies, we identified the chemokine CCL3 (MIP-1α) as a crucial component of this inflammatory response. PVM not only elicits production of CCL3 by infected bronchial epithelial cells , mice devoid of CCL3 or its receptor, CCR1, recruit dramatically fewer neutrophils to airways . Blockade of the CCL3/CCR1 proinflammatory signaling pathway in conjunction with antiviral therapy resulted in improved survival in response to an otherwise lethal virus inoculum [14, 15]. As CCL3 is only one of several major pro-inflammatory signaling pathways activated by PVM infection , there is certainly the possibility of additive, synergistic, or hierarchical means to promote and to amplify the ongoing inflammatory response.
Although first identified as a component of the antiviral response to Sindbis virus , the role of the Th1 cytokine, interferon-γ (IFNγ) in pneumovirus infection remains uncertain. IFNγ is readily detected in bronchoalveolar lavage fluid and nasal washings from RSV-infected infants [17, 18], and minimal or absent response has been correlated with poor clinical outcome [19–24]. IFNγ is also detected in BAL fluid of BALB/c mice in response to challenge with RSV virions [25, 26] and plays a role in limiting the inflammatory response to secondary challenge and in generating the allergic histopathology in response to formalin-fixed RSV vaccine antigens and virion components [27, 28]. Likewise, local production of IFNγ is a prominent response to PVM infection [12, 29, 30], although its role in modulating the primary inflammatory response has not yet been fully explored.
In this manuscript, we explore the role of IFNγ in modulating the inflammatory response to PVM infection, and utilize overexpression analysis to begin a dissection of the independent and interdependent contributions of both IFN-γ and CCL3 to the process of neutrophil recruitment in vivo.
Microarray profiling of IFNγ expression in response to PVM infection
Expression profiles that correlate with IFN-γ in PVM-infected mouse lung tissue.
Interferon-γ and related transcripts
Interferon inducible protein 1
Interferon-γ induced GTPase
Interferon-induced transmembrane protein 6
CXC chemokine ligand 11 (IP-9)
Interferon inducible protein 47
Interferon activatible protein 203
Interferon activated gene 205
Interferon induced protein with tetratricopeptide repeats 1
Interferon consensus sequence binding protein 1
Interferon regulatory factor 7
Interferon activated gene 205
Interferon regulatory factor 5
Interferon activated gene 203
Interferon-induced protein with tetratricopeptide repeats, 3
Interferon induced protein with tetratricopeptide repeats 2
Other inflammation-associated transcripts
CC Chemokine ligand 3 (MIP-1α)
Toll-like receptor 2
Interleukin-13 receptor alpha 1
Suppressor of cytokine signaling 3
Interleukin-1 receptor antagonist
Regulator of G-protein signaling 19 interacting protein 1
CD244 natural killer cell receptor 2B4
Fc receptor, IgG, high affinity, I
CC chemokine receptor 1
CXC chemokine ligand 13 (BLC)
CXC chemokine ligand 2 (MIP-2α)
CXC chemokine ligand 5 (ENA-78)
Signal transducer and activator of transcription 1
Regulator of G-protein signaling 1
CC chemokine receptor-like 2
Membrane-spanning 4-domains, subfamily A, member 8A
Chondroitin sulfate proteoglycan 2
Fas death domain-associated protein
O-acyltransferase domain containing 1
Mitogen activated protein kinase kinase kinase kinase 1
Lymphocyte cytosolic protein 2
Solute carrier family 15, member 3
Indoleamine-pyrrole 2,3 dioxygenase
Proteosome subunit beta type 9
Phospholipase A1 member A
Methylene tetrahydrofolate dehydrogenase
Pre-B colony enhancing factor 1
Thioredoxin reductase 1
CGG triplet repeat binding protein 1
Sphingosine kinase 1
2'-5' oligoadenylate synthetase 1G
Membrane-spanning 4-domains, subfamily A, member 4C
AT rich interacting domain 5A
Hemopoietic cell kinase
Histocompatibility 2, complement component factor B
ATP binding cassette
BING 4 protein
Thymidylate kinase, LPS inducible
Tripartite motif protein 30
Tissue specific transplantation antigen 30
Syndecan binding protein
Prostaglandin-endoperoxide synthase 2
Traf binding protein
Two pore segment channel 2
Early growth response 2
