Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes

Sigrid P. Matzer1, Franz Rödel2, Robert M. Strieter3, Martin Röllinghoff1 and H. Ulrich Beuscher1

1 Institute for Clinical Microbiology, Immunology and Hygiene and 2 Department of Radiooncology, University of Erlangen, Erlangen, Germany
3 Department of Medicine and Pathology and Laboratory Medicine, UCLA, School of Medicine, Los Angeles, CA, USA

Corresponding author: H. U. Beuscher; E-mail: beuscher{at}mikrobio.med.uni-erlangen.de


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CXCL2/macrophage inflammatory protein (MIP)-2 is an inducible murine chemokine involved in attraction of polymorphonuclear granulocytes to sites of infection. In comparison, its role as constitutive produced chemokine in mice is unclear. The present study aimed to specify the cellular source of constitutively produced CXCL2/MIP-2 and to examine its expression pattern in comparison to other chemokines in peripheral lymphoid tissues as well as bone marrow (BM) of normal mice. The results showed that constitutive expression of CXCL2/MIP-2 mRNA was restricted to BM. As revealed by RT–PCR and FACS analysis, CXCL2/MIP-2 production was restricted to a specialized subset of BM derived Gr-1high granulocytes. This subset was characterized by surface expression of CD11b+, CD62Lhigh and CXCR2+ and accounted for 4–6% of total BM cells. In vitro stimulation of BM cells did not increase the number of CXCL2/MIP-2+ granulocytes. Intracellular CXCL2/MIP-2 was not strictly correlated to surface expression of its receptor, as the majority of the CXCR2+/Gr-1high cells lacked CXCL2/MIP-2 staining. In controls, CXCL1/KC expression was not detected in BM but was found in peripheral tissues in the absence of CXCL2/MIP-2. Together, our results show that CXCL2/MIP-2 and CXCL1/KC are differentially expressed in a tissue specific manner in normal mice and that CXCL2/MIP-2 is produced in a well-defined CD11b+, CD62Lhigh, Gr-1high subset of BM granulocytes, thereby providing a possible explanation for the independent regulation of both chemokines.

Keywords: chemokines, neutrophils, Peyer's patches, spleen


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Neutrophils are the most abundant leukocyte population in blood and are constantly released from the bone marrow (BM) into the circulation (1). Hence, they are rapidly recruited to sites of infection and their main function resides in protecting the host from invading microorganisms by phagocytosis and the release of toxic molecules from their granules (2). In addition, evidence has accumulated indicating that neutrophils play an important role as regulators of immune responses through release of cytokines such as interleukin (IL)-1, IL-3, IL-6, IL-12, tumor necrosis factor (TNF)-{alpha} or tumor growth factor (TGF)-ß as well as chemokines such as CCL2/monocyte chemoattractant protein (MCP)-1, CCL3/macrophage inflammatory protein (MIP)-1{alpha}, CXCL8/IL-8, CCL19/MIP-3ß or CCL20/MIP-3{alpha} (3,4). Chemokines are small basic chemotactic proteins, which exert their function through seven-transmembrane G protein-coupled receptors on the surface of target cells. They can broadly be divided into two categories (5,6), one of which is expressed constitutively. They sustain homeostasis of the lymphoid system and are involved in embryonic development of secondary lymphoid organs. The second group comprises inducible chemokines which direct leukocyte migration in response to endogenous signals or exogenous stimuli evoking inflammatory processes (5,7). Members of the CXC-subclass of chemokines, particularly those containing a glutamic acid–leucine–arginine (ELR) motif, are induced at early stages of acute inflammation (8,9). These ELR+CXC chemokines act selectively on neutrophils (10,11). The most prominent ELR+CXC chemokines are human CXCL8/IL-8 and, in the murine system, CXCL2/MIP-2 and platelet-derived growth factor-inducible chemokine KC (KC, CXCL1) (1214). CXCL2/MIP-2 and CXCL1/KC were shown to bind to the murine CXC receptor 2 (CXCR2), which is abundantly expressed on granulocytes and on NKT cells (15,16). When neutrophils from CXCR2 knock out mice were exposed to CXCL2/MIP-2 or CXCL1/KC no migration was observed, supporting the view that CXCR2 is the principal receptor for CXC chemokines on murine neutrophils (17,18).

CXCL2/MIP-2 is considered a major inducible chemokine. Macrophages for example were shown to express large amounts of CXCL2/MIP-2 after stimulation with whole bacteria or bacterial cell wall components, e.g. lipopolysaccharide (LPS) (13,19,20). CXCL2/MIP-2 induction was also observed in epithelial cells, vascular endothelial cells, astrocytes, mast cells and neutrophils (2125). Additionally, proinflammatory cytokines like IL-1 and TNF-{alpha} were shown to induce CXCL2/MIP-2 expression in vitro in murine endothelial cells (26). In vivo, in a model of ischemia/reperfusion, IL-1 was shown to be an important inducer of CXCL2/MIP-2 expression and subsequent hepatic neutrophil recruitment (26,27).

