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*
Institute for Clinical Microbiology, Immunology, and Hygiene, University of Erlangen, Erlangen, Germany; and
Departments of Pathology and Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-1 receptor antagonist (1, 2). Some severe clinical disorders, e.g. inflammatory bowel
disease (3) and glomerulonephritis (4), are
associated with infiltration of PMNs and elevated tissue levels of
proinflammatory cytokines such as IL-1. Because treatment with IL-1R
antagonist reduced the severity of the inflammation in inflammatory
bowel disease (5), these and other studies suggest that
PMN and PMN-derived cytokines could play a role in the pathogenesis of
inflammatory diseases.
Migration and extravascular accumulation of PMNs is a multistep process
and requires a series of regulatory signals that include expression and
activation of adhesion molecules (6) as well as the
generation of chemotactic gradients by cells of the extravascular
compartment (4). Chemotaxis of immune cells is mediated by
a large group of small basic proteins, commonly termed chemokines
(7, 8). They are categorized into four families C, CC,
CXC, and CX3C, based on the position of one or two conserved cysteine
residues in the amino (N) terminus. Migration of PMNs is mediated by
members of the growing CXC chemokine family, including human IL-8
(9)(=CXCL8; Refs. 10, 11), the human
growth-related oncogenes (Gro 
-
/CXCL1–3) and
their murine homologues macrophage-inflammatory protein (MIP) 2/CXCL1
and platelet-derived growth factor-inducible chemokine KC (KC/CXCL1)
(12, 13). The potent neutrophil chemotactic activity of
each of the three chemokines requires the presence of a
glutamate-leucine-arginine (ELR) motif near the N terminus, and
preceding the first conserved cysteine residue. Both MIP-2 and KC were
shown to bind to the murine CXCR2, which is abundantly expressed on
granulocytes and as recently shown on NKT cells (14, 15).
When neutrophils from CXCR2 knockout mice were exposed to MIP-2 or KC,
no migration was observed and specific blocking of CXCR2 using a
chemokine antagonist resulted in inhibition of neutrophil recruitment
in response to TNF, IL-1, or LPS, supporting the view that CXCR2 is the
principal receptor for CXC chemokines on neutrophils (16, 17).
MIP-2 has been shown to be one of the major inducible chemokines with the ability to attract neutrophils to the site of inflammation (18, 19), although other mediators such as C5a need to be considered (20). Expression of MIP-2 mRNA has been observed in response to microbial infection, injection of LPS, and stimulation of cells with proinflammatory mediators such as IL-1 and TNF (21, 22, 23). Several cell types including macrophages (24), epithelial cells (23), bone marrow (BM) endothelial cells (25), astrocytes (26), and mast cells (27) have been identified as cellular sources of MIP-2. In addition, like one of its functional homologues, such as human IL-8, MIP-2 has been suggested to be produced by neutrophils (28, 29), although available data to support this conclusion are rather limited. The concept of MIP-2 production by neutrophils, however, is intriguing because neutrophils are the first cells recruited to sites of infection and the release of chemokines by these cells may have an important impact on the development of early immune responses. For example, the early release of MIP-2 could promote recruitment of more neutrophils or even modulate the type of leukocyte infiltration. In view of these considerations, the present study aimed to characterize the production of MIP-2 in response to an acute bacterial infection. As a model we used the i.p. infection of mice with the Gram-negative Yersinia enterocolitica (30). In humans and rodents, this enteric pathogen causes inflammatory diseases that range from gastroenteritis to ileitis and lymphadenitis. Replication of Y. enterocolitica takes place in lymphoid tissues and depends on the expression of plasmid-encoded, secreted virulence proteins to evade the immune system. The host response to Yersinia infection is characterized by a massive infiltration of granulocytes and monocytes into infected tissues and application of Y. enterocolitica into the peritoneal cavity of mice leads to abscess formation in spleen, liver, and the peritoneal wall. Our results show that Gr-1-positive granulocytes rapidly accumulate in infected spleens and a subpopulation of these cells displays MIP-2 protein. In the absence of an ongoing infection, these MIP-2-positive granulocytes were found in spleens and BM; however, MIP-2 mRNA was only detectable in BM, but not in spleens. These results suggest that MIP-2 is produced by PMNs during maturation in BM and then stored.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The avirulent strain (NCTC 10598), here named O:8-, and the isogenic virulent strain (NCTC 10938), here named O:8+, of Y. enterocolitica O:8 were obtained from the National Collection of Type Cultures (Central Public Health Laboratory, London, U.K.). Bacteria were routinely grown overnight in defined trypton yeast extract glucose medium at 26°C (31). 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 PBS (Biochrom, Berlin, Germany).