GLI pathogenesis-related 2
Cytochrome p450, family 7, subfamily b
Rab20, Ras oncogene
Solute carrier 39
Dual specificity phosphatase 3
Ribosome binding protein 1
Ubiquitin-specific protease 18
Guanylate nucleotide binding protein 3
Pre-B cell colony-enhancing factor 1
Membrane-spanning 4-domains subfamily A, member 6B
SLAM family member 7
Ras and Rab interactor 1
Class II transactivator
Myxovirus resistance I
Chloride channel calcium activated 2
Rap2C, member of RAS oncogene family
Tumor necrosis factor, alpha induced protein 2
SLAM family member 8
Detection IFNγ+ NK and T cells in PVM infected lung tissue
Both total and IFNγ+ subsets of NK cells, CD4+ and CD8+ T cells were enumerated in single cell suspensions of lung tissue from PVM-infected BALB/c mice evaluated at day 6 after inoculation with 10 pfu PVM strain J3666 [Figure 1C]. Only a small fraction (<2%) of the CD4+ T cells detected at this time point stained positively for IFNγ, in contrast to the larger fraction of IFNγ+CD8+ T cells detected (9.9 ± 0.6 × 103 cells, 11% of total CD8+ T cells). Interestingly, 23% of the total NK cells (3.4 ± 0.9 × 103 cells) stained positively for IFNγ, an increase from 0.3 ± 0.08 × 103 cells, or 4% of the total NK cells detected in a single lung from uninfected mice (data not shown).
IFNγ-dependent responses to PVM infection
Receptor expression and responses of neutrophils from IFNγR gene-deleted mice
Overexpression of CCL3
Inflammatory responses to IFNγ and CCL3
In previous work, we demonstrated that the actions of the chemokine, CCL3, signaling via its receptor CCR1, were crucial for granulocyte recruitment to the lungs in response to PVM infection [13–15]; CCL3 has also been shown to be a crucial mediator of granulocyte recruitment in mouse models of influenza . Paradoxically, CCL3 gene-deletion results in augmented neutrophil and eosinophil recruitment in response to Cryptococcus neoformans infection . Here we show that CCL3-mediated neutrophil recruitment depends directly on IFNγ signaling, both in the setting of acute virus infection and in response to heterologous CCL3 expression in the respiratory epithelium.
Granulocyte recruitment is a primary finding in severe respiratory virus infection; activation of granulocytes can result in the release of proinflammatory cytokines and proteolytic enzymes that can contribute to the ongoing lung damage [33–37]. Interestingly, although neutrophils are recruited to the lung parenchyma in response to CCL3 via coordination by IFNγ, these cytokines alone clearly are not sufficient to induce the inflammatory state that ultimately promotes lung damage and respiratory failure. Thus, despite our findings demonstrating improved survival from PVM infection with CCR1 blockade , and those of He and colleagues , who likewise demonstrated that CCR1 antagonism provided protection against neutrophil-mediated lung injury in a mouse model of acute pancreatitis, the results presented here, in which we observe neutrophil recruitment but minimal clinical disease, suggest that neutrophil recruitment and neutrophil activation are to some extent distinct and discrete signaling events. It will be crucial to identify the proinflammatory mediators that activate and well as those that recruit neutrophils in order to have a complete picture of the proinflammatory state characteristic of PVM infection.
The experimental studies performed in this manuscript utilize both IFNγ and IFNγR gene-deleted mice, which are in BALB/c and C57BL/6 background strains, respectively. PVM infection has been explored systematically in several inbred strains of mice by Anh and colleagues  who determined that the C57BL/6 strain is somewhat more resistant to infection than BALB/c, but that both of these inbred strains can ultimately succumb to the sequelae of severe disease. We have used both of these strains extensively for our studies (reviewed in [7–9]) and both respond to PVM infection with robust virus replication in lung tissue, granulocyte recruitment and local production of proinflammatory cytokines, including CCL3 and IFNγ; no systematic differences, other than the aforementioned susceptibility to infection, have been detected.