Comparatively little is known about the constitutive expression of CXCL2/MIP-2 and consequently, its role in homeostasis. CXCL2/MIP-2 expression was described in several tissues during normal embryonic development of healthy mice (28). In adult mice constitutive CXCL2/MIP-2 mRNA expression was detected in mouse gallbladder cells (29). Finally, our group recently reported that CXCL2/MIP-2 is constitutively produced in BM granulocytes (30). The present study now attempts to examine the expression pattern of CXCL2/MIP-2 in comparison to other chemokines and aims to define the cellular source of CXCL2/MIP-2 in BM. The results show that CXCL2/MIP-2 expression is restricted to BM when compared to peripheral lymphoid tissues or blood leukocytes of normal mice. CXCL2/MIP-2 synthesis occurred independently of CXCR2 surface expression in a small subpopulation of CD11b+/CD62Lhigh/Gr-1high BM granulocytes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Bacteria
The virulent strain (NCTC 10938), here named O:8+, of Yersinia enterocolitica O:8 was obtained from the National Collection of Type Cultures (Central Public Health Laboratory, London, UK). Bacteria were routinely grown overnight in defined tryptone yeast extract glucose medium at 26°C. For infection, Yersinia cultures grown overnight were diluted 1:20 in Luria–Bertani medium and incubated at 37°C for 2 h. Bacteria were harvested and washed twice with either PBS (Biochrom, Berlin, Germany) for i.p. infection or tryptone yeast extract glucose medium for oral infection.

Experimental infection of mice
Female BALB/c mice (6–10 weeks of age) were purchased from Charles River (Sulzfeld, Germany). Mice were infected orally with 0.1 ml of Yersinia suspension containing 1 x 107 CFU (LD50 = 5.6 x 106) or i.p. with 1 ml of Yersinia suspension containing 5 x 103 CFU (LD50 = 2.6 x 103). Mice were sacrificed at day 5 after infection and Peyer's patches (PPs) or spleens and BM cells were harvested.

Cells and cell culture
BM cells were obtained by flushing femurs and tibiae of BALB/c mice with culture medium RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Biochrom) and 10% FCS (Sigma, Deisenhofen, Germany). For cell sorting, contaminating erythrocytes were removed by suspending the BM cells in NH4Cl solution (168 mM) for 10 min at room temperature. For in vitro stimulation, cells were incubated at a density of 1 x 107 cells/ml and stimulated for 4 h (37°C, 5% CO2, 95% humidity) with either LPS (10 µg/ml) or IL-1{alpha} and TNF-{alpha} (100 ng/ml each). To obtain peripheral blood leukocytes (PBL), EDTA–blood was centrifuged and erythrocytes were lysed using NH4Cl as described above.

FACS analysis
Single cell suspensions of BM (1 x 106 cells/ml) were preincubated with Fc-Block (Becton-Dickinson, San Jose, CA) for 10 min followed by PE-conjugated rat anti-murine Gr-1 antibody (clone RB6-8C5, Becton-Dickinson) and goat anti-CXCR2 serum (kindly provided by M. Burdick), FITC-conjugated rat anti-murine CD11b or PE-conjugated rat anti-murine CD62L (both Becton-Dickinson). After incubation for 30 min cells were thoroughly washed with PBS/1% FCS. For CXCR2 staining, cells were additionally incubated with Cy5-conjugated donkey anti-goat secondary antibodies (Dianova, Hamburg, Germany) for 20 min and again washed. For intracellular staining, cells were then fixed for 10 min with 4% paraformaldehyde and stained with anti-CXCL2/MIP-2 serum (kindly provided by N. W. Lukacs; Ann Arbor, MI) (31) or normal rabbit serum for 45 min. FITC-conjugated donkey anti-rabbit antibodies (Dianova) were used as secondary antibodies. All washing steps after fixation of the cells were carried out using PBS containing 1% FCS and 0.5% saponin (Sigma). Samples were analyzed by flow cytometry using a FACScalibur and CellQuest software (Becton-Dickinson). As a control, CXCL2/MIP-2 staining was partially blocked by preincubation of rabbit anti-murine CXCL2/MIP-2 serum with recombinant CXCL2/MIP-2 (10 µg/ml; PeproTech, London, UK) for 16 h.

Cell sorting
Single cell suspensions of freshly isolated BM were depleted from erythrocytes using NH4Cl, stained with PE-conjugated rat anti-murine Gr-1 antibody (clone RB6-8C5, Becton-Dickinson) and passed through a cell strainer (70 µm, Becton Dickinson). Subsequently, cells were injected into the MoFlo® cell sorter (DakoCytomation, Freiburg, Germany) for phenotypic isolation using gates for Gr-1high expressing BM cells and Gr-1 BM cells. The sorted populations were of 99% purity. The viability of the sorted cells was determined by propidium iodide staining. The cells were kept on ice until use.