Experimental infection of mice
Female BALB/c mice (6–10 wk of age) were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany). Mice were infected i.p. with 1 ml of Yersinia suspension containing 5 x 103 CFU. Mice were sacrificed at different time points (0–6 days) after infection. Spleens, lungs, BM, and blood cells were harvested. For determination of bacterial content, tissues were homogenized in saline containing 0.1% deoxycholic acid (Sigma, Deisenhofen, Germany) using a Potter homogenizer. Appropriate dilutions of the homogenates were plated onto Yersinia-selective agar containing the selective supplement SR 109 (Oxoid, Basingstoke, U.K.). After 2 days of incubation at room temperature, the numbers of CFU were counted.
Cells and cell culture
Peritoneal exudate cells were prepared from peritoneal cavities of mice 3 days after a single i.p. injection of 1 ml of 10% thioglycolate (Difco, Offenbach, Germany) and cultured in 6-well-plates at a density of 4 x 106 cells/well (21). The culture medium Clicks/RPMI 1640 was supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (Biochrom), and 10% FCS (Sigma). After 3 h of incubation (37°C, 5% CO2, 95% humidity), nonadherent cells were washed off. Subsequently, the peritoneal macrophages were stimulated for 2 h with LPS (10 µg/ml) or with Y. enterocolitica O:8+ at a multiplicity of infection (MOI) of 10.
Casein-elicited granulocytes were prepared from peritoneal cavities of mice 3 h after a single i.p. injection of 2 ml of 0.2% casein (Sigma) (32) and cultured in 6-well plates as described for preparation of peritoneal exudate cells. After 4 h of incubation (37°C, 5% CO2, 95% humidity), the granulocyte containing nonadherent cell fraction was recovered. Subsequently, granulocytes were incubated in 12-well plates at a density of 1 x 106 cells/well with Y. enterocolitica O:8+ at a MOI of 10. After 1 h of incubation, the cell-free supernatants were sterile filtered and subjected to a MIP-2 ELISA.
Single-cell suspensions of spleens were prepared as described previously (31). Briefly, spleens were excised and gently passed through a net. BM cells were obtained by flushing femurs and tibiae with culture medium. Contaminating erythrocytes were removed by suspending the splenic and BM cells in NH4Cl solution (168 mM) for 10 min at room temperature.
To obtain peripheral blood leukocytes (PBL), heparinized blood was diluted at a ratio of 2:1 with PBS and applied onto a NIM-2 two-step density gradient (Cardinal Associates, Santa Fe, NM) according to the manufacturer’s instructions to gently remove erythrocytes. After centrifugation (1000 x g, 25 min, 20°C) cells were collected from both the upper and interphase of the gradient, washed with PBS, and resuspended in culture medium. In some experiments, BM was treated similarly. BM-derived granulocytes were prepared using the same procedure as described for blood leukocytes, except that granulocytes were collected only from the interphase.
To prepare cytospin preparations, single-cell suspensions of spleens, BM, and PBL were adjusted to a density of 1 x 106 cells/ml and placed onto microscopic slides by centrifugation (21 x g, 2 min) in a cytocentrifuge (Shandon, Pittsburgh, PA). The slides were dried overnight at room temperature and fixed in acetone (10 min, -20°C) for subsequent immunostaining.