Both CCL3 and IFNγ have been detected in human studies and in mouse models of other severe respiratory virus infections, including avian influenza, SARS coronavirus, and human respiratory syncytial virus [17, 18, 40–47], although the potential for interplay between these specific signaling pathways has not been considered previously. Our data suggest that that IFNγ and CCL3 signaling pathways, both crucial features of the response to pneumovirus infection, interact in a hierarchical fashion, as IFNγ does not elicit neutrophil recruitment on its own [Figure 5A], but is crucial for CCL3 to function effectively. Interactions between IFNγ and CCL3 may occur at the level of signal transduction, or via alterations to the neutrophil itself. As has been documented clearly, CCL3 can function alone to induce changes in calcium concentration and chemotactic responses in mouse neutrophils in vitro . The current literature on interactions of IFNγ with granulocytes was recently reviewed . Among the possibilities that may address our findings, Hansen and Finbloom  reported that human neutrophils express IFNγ receptors and Bonecchi and colleagues  have shown that human neutrophils respond to IFNγ with increased expression of a variety of mediators and receptors, including the primary CCL3 receptor, CCR1. It is unclear whether mouse neutrophils respond in a similar fashion, and whether or not these defined molecular responses take place in vivo, although we have shown here that neutrophils from IFNγR gene-deleted mice express transcripts for CCR1 and mobilize intracellular calcium in response to CCL3 when examined ex vivo. We have not yet explored the possibility that the IFNγ coordinates neutrophil recruitment in response to CCL3 in a more indirect fashion, possibly via one or more intermediary cytokines. An example of this phenomenon was reported by Khader and colleagues , who demonstrated that Mycobacterium tuberculosis-infected dendritic cells from IL-12p40 gene-deleted mice that were unresponsive to a CCL19 gradient were also overproducing the cytokine IL-10. Most intriguing, addition of IL-10 to wild-type dendritic cells reproduced the inhibited chemotaxis response.
In summary, we demonstrate here that CCL3, a proinflammatory mediator produced in response to RSV and shown to be a crucial in recruiting neutrophils in response to the mouse pneumovirus, PVM, functions via a hierarchical relationship with IFNγ. Specifically, CCL3 recruits neutrophils to the lung in vivo only in coordination with IFNγ-mediated signaling pathways. The mechanism via which IFNγ modulates neutrophil responses to CCL3 is an intriguing subject for future exploration.
Generation of gene microrarray data was as described previously . Data collected were evaluated using the microarray software analysis package Genespring GX 7.3. The 'find similar' function was used to inspect all 45,101 transcript profiles in order to detect kinetic profiles similar to that of IFNγ. The minimum correlation to be considered a similar profile was set at 0.900. The higher the correlation coefficient (maximum 1.000 for complete overlap), the more similar the gene expression profiles.
Mouse, virus and vector stocks
BALB/c and C57BL/6 mice were purchased from Taconic Laboratories (Germantown, NY and Rockville, MD). Homozygous IFNγ gene-deleted (IFNγ -/-) mice  on a BALB/c background and IFNγ receptor gene-deleted (IFNγR -/-) mice  on a C57BL/6 background were purchased from Jackson Laboratories, Bar Harbor, ME. All animal studies were performed as per approved protocols CHUA #634 (SUNY Upstate) or LAD 8E (NIAID). PVM strain J3666 was passaged, stored and quantitated as described previously . Mice were anaesthetized and inoculated by intranasal challenge with 30 – 100 plaque forming units (pfu) PVM also as previously described. For challenge with recombinant vectors (described as follows), dilutions of secondary stock aliquots of vCCL3 and vctrl (described in the section to follow) were prepared in RPMI cell culture medium. Under brief anaesthesia, mice were inoculated with 150 μl of stock (50 μl/dose × 3 doses) to achieve challenges of 1.0 – 1.5 × 1011 pfu per mouse. On days indicated, mice in each challenge group were sacrificed by cervical dislocation and bronchoalveolar lavage (BAL) fluid, total lung protein and total lung RNA were harvested. For some experiments, mice received 15 μg recombinant murine IFNγ (R&D Systems, Minneapolis, MN) diluted in tissue culture medium (RPMI + 10% fetal calf serum) or tissue culture medium (vehicle) via intraperitoneal injection one day prior to intranasal challenge with the vCCL3 or vctrl which yielded 323 ± 28 pg IFNγ/mg lung on day 4 post-inoculation.