Reverse transcription PCR
Total RNA was prepared from frozen tissues or single-cell suspensions by using the guanidine–thiocyanate extraction procedure followed by acid–phenol extraction (32). For small samples, RNA was also isolated using the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). Residual genomic DNA was removed using DNAfree following the manufacturer's instructions (Ambion, Cambridgeshire, UK). The cDNA was synthesized from 2 µg of total RNA using 2.5 µM oligo(dT)12–18, 250 µM dNTP (Qiagen), 12 U RNAguard (Amersham Pharmacia Upjohn, Freiburg, Germany), 4 U Omniscript RT reverse transcriptase (Qiagen) to a final volume of 25 µl at 37°C for 60 min. For gene specific PCR, equal amounts of sample cDNA were amplified in 25-µl reaction volumes containing 200 µM dNTP, 1 U Taq DNA polymerase (Qiagen) and 1 µM primers during 35 cycles (30 s denaturation, 94°C; 30 s annealing, 55°C or 58°C depending on the primer pair; 30 s polymerization, 72°C) with an Omnigene temperature cycler (Hybaid, Ashford, UK). The primers were purchased from Amersham Pharmacia Upjohn, MWG Biotech (Ebersberg, Germany) and Qiagen Operon (Köln, Germany). Primer sequences are listed in Table 1 (33,34). The PCR products were separated on 2% agarose gels, visualized by ethidium bromide staining and photographed on an ImageMaster VDS (Amersham Pharmacia Upjohn).


View this table:
[in this window]
[in a new window]
 
Table 1. Primer sequences used for RT–PCR

 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lack of CXCL2/MIP-2 expression in peripheral lymphoid tissues and blood leukocytes
The expression patterns of CXCL2/MIP-2 and chemokines like CXCL1/KC, CXCL5/LIX, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN and CCL3/MIP-1{alpha} were investigated in PPs, spleen and PBLs of healthy mice using RT–PCR analysis. In PPs, constitutive mRNA expression was detectable for CXCL1/KC and most other chemokines, but not for CXCL2/MIP-2 (Fig. 1A). In controls, however, CXCL2/MIP-2 mRNA expression was readily detectable in PP after oral infection of mice with Y. enterocolitica bacteria (Fig. 1A, + Y. ent.). In addition, analysis of chemokine mRNA expression in splenic tissue (Fig. 1B) as well as blood leukocytes (Fig. 1C) confirmed that CXCL2/MIP-2 mRNA remained undetectable, while CXCL1/KC mRNA is constitutively expressed in peripheral lymphoid tissues.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 1. Analysis of chemokine mRNA expression pattern in peripheral lymphatic tissues and blood. Total RNA was prepared from PPs, spleens and PBLs of female BALB/c mice and subjected to RT–PCR with primers for CXCL2/MIP-2, CXCL5/LIX, CXCL1/KC, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha} and ß-actin. (A) Chemokine mRNA expression pattern of PPs of mice that were either challenged orally with 1 x 107 Y. enterocolitica O:8+ (+ Y. ent.) or left untreated (constitutive) and sacrificed on day 5 post infection. Lanes 1–8: CXCL1/KC, CXCL5/LIX, CXCL2/MIP-2, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha}, ß-actin; lane 9: CXCL2/MIP-2. (B) Chemokine mRNA expression pattern of splenic tissue of healthy mice. (C) Chemokine mRNA expression pattern of PBLs of healthy mice. (B and C) Lanes 1–8: CXCL2/MIP-2, CXCL5/LIX, CXCL1/KC, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha}, ß-actin. Data are representative of at least three different experiments.

 
CXCL2/MIP-2 is constitutively expressed in BM
To investigate the expression of CXCL2/MIP-2 in primary lymphoid organs, total RNA was prepared from BM and subjected to RT–PCR analysis. Results showed that CXCL2/MIP-2 as well as CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN and CCL3/MIP-1{alpha} mRNA were constitutively expressed in BM. In comparison, no CXCL1/KC mRNA was detectable in BM (Fig. 2A). Lack of CXCL1/KC mRNA expression in BM was confirmed by using two other published primer pairs (data not shown). In addition, CXCL1/KC mRNA expression in BM was inducible through systemic Yersinia infection (Fig. 2B). The data indicate that CXCL2/MIP-2 is constitutively expressed in BM and further imply that the CXC chemokines CXCL2/MIP-2 and CXCL1/KC are differentially regulated.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. Analysis of chemokine mRNA expression pattern in BM. Total RNA was prepared from BM of BALB/c mice and subjected to RT–PCR with primers for CXCL2/MIP-2, CXCL5/LIX, CXCL1/KC, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha} and ß-actin. (A) Chemokine mRNA expression pattern of BM of healthy mice. (B) Chemokine mRNA expression pattern of BM of BALB/c mice that were challenged i.p. with 5 x 103 Y. enterocolitica O:8+ (+ Y. ent.) and sacrificed on day 5 post infection. Lanes 1–8: CXCL2/MIP-2, CXCL5/LIX, CXCL1/KC, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha}, ß-actin. Data are representative of at least three different experiments.