FACS analysis
Splenic single-cell suspensions (1 x 106 cells/ml) were preincubated with Fc-Block (1.25 µg/106 cells; BD Biosciences, San Jose, CA) for 10 min followed by PE-conjugated rat anti-murine Gr-1 Ab (clone RB6-8C5; BD Biosciences). The Gr-1 (Ly-6G) Ag is expressed on granulocytes including neutrophils (PMNs) as well as eosinophils although the neutrophils represent the vast majority of Gr-1-positive cells in the periphery. A further discrimination between neutrophils and eosinophils was not approached because the neutrophil-specific mAb 7/4 (Serotec, Oxford, U.K.) does not react with neutrophils from BALB/c mice sufficiently as described by the manufacturer (33). After incubation for 45 min, cells were thoroughly washed with PBS/1% FCS. All steps were conducted at 4°C. Samples were analyzed by flow cytometry using a FACSCalibur and CellQuest software (BD Biosciences).
Immunohistochemistry
Preparation of frozen tissue sections and immunoperoxidase staining were performed as described elsewhere (34). Briefly, cryostat sections (5 µm) and cytospin preparations were incubated overnight with appropriate dilutions of either rat anti-murine Gr-1 Ab (clone RB6-8C5; BD Biosciences) or rabbit anti-murine MIP-2 Ab purchased from PeproTech (London, U.K.) or prepared as described previously (35). To block staining, recombinant MIP-2 (100 µg/ml; PeproTech) was preincubated with rabbit anti-murine MIP-2 for 8 h. After thoroughly washing, the slides were incubated with biotin-conjugated secondary stage Abs: mouse anti-rat IgG and donkey anti-rabbit IgG (Dianova, Hamburg, Germany). Streptavidin-biotinylated HRP kits were used according to the suppliers’ recommendations (DAKO, Hamburg, Germany) and the peroxidase was visualized with 3-amino-9-ethylcarbazole (Sigma). Nuclei were stained using Meyer’s hematoxylin (Dr. K. Hollborn und Söhne, Leipzig, Germany). For immunofluorescence, double-staining tissue sections and cytospin preparations were blocked in PBS containing 0.1% saponin (Sigma) and 20% FCS and incubated simultaneously with both primary Abs described above. After extensive washing with PBS/0.1% Tween 20, FITC-conjugated donkey anti-rabbit IgG (BD Biosciences) and Rhodamine Red X-conjugated donkey anti-rat IgG (Dianova) secondary Abs were applied for 45 min at room temperature. Cytospin preparations were additionally counterstained with the DNA stain 4',6'-diamidine-2'-phenylindole dihydrochloride (DAPI, 1 µg/ml PBS; Boehringer Mannheim, Mannheim, Germany). Slides were mounted in Mowiol (Hoechst, Frankfurt, Germany) containing 1,4-diazabicyclo[2,2,2]octane (Sigma) as an antifading reagent. Cells and tissue sections were examined by using an Axiophot microscope (Zeiss, Oberkochen, Germany) equipped with appropriate filters to gate FITC, Rhodamine Red X and DAPI fluorescence. Photographs were acquired and processed using a Spot camera and Metaview software (Diagnostic Instruments, Burroughs, MI).
ELISA
MIP-2 protein levels in serum, spleen, or cell culture supernatants were quantitated using the murine Quantikine M MIP-2 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Spleens were excised, immediately snap frozen in liquid nitrogen, and stored at -70°C until homogenization in PBS containing complete protease inhibitor mixture (Roche Diagnostic Systems, Mannheim, Germany) using a Potter homogenizer. ELISA results from splenic homogenates were normalized to sample weight to correct for differences in sample size.