Flow cytometric determination of IFNγ+ NK and T cells in mouse lung tissue
Whole lungs of BALB/c mice (uninfected or day 6 after inoculation with 10 pfu PVM, n = 5 per datapoint) were cut into ~3 mm3 pieces in HBSS buffer (Invitrogen) and pressed through a 100 micron cell strainer (BD Biosciences, San Jose, CA) to obtain single cell suspensions. Cells were suspended in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, 100 U/mL streptomycin, 50 μM 2-mercaptoethanol, 1 mM sodium pyruvate, and nonessential amino acids (all from Invitrogen) and incubated for 6 hrs at 37°C at a density of 1 × 106 cells/ml with 1 μM ionomycin, 20 ng/ml phorbol-12-myristate acetate (EMD Biosciences, San Diego, CA) and 10 μg/ml brefeldin A (Sigma-Aldrich Co., St. Louis, MO). DNAse I (Sigma) was added for 5 minutes and then cells were washed once and stained with violet LIVE/DEAD Fixable Dead Cell stain (Invitrogen) for 30 minutes on ice, washed in PBS, fixed in 4% PFA, and stored at -80°C until analysis. Intracellular cytokine staining was performed as described previously . Cells were stained with I-Ad FITC, DX5-PE, CD3-PE-Cy5, CD4 PerCP/Cy5.5, IFNγ PE-Cy7, and CD8 APC-Cy7 (BD Biosciences) in PBS with 0.1% BSA, 0.1% saponin (Sigma) and 5% nonfat dry skim milk. Controls were stained with isotype matched antibodies. Samples were acquired with a 4-laser LSR II flow cytometer (BD Biosciences) and analyzed on FlowJo software (Tree Star, Inc., San Carlos, CA). Viable lymphocytes were identified by typical forward and side scatter and negative staining for LIVE/DEAD violet. T cells were identified as I-A-, CD3+ and either CD4+ or CD8+ as indicated; NK cells were identified as I-A-, CD3-, DX5+. Quadrant statistical markers were based on corresponding isotype matched controls. Samples consisted of a known fraction (typically 1/4) of the cells obtained from a whole lung, and the entire sample was analyzed (typically 2 – 4 × 105 events) yielding the absolute number of cells per lung.
Isolation of neutrophils from wild type and IFNγR-/- mice
Neutrophils were isolated from wild type and IFNγR-/- mice as described . Briefly, 2 mL intraperitoneal injection of thioglycollate was administered and 4 hours later, mice were sacrificed and cells were harvested by peritoneal flush with 10 mL PBS (without calcium or magnesium). Cells were washed, red blood cells lysed with distilled water, and viability determined at >95% by trypan blue exclusion. Further isolation via Ficoll/Hypaque density gradient centrifugation yielded neutrophil purities of 85 – 99% as determined by modified Giemsa staining of cytospin preparations.
Absolute quantification of CCR1 expression
Total RNA was isolated from neutrophils elicited from wild type and IFNγR -/- mice using the RT2 qPCR-Grade RNA Isolation Kit (SuperArray Bioscience Corporation). The cDNA was prepared using the 1st strand cDNA Synthesis Kit for RT-PCR (AMV; Roche Applied Science). QPCR was performed using the TaqMan Universal PCR Maser Mix (Applied BioSystems) with primer-probe pairs for GAPDH (TaqMan Rodent GAPDH Control Reagents VIC probe Applied Biosysystems) or CCR1 (chemokine (C-C motif) receptor 1 Mm00438260_s1 FAM labled, Applied Biosystems). The standard curve for mouse GAPDH included serial dilutions of the DECA template GAPDH-Mouse probe (Ambion); the standard curve for mouse CCR1 included serial dilutions of the coding sequence (GenBank Accession # U28404) in pCEP4. Reactions were run in triplicate in the 7500 RealTime PCR System (Applied Biosystems); data presented as copies of CCR1 per 109 copies GAPDH.
Intracellular calcium measurements
Intracellular calcium measurements were performed in a Benchtop Scanning Fluorometer and Integrated Fluid Transfer Workstation (Flexstation; Molecular Devices) as described . Briefly, 2.5 × 105 thioglycollate-elicited neutrophils were suspended in 100 μl of Hank's buffered saline solution with 20 mM HEPES and 100 μl fluorescent dye (FLIPPER calcium 3 assay kit component A; Molecular Devices) in a 96 well plate. The cells were incubated at 37°C for 30 minutes, centrifuged for 5 minutes, and challenged with various concentrations of CCL3, buffer alone (negative control) or f-MLF (positive control). Changes in intracellular calcium concentration were recorded as relative fluorescence units (RFU).