 
In the BM CXCL2/MIP-2 mRNA is expressed in Gr-1+ granulocytes
Previously we showed that Gr-1+ BM granulocytes carry CXCL2/MIP-2 protein (30). Therefore, BM granulocytes were now analyzed for CXCL2/MIP-2 mRNA synthesis using RT–PCR analysis. Gr-1 labeled cells were fractionated via Mo-Flo® in two populations, those strongly expressing Gr-1 antigen (Gr-1positive) and those lacking Gr-1 surface expression (Gr-1negative). As shown in Fig. 3(A), Gr-1+ cells (left panel) were of 99.5% purity. Each of the two cell populations was subjected to RT–PCR analysis using primers for CXCL2/MIP-2, CXCL1/KC, CXCL5/LIX, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN and CCL3/MIP-1{alpha}. The results in Fig. 3(B) show that Gr-1+ BM cells expressed CXCL2/MIP-2 and CXCL10/IP-10 mRNA (upper panel). As expected, no CXCL1/KC mRNA expression was found in Gr-1+ cells. However, upon stimulation with IL-1 and TNF-{alpha}, Gr-1+ BM granulocytes were found to be able to express CXCL1/KC and CCL3/MIP-1{alpha} mRNA (Fig. 3B, lower panel). In contrast, no chemokine expression was observed in the Gr-1 population (Fig. 3C, upper panel). In controls, CXCL2/MIP-2 and CXCL1/KC as well as CXCL10/IP-10 and CCL3/MIP-1{alpha} mRNAs were expressed by Gr-1 cells after stimulation with IL-1 and TNF-{alpha} (Fig. 3C, lower panel). Chemokine expression patterns were similar in both cell populations when using LPS as a stimulus (data not shown). The data indicate that Gr-1+ granulocytes are the principal source of CXCL2/MIP-2 in BM.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3. RT–PCR analysis of chemokine expression in Gr-1+ granulocytes of the BM. BM cells were prepared from untreated BALB/c mice, stained for granulocytes with PE-conjugated rat anti-mouse Gr-1 antibody and subjected to cell sorting. Cells strongly expressing the Gr-1 antigen (Gr-1positive) or unlabeled cells (Gr-1negative) were collected. The sorted fractions where either stimulated with TNF-{alpha} and IL-1 (100 ng/ml each) simultaneously for 4 h (+ IL-1/TNF) or left untreated. Total RNA was extracted from all populations and subjected to RT–PCR. Identical results were obtained using LPS (10 µg/ml) as a stimulus. (A) The purity of both populations was determined by FACS analysis. (B) RT–PCR analysis of Gr-1+ BM cells. (C) RT–PCR analysis of Gr-1 BM cells. (D) As a control for cDNA quality and amount, ß-actin expression of all analyzed cell populations is shown. Data are representative for two or three separate experiments. (B and C) Lanes 1–7: CXCL2/MIP-2, CXCL5/LIX, CXCL1/KC, CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN, CCL3/MIP-1{alpha}. (D) Lanes 1/2: upper/lower panel (B); lanes 3/4: upper/lower panel (C).

 
Phenotypical characterization of Gr-1high BM granulocytes
To characterize the CXCL2/MIP-2+/Gr-1+ phenotype of BM granulocytes, BM cells were subjected to FACS analysis using fluorescence labeled antibodies to various cell surface molecules including CD11b, CD62L, Gr-1 and CXCR2. Results identified granulocyte populations with high (Gr-1high) and low (Gr-1low) levels of Gr-1 expression (Fig. 4A). CXCR2 expression was restricted to the Gr-1high population (Fig. 4B). CD62L was strongly expressed on the surface of Gr-1high when compared to Gr-1low granulocytes (Fig. 4C). In comparison, CD11b was found evenly expressed on both the Gr-1high and Gr-1low granulocytes (Fig. 4D). Together, cell surface staining of each Gr-1 subpopulation revealed that only the expression of CXCR2 was restricted to the Gr-1high subpopulation. The data indicate that in the BM only Gr-1high, mature granulocytes express CXCR2.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4. FACS analysis of the phenotype of BM granulocytes. BM cells were isolated from healthy BALB/c mice and double staining was performed for Gr-1 antigen and cell surface markers CXCR2, CD62L or CD11b. (A) Gates were set for Gr-1low (gate R1) or Gr-1high (gate R2) expressing BM cells. (B–D) FACS analysis of BM cells expressing low (Gr-1low) or high (Gr-1high) levels of Gr-1 antigen for surface expression of CXCR2 (B), CD62L (C) and CD11b (D). Data are representative for three to five experiments. Numbers indicate the mean fluorescence intensity (MFI). Line: negative control; filled: specific staining; SSC: side scatter.

 
CXCL2/MIP-2 synthesis occurs in a subpopulation of Gr-1high BM cells
To further characterize and quantify granulocytes capable of CXCL2/MIP-2 protein synthesis, BM cells were subjected to intracellular immunostaining using anti-CXCL2/MIP-2 antiserum in combination with anti-Gr-1 antibodies and subsequent FACS analysis. As shown in Fig. 5(A), CXCL2/MIP-2 protein was only detectable in 25–35% of Gr-1high granulocytes, but not in granulocytes expressing low levels of Gr-1 (Gr-1low) or Gr-1 BM cells (data not shown). With respect to the total number of BM cells, only 4–6% show detectable levels of CXCL2/MIP-2 protein. Interestingly, in vitro stimulation of BM cells with IL-1 and TNF-{alpha} did not change the number of CXCL2/MIP-2+/Gr-1high granulocytes (Fig. 5B). The data therefore indicate that synthesis of CXCL2/MIP-2 is restricted to a subpopulation of Gr-1high granulocytes in BM.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5. Two-color immunofluorescence analysis of CXCL2/MIP-2 expression in BM granulocytes. BM cells from healthy BALB/c mice were analyzed by FACS. Granulocytes were determined with PE-conjugated anti-mouse Gr-1 mAbs. Cells were gated for Gr-1high or Gr-1low BM cells as shown in Fig. 3(A). (A) Cells were stained intracellularly for CXCL2/MIP-2 using rabbit anti-mouse CXCL2/MIP-2 antiserum or with normal rabbit serum (NRS) as control and FITC conjugated secondary antibodies. Data are representative of five separate experiments. (B) BM cells were stimulated in vitro with IL-1 and TNF-{alpha} (100 ng/ml each) simultaneously for 4 h (+ IL-1/TNF) or left untreated (constitutive) prior to immunostaining performed as described in (A). Only Gr-1high cells are displayed. Data are representative of two separate experiments.

 
CXCL2/MIP-2 synthesis is independent of CXCR2 expression
Cell surface expression of CXCR2 was analyzed to further characterize the population of granulocytes CXCL2/MIP-2+/Gr-1high. As revealed by FACS analysis, CXCR2 expression was detectable on ~60% of the Gr-1high cell population (Fig. 6A). However, intracellular immunostaining and FACS analysis using a combination of anti-CXCR2 and anti-CXCL2/MIP-2 antibodies identified <20% of the Gr-1high cells as double positive granulocytes. The majority of the CXCR2+/Gr-1high cells failed to express CXCL2/MIP-2 (Fig. 6B). This suggests that CXCL2/MIP-2 and its receptor CXCR2 are not coordinately regulated at the cellular level.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6. Immunofluorescence analysis of CXCR2 expression in Gr-1high/CXCL2/MIP-2+ BM granulocytes. BM cells from healthy BALB/c mice were subjected to cell surface immunostaining with PE-conjugated anti-mouse Gr-1 mAbs and goat anti-mouse CXCR2 serum or normal goat serum together with Cy5-conjugated secondary antibodies. Cells were subsequently stained intracellularly for CXCL2/MIP-2 using rabbit anti-mouse CXCL2/MIP-2 antiserum or with normal rabbit serum as control and FITC conjugated secondary antibodies. Cells were subjected to FACS analysis and gated for high levels of Gr-1 expression as shown in Fig. 3(A) gate R2. Serum controls were used for positioning the quadrants. (A) CXCR2 expression of Gr-1high BM cells. (B) CXCR2 and CXCL2/MIP-2 expression of Gr-1high BM cells. Data are representative of four separate experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have demonstrated that CXCL2/MIP-2 is constitutively expressed in BM, but absent in other lymphoid organs such as spleen and PP. Furthermore, CXCL2/MIP-2 mRNA expression and protein synthesis in BM is restricted to a subset of BM granulocytes. They are characterized by the expression of cell surface proteins Gr-1high, CD11b+ and CD62Lhigh. This subset of granulocytes accounted for <6% of the total number of BM cells in normal mice (Fig. 5A). Moreover, in vitro stimulation of Gr-1high BM granulocytes with IL-1 and TNF-{alpha} up-regulated CXCL2/MIP-2 mRNA levels but not the number of cells staining positive for CXCL2/MIP-2 protein, indicating a well-defined subpopulation of granulocytes (Figs 3B and 5B). From these data we suggest a model in which granulocytes may be separated into distinct subsets distinguished by their immunomodulatory mediators. It is possible that such subsets of granulocytes could serve different biological functions. This conclusion is supported by studies showing constitutive IL-12 production in a subset of peripheral and peritoneal neutrophils, pointing to a role for granulocytes in directing a TH1 type of immune response (35,36). Additionally, granulocytes are thought to be involved in driving TH2 responses because of their ability to produce IL-4 (37,38). Also, production of IL-10 and interferon-{gamma} (IFN-{gamma}) by granulocytes underscores their immunomodulatory role (39,40). For example, in human neutrophils, MHC class II antigen was shown to be synthesized and expressed following stimulation with IFN-{gamma}. Interestingly enough, MHC class II expression was only induced in a subset of neutrophils (41,42). However, whether there are granulocyte subsets carrying exclusively only one or more specific cytokines or chemokines remains to be explored. To this end, it is unknown whether such subsets originate from distinct granulocyte lineages or represent different maturation or activation stages of granulocytes. CXCL2/MIP-2, IL-12 and IL-6 were found exclusively in mature granulocytes expressing high levels of Gr-1, thus pointing to a developmental regulation of cytokine or chemokine production in granulocytes (Fig. 3B and 5A) (35,43,44).

The biological role of constitutive CXCL2/MIP-2 expression remains ill-defined. For peripheral tissues, CXCL2/MIP-2 expression was observed during embryonic development in the cardiovascular system, lung, bone and gut endoderm and was supposed to recruit leukocytes to infected tissues or to maintain homeostasis of gut epithelium (28). In adult mice CXCL2/MIP-2 mRNA was detected in mouse gall bladder, mammary gland and vaginal tissue (29,45,46). In BM the expression of CXCL2/MIP-2 is likely to contribute to retention of granulocytes in BM and their sequestration from circulation to BM. Consistent with this idea, murine BM granulocytes as well as blood neutrophils were shown to home to BM when infused intravenously in mice (47). In addition, CXCR2-deficient mice were shown to develop neutrophilia when not housed under gnothobiotic conditions, indicating a correlation between CXCR2 expression levels and mobilization of granulocytes from BM (28). CXCR2 expression was found on ~60% of the Gr-1high granulocytes, but only 20% thereof showed additional CXCL2/MIP-2 production (Fig. 6). These data show that expression of either CXCL2/MIP-2 or CXCR2 is independently regulated between granulocyte subsets.

Expression patterns of CXC, CC and C chemokines in peripheral lymphoid tissues as well as BM demonstrated restriction of CXCL1/KC mRNA expression to the periphery while CXCL2/MIP-2 was exclusively found in BM (Figs 1 and 2). The differential expression appeared to be exclusive for CXCL1/KC and CXCL2/MIP-2, because other chemokines, i.e. CXCL10/IP-10, CXCL14/MIP-2{gamma}, XCL1/LTN and CCL3/MIP-1{alpha} were detected in both peripheral tissues as well as BM. Hence, it appears that expression of CXCL2/MIP-2 and CXCL1/KC are independently regulated in response to either granulocyte specific maturational changes and/or local BM/cell specific interactions. It is possible that this dichotomy reflects a cell-type specific expression of either chemokine. However, to this end we were unable to identify the cell type responsible for peripheral constitutive CXCL1/KC production in vivo. Others have shown that fibroblasts are likely candidates (25,48). In vitro, both chemokines were shown to have similar activities to induce granulocyte chemotaxis (11,48,49). In vivo however, data revealed remarkable differences in the biological relevance of either chemokine. On the one hand, there are data showing that CXCL2/MIP-2 and CXCL1/KC were both induced in mouse liver by Fas ligation. Subsequent blocking experiments revealed that granulocyte recruitment to the liver was primarily accomplished by CXCL1/KC (50). On the other hand, a cooperative activity for both chemokines was found in a model of surgical injury, in that CXCL1/KC was detected at very early stages of inflammation, whereas CXCL2/MIP-2 was found to be expressed at later time points and for a longer time period. This suggests a role for CXCL1/KC as an immediate early chemokine (25,51). This function is definitely supported by its constitutive expression in peripheral tissues.

In sum, our results show that CXCL2/MIP-2 and CXCL1/KC are differentially expressed in the BM granulocytes of normal mice and that CXCL2/MIP-2 is produced in a well-defined CD11b+, CD62Lhigh, Gr-1high subset of BM granulocytes, thereby providing a possible explanation for the independent regulation of both chemokines.


    Acknowledgements
 
This work was supported by grants of the Deutsche Forschungsgemeinschaft BE1005/8-2 and the Federal Ministry of Education and Research (BMBF) and the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg. We gratefully acknowledge the expert technical assistance of Claudia Feulner and Anne-Kathrin Lehner. We thank Peter Rohwer (Medizinische Klinik III und Polyklinik, University of Erlangen, Erlangen, Germany) for cell sorting. We thank Nicholas W. Lukacs (Department of Pathology and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI) for kindly providing the rabbit anti-CXCL2/MIP-2 antiserum.


    Abbreviations
 
BM   bone marrow
CCL   CC chemokine
CXCL   CXC chemokine
CXCR   CXC chemokine receptor
IP-10   inducible protein 10
KC   platelet-derived growth factor-inducible chemokine KC
LIX   LPS-induced chemokine
LTN   lymphotactin
MIP   macrophage inflammatory protein
NRS   normal rabbit serum
PBL   peripheral blood leukocytes
PP   Peyer's patches
XCL   XCL chemokine

    Notes
 
Transmitting editor: A. Falus

Received 7 July 2004, accepted 30 August 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Thomas, J., Liu, F. and Link, D. C. 2002. Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor. Curr. Opin. Hematol. 9:183.[CrossRef][ISI][Medline]
  2. Borregaard, N. and Cowland, J. B. 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503.[Free Full Text]
  3. Cassatella, M. A. 1995. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 16:21.[CrossRef][ISI][Medline]
  4. Galligan, C. and Yoshimura, T. 2003. Phenotypic and functional changes of cytokine-activated neutrophils. Chem. Immunol. Allergy 83:24.[Medline]
  5. Muller, G., Hopken, U. E., Stein, H. and Lipp, M. 2002. Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors. J. Leukoc. Biol. 72:1.[Abstract/Free Full Text]
  6. Zlotnik, A. and Yoshie, O. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121.[ISI][Medline]
  7. Murphy, P. M., Baggiolini, M., Charo, I. F., Hebert, C. A., Horuk, R., Matsushima, K., Miller, L. H., Oppenheim, J. J. and Power, C. A. 2000. International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol. Rev. 52:145.[Abstract/Free Full Text]
  8. Schluger, N. W. and Rom, W. N. 1997. Early responses to infection: chemokines as mediators of inflammation. Curr. Opin. Immunol. 9:504.[CrossRef][ISI][Medline]
  9. Rossi, D. and Zlotnik, A. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18:217.[CrossRef][ISI][Medline]
  10. Lukacs, N. W., Hogaboam, C., Campbell, E. and Kunkel, S. L. 1999. Chemokines: function, regulation and alteration of inflammatory responses. Chem. Immunol. 72:102.[Medline]
  11. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565.[CrossRef][ISI][Medline]
  12. Walz, A., Peveri, P., Aschauer, H. and Baggiolini, M. 1987. Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Commun. 149:755.[ISI][Medline]
  13. Wolpe, S. D., Sherry, B., Juers, D., Davatelis, G., Yurt, R. W. and Cerami, A. 1989. Identification and characterization of macrophage inflammatory protein 2. Proc. Natl Acad. Sci. USA 86:612.[Abstract]
  14. Cochran, B. H., Reffel, A. C. and Stiles, C. D. 1983. Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell 33:939.[ISI][Medline]
  15. Bozic, C. R., Gerard, N. P., von Uexkull-Guldenband, C., Kolakowski, L. F., Conklyn, M. J., Breslow, R., Showell, H. J. and Gerard, C. 1994. The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization. J. Biol. Chem. 269:29355.[Abstract/Free Full Text]
  16. Faunce, D. E., Sonoda, K. H. and Stein-Streilein, J. 2001. MIP-2 recruits NKT cells to the spleen during tolerance induction. J. Immunol. 166:313.[Abstract/Free Full Text]
  17. Lee, J., Cacalano, G., Camerato, T., Toy, K., Moore, M. W. and Wood, W. I. 1995. Chemokine binding and activities mediated by the mouse IL-8 receptor. J. Immunol. 155:2158.[Abstract]
  18. McColl, S. R. and Clark-Lewis, I. 1999. Inhibition of murine neutrophil recruitment in vivo by CXC chemokine receptor antagonists. J. Immunol. 163:2829.[Abstract/Free Full Text]
  19. Tannenbaum, C. S., Major, J. A., Poptic, E. J., DiCorleto, P. E. and Hamilton, T. A. 1990. Lipopolysaccharide induces competence genes JE and KC in Balb/C 3T3 cells. J. Cell. Physiol. 144:77.[ISI][Medline]
  20. Huang, S., Paulauskis, J. D. and Kobzik, L. 1992. Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts. Biochem. Biophys. Res. Commun. 184:922.[ISI][Medline]
  21. Zhao, M. Q., Stoler, M. H., Liu, A. N., Wei, B., Soguero, C., Hahn, Y. S. and Enelow, R. I. 2000. Alveolar epithelial cell chemokine expression triggered by antigen-specific cytolytic CD8(+) T cell recognition. J. Clin. Invest. 106:R49.[ISI][Medline]
  22. Mancardi, S., Vecile, E., Dusetti, N., Calvo, E., Stanta, G., Burrone, O. R. and Dobrina, A. 2003. Evidence of CXC, CC and C chemokine production by lymphatic endothelial cells. Immunology 108:523.[ISI][Medline]
  23. Nygardas, P. T., Maatta, J. A. and Hinkkanen, A. E. 2000. Chemokine expression by central nervous system resident cells and infiltrating neutrophils during experimental autoimmune encephalomyelitis in the BALB/c mouse. Eur. J. Immunol. 30:1911.[CrossRef][ISI][Medline]
  24. Biedermann, T., Kneilling, M., Mailhammer, R., Maier, K., Sander, C. A., Kollias, G., Kunkel, S. L., Hultner, L. and Rocken, M. 2000. Mast cells control neutrophil recruitment during T cell-mediated delayed-type hypersensitivity reactions through tumor necrosis factor and macrophage inflammatory protein 2. J. Exp. Med. 192:1441.[Abstract/Free Full Text]
  25. Armstrong, D. A., Major, J. A., Chudyk, A. and Hamilton, T. A. 2004. Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury. J. Leukoc. Biol. 2:2.
  26. Liu, Q., Wang, Y. and Thorlacius, H. 2000. Dexamethasone inhibits tumor necrosis factor-alpha-induced expression of macrophage inflammatory protein-2 and adhesion of neutrophils to endothelial cells. Biochem. Biophys. Res. Commun. 271:364.[CrossRef][ISI][Medline]
  27. Kato, A., Gabay, C., Okaya, T. and Lentsch, A. B. 2002. Specific role of interleukin-1 in hepatic neutrophil recruitment after ischemia/reperfusion. Am. J. Pathol. 161:1797.[Abstract/Free Full Text]
  28. Luan, J., Furuta, Y., Du, J. and Richmond, A. 2001. Developmental expression of two CXC chemokines, MIP-2 and KC and their receptors. Cytokine 14:253.[CrossRef][ISI][Medline]
  29. Savard, C. E., Blinman, T. A., Choi, H. S., Lee, S. K., Pandol, S. J. and Lee, S. P. 2002. Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-alpha by gallbladder epithelial cells: response to bacterial lipopolysaccharides. BMC Gastroenterol. 2:23.[CrossRef][Medline]
  30. Matzer, S. P., Baumann, T., Lukacs, N. W., Röllinghoff, M. and Beuscher, H. U. 2001. Constitutive expression of macrophage-inflammatory protein 2 (MIP-2) mRNA in bone marrow gives rise to peripheral neutrophils with preformed MIP-2 protein. J. Immunol. 167:4635.[Abstract/Free Full Text]
  31. Kasama, T., Strieter, R. M., Lukacs, N. W., Lincoln, P. M., Burdick, M. D. and Kunkel, S. L. 1995. Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis. J. Clin. Invest. 95:2868.[ISI][Medline]
  32. Chomczynski, P. and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem 162:156.[CrossRef][ISI][Medline]
  33. Ajuebor, M. N., Das, A. M., Virag, L., Flower, R. J., Szabo, C. and Perretti, M. 1999. Role of resident peritoneal macrophages and mast cells in chemokine production and neutrophil migration in acute inflammation: evidence for an inhibitory loop involving endogenous IL-10. J. Immunol. 162:1685.[Abstract/Free Full Text]
  34. Luo, Y., Lloyd, C., Gutierrez-Ramos, J. C. and Dorf, M. E. 1999. Chemokine amplification in mesangial cells. J. Immunol. 163:3985.[Abstract/Free Full Text]
  35. Bliss, S. K., Butcher, B. A. and Denkers, E. Y. 2000. Rapid recruitment of neutrophils containing prestored IL-12 during microbial infection. J. Immunol. 165:4515.[Abstract/Free Full Text]
  36. Romani, L., Mencacci, A., Cenci, E., Del Sero, G., Bistoni, F. and Puccetti, P. 1997. An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis. J. Immunol. 158:2356.[Abstract]
  37. Brandt, E., Woerly, G., Younes, A. B., Loiseau, S. and Capron, M. 2000. IL-4 production by human polymorphonuclear neutrophils. J. Leukoc. Biol. 68:125.[Abstract/Free Full Text]
  38. Denkers, E. Y., Del Rio, L. and Bennouna, S. 2003. Neutrophil production of IL-12 and other cytokines during microbial infection. Chem. Immunol. Allergy 83:95.[Medline]
  39. Yeaman, G. R., Collins, J. E., Currie, J. K., Guyre, P. M., Wira, C. R. and Fanger, M. W. 1998. IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J. Immunol. 160:5145.[Abstract/Free Full Text]
  40. Romani, L., Mencacci, A., Cenci, E., Spaccapelo, R., Del Sero, G., Nicoletti, I., Trinchieri, G., Bistoni, F. and Puccetti, P. 1997. Neutrophil production of IL-12 and IL-10 in candidiasis and efficacy of IL-12 therapy in neutropenic mice. J. Immunol. 158:5349.[Abstract]
  41. Gosselin, E. J., Wardwell, K., Rigby, W. F. and Guyre, P. M. 1993. Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/macrophage colony-stimulating factor, IFN-gamma and IL-3. J. Immunol. 151:1482.[Abstract/Free Full Text]
  42. Hansch, G. M. and Wagner, C. 2003. Expression of MHC class II antigen and coreceptor molecules in polymorphonuclear neutrophils. Chem. Immunol. Allergy 83:45.[Medline]
  43. Terebuh, P. D., Otterness, I. G., Strieter, R. M., Lincoln, P. M., Danforth, J. M., Kunkel, S. L. and Chensue, S. W. 1992. Biologic and immunohistochemical analysis of interleukin-6 expression in vivo. Constitutive and induced expression in murine polymorphonuclear and mononuclear phagocytes. Am. J. Pathol. 140:649.[Abstract]
  44. Fleming, T. J., Fleming, M. L. and Malek, T. R. 1993. Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family. J. Immunol. 151:2399.[Abstract/Free Full Text]
  45. Available at: http://www.tigr.org/docs/tigr-scripts/tgi/tc_report.pl?species=mouse&tc=TC1189325
  46. Available at: http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Mm&CID=4979
  47. Suratt, B. T., Young, S. K., Lieber, J., Nick, J. A., Henson, P. M. and Worthen, G. S. 2001. Neutrophil maturation and activation determine anatomic site of clearance from circulation. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L913.[Abstract/Free Full Text]
  48. Bozic, C. R., Kolakowski, L. F., Gerard, N. P., Garcia-Rodriguez, C., von Uexkull-Guldenband, C., Conklyn, M. J., Breslow, R., Showell, H. J. and Gerard, C. 1995. Expression and biologic characterization of the murine chemokine KC. J. Immunol. 154:6048.[Abstract/Free Full Text]
  49. Call, D. R., Nemzek, J. A., Ebong, S. J., Bolgos, G. R., Newcomb, D. E., Wollenberg, G. K. and Remick, D. G. 2001. Differential local and systemic regulation of the murine chemokines KC and MIP2. Shock 15:278.[ISI][Medline]
  50. Faouzi, S., Burckhardt, B. E., Hanson, J. C., Campe, C. B., Schrum, L. W., Rippe, R. A. and Maher, J. J. 2001. Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway. J. Biol. Chem. 276:49077.[Abstract/Free Full Text]
  51. Endlich, B., Armstrong, D., Brodsky, J., Novotny, M. and Hamilton, T. A. 2002. Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury. J. Immunol. 168:3586.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
16/11/1675    most recent
dxh169v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Matzer, S. P.
Articles by Beuscher, H. U.
PubMed
PubMed Citation
Articles by Matzer, S. P.
Articles by Beuscher, H. U.