RNase protection assay
Total RNA was prepared from frozen tissues or single-cell
suspensions by using the guanidine-thiocyanate extraction procedure
followed by acid-phenol extraction (36). RNase protection
assays were performed using RiboQuant assay kits purchased from BD
Biosciences according to the manufacturer’s instructions. Briefly,
radiolabeled antisense RNA probes were generated from the murine cDNA
template sets mCK-5 and mCR-6 (containing lymphotactin/XCL1,
RANTES/CCL5, eotaxin/CCL11, MIP-1/CCL4, MIP-1/CCL3, MIP-2,
IFN--inducible protein 10 (IP-10)T/CXCL10, monocyte chemoattractant
protein 1 (MCP-1)/CCL2, T cell activation gene 3 (TCA-3)/CCL1, and
CXCR2, CXCR4, Burkitt lymphoma receptor 1/CXCR5, respectively) and
hybridized to target RNA. ssRNA was digested with RNase and protected
probes were separated on 5% polyacrylamide sequencing gels. Gels were
dried and visualized by autoradiography or using a phosphoimager (Fuji,
Elmsford, NY). Quantification was performed using AIDA software (Ray
Test, Straubenhardt, Germany).
Reverse transcription and semiquantitative PCR
The cDNA was synthesized from 2 µg of total RNA using 2.5 µM
oligo(dT)12–18, 1 mM dNTP, 12 U RNAguard
(Amersham Pharmacia Upjohn, Freiburg, Germany), 200 U Superscript II
reverse transcriptase, 80 mM DTT (Life Technologies, Karlsruhe,
Germany), and 1 mM MgCl2 (Peqlab, Erlangen,
Germany) to a final volume of 25 µl at 42°C for 60 min. For MIP-2
and -actin-specific PCR, equal amounts of sample cDNA were amplified
in 25-µl reaction volumes containing 1 mM dNTP (Amersham Pharmacia
Upjohn), 1 U Taq DNA polymerase (Peqlab), and 20 µM
primers during 35 cycles (30 s denaturation, 94°C; 30 s
annealing, 58°C for -actin, 55°C for MIP-2; 30 s
polymerization, 72°C) with an Omnigene temperature cycler (Hybaid,
Ashford, U.K.) to allow quantitative considerations. The primers
for -actin and MIP-2 were purchased from Amersham Pharmacia Upjohn
and MWG Biotech (Ebersberg, Germany), respectively, and were as
follows: -actin: sense primer, 5'-CACCCGCCACCAGTTCGCCA-3';
antisense primer, 3'-CAGGTCCCGGCCAGCCAGGT-5' (amplified fragment 574
bp); MIP-2: sense primer, 5'-CCACTCTCAAGGGCGGTCAA-3'; antisense
primer, 3'-CCCCTTATCCCCAGTCTCTTTCAC-5' (amplified fragment 515
bp). PCR samples were separated on 1% agarose gels and visualized by
ethidium bromide staining and photographed on an ImageMaster VDS
(Amersham Pharmacia Upjohn).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the kinetics of induction of chemokine genes, mice
were challenged i.p. with virulent Y. enterocolitica
O:8+, and total RNA was prepared from spleens and
lungs on days 0–6 postinfection. RNase protection assays were used to
compare mRNA levels of lymphotactin, RANTES, eotaxin, MIP-1,
MIP-1, MIP-2, IP-10, MCP-1, and TCA3. As shown in Fig. 1
A, in the spleen low levels
of transcripts were detectable for eotaxin, MIP-1, MIP-1, IP-10,
and MCP-1 from days 2 to 6. Constitutive mRNA expression was only found
for RANTES, the levels of which slightly increased by day 2. In
comparison, only MIP-2 mRNA, though not detectable until day 2,
preferentially increased during infection, i.e., days 4–6. Similar
results were obtained when analyzing the expression of MIP-2 in lungs
(Fig. 1A). In comparison, MIP-2 mRNA was not found in
spleens of mice challenged with the avirulent strain Y.
enterocolitica O:8- (Fig. 1B),
indicating that MIP-2 expression is associated with the disease. To
further assess the potency of Yersiniae as stimulants of
chemokine expression, macrophage cultures were incubated in parallel
with virulent bacteria or LPS. Subsequent RNase protection assays of
total RNA revealed both stimuli as equally potent for up-regulation of
RANTES, MIP-1, MIP-1, MIP-2, IP-10, and MCP-1 mRNA expression. No
transcripts were detected for eotaxin and TCA3 (Fig. 1C).
The data show that the pattern of chemokine mRNA induction is much more
restricted during infection of mice as compared with infection of
macrophages in tissue culture.
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To correlate MIP-2 expression with leukocyte migration, we next
assessed the kinetics of appearance of Gr-1-positive granulocytes
(granulocytesGr-1+) in
Yersinia-infected spleens. Results (Fig. 2A) obtained by flow
cytometric analysis show that the relative numbers of granulocytes
gradually increased over time, reaching a plateau on days 5–6 after
infection. In addition, RNase protection assays were performed with
total splenic mRNA to determine the expression of CXCR2, the
established neutrophil receptor for MIP-2. As compared with mRNA levels
from uninfected mice, there was a 24-fold increase in CXCR2 mRNA on day
6 after infection (Fig. 2B). Also, bacterial burden
increased over time such that by days 4 and 5 postinfection a 100-fold
excess of the inoculated CFU could be recovered from infected spleens
(Fig. 2C). The data indicate that recruitment of leukocytes
into infected spleens is temporally coordinated with the appearance of
MIP-2 mRNA shown in Fig. 1
A.
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To determine whether Yersinia infection causes the
release of MIP-2, systemic as well as local MIP-2 protein levels were
determined. Therefore, the amount of MIP-2 protein was measured using
an ELISA. Serum and splenic tissue samples of infected and uninfected
mice were collected 6 days after infection. The data summarized in Fig. 3 show that infection caused elevated
levels of MIP-2 in serum as well as in splenic homogenates.
Interestingly, the levels of MIP-2 in those specimen correlated with
the relative number of bacteria (Fig. 3C). The data
therefore suggest that the induction of MIP-2 synthesis relates to the
severity of the infection.
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To define the cellular sources of MIP-2, consecutive
tissue sections of infected spleens were subjected to immunoperoxidase
staining. The anti-Gr-1 Ab identified a discrete abscess area (Fig. 4A) in which only a few cells
stained positive with the anti-MIP-2 antiserum (Fig. 4B). Immunofluorescence double staining using
anti-MIP-2-FITC (green) and anti-Gr-1-Rhodamine Red X (red)
confirmed that MIP-2 colocalized with
granulocytesGr-1+ (yellow), indicating that MIP-2
is produced by a subset of recruited
granulocytesGr-1+ (Fig. 4C).
Apparently no other cell types were identified to synthesize MIP-2. In
controls, immunostaining for MIP-2 was blocked by an excess (100
µg/ml) of recombinant MIP-2 (data not shown), thus confirming
the specificity of MIP-2 immunodetection. Finally, immunoperoxidase
staining (Fig. 4D) and immunofluorescence double staining
(Fig. 4E) revealed MIP-2 protein in
granulocytesGr-1+ of spleens of uninfected mice
as well. However, when counting MIP-2-positive granulocytes
(granulocytesGr-1+/MIP-2+) in splenic cell
suspensions of control and diseased mice, it became evident that
infection with Y. enterocolitica caused a 4-fold increase in
the number of granulocytesGr-1+/MIP-2+. These
data argue for a production of MIP-2 by newly
recruitedgranulocytesGr-1+, but also
raise the issue of a constitutive MIP-2 expression.
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To address the discrepancy between detection of MIP-2 protein
(Fig. 4) and lack of MIP-2 mRNA (Fig. 1A) in spleens of
uninfected mice, expression of MIP-2 mRNA was analyzed by PCR. No MIP-2
mRNA