Construction vCCL3 and control (vctrl) overexpression vectors
Generation of overexpression vectors was accomplished using the commercially available AdEasy XL vector system (Stratgene, La Jolla CA) according to the manufacturer's instructions. Briefly, murine CCL3 (GenBank Accesion No. NM 011337) was ligated into the multiple cloning site of the shuttle vector, pShuttleCMV; the corresponding control plasmid, containing the β-galactosidase gene, was supplied with the kit. The constructs were linearized with Pme I and transformed into an E. coli strain, (BJ5183) which contains the replication-incompetent pAD-1 backbone. Transformants were selected for kanamycin resistance, and recombinants subsequently identified by restriction digestion. Once recombinants were identified, they were produced in bulk using the recombination-deficient bacterial strain, XL-10 Gold. Purified recombinant plasmid DNA was digested with Pac I to expose inverted terminal repeats and used to transfect AD-293 cells in which the deleted viral assembly genes are complemented in vivo. The resulting constructs, vctrl and vCCL3 were harvested from the transfected AD-293 cells when more than 90% of the monolayer exhibited cytopathic effects. Secondary stocks were produced in a similar fashion. Titration was performed by standard plaque assay. The concentration of secondary stocks reached titers of ~1012 pfu/ml for each construct. Viral stocks were stored at -80°C prior to use.
Bronchoalveolar lavage (BAL) and differential cell counts
At time points indicated, BAL fluids were harvested from 5 mice by trans-tracheal instillation and removal of pre-chilled phosphate-buffered saline with 0.25% bovine serum albumin (BSA; 0.80 ml instillation with recovery of 0.5 to 0.6 ml per mouse). Neutrophil counts were determined by visual inspection of methanol-fixed cytospin preparations stained with modified Giemsa (DiffQuik, Fisher Scientific, Pittsburgh PA).
Chemokine and cytokine determinations
Concentrations of CCL3 and IFNγ were determined in BAL fluid (pg/ml) isolated as previously described  from five mice per datapoint, using commercially available ELISA kits (R&D Systems, Minneapolis, MN). Total protein was determined the Bradford colorimetric assay using bovine serum albumin standards
Gross and microscopic pathology and immunohistochemical detection of CCL3
Paraffin blocks of formalin-fixed lung tissue from mice challenged with vctrl or vCCL3 were paraffin-embedded and sectioned. Standard hematoxylin and eosin staining of formalin-fixed tissue was performed by American Histolabs (Gaithersburg, MD). To detect CCL3 protein expression in situ, slides were incubated with a 1:50 dilution of goat anti-CCL3 (R&D Systems, Minneapolis, MN) followed by a 1:400 dilution of biotinylated rabbit anti-goat Ig and developing reagents (performed by Histoserv, Inc., Germantown, MD).
Quantitative reverse transcriptase PCR to document PVM titer in mouse lung tissue was as described previously . Datapoints are presented as copies of PVM SH gene per 109 copies GAPDH.
Experimental datapoints were from triplicate samples, experiments replicated two to three times. Data were evaluated by Student's t-test or Mann-Whitney U-test as appropriate.
Joseph B. Domachowske, M. D. is Professor of Pediatrics, Microbiology and Immunology at SUNY Upstate Medical University, Syracuse, New York. Helene F. Rosenberg, M. D. is Senior Investigator and Section Chief in the Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Drs. Domachowske and Rosenberg have collaborated extensively, and together have developed the pneumonia virus of mice (PVM) model for the study of the sequelae severe respiratory virus infection in a rodent host.
CC chemokine ligand 3
pneumonia virus of mice
respiratory syncytial virus
CC chemokine receptor 1
CC chemokine ligand 19
plaque forming units
relative fluorescence units.
The authors thank Ms. Leslie Pesnicak for assistance with the intraperitoneal inoculations. Grant support provided by Children's Miracle Network of NY to JBD and NIAID DIR funding to HFR.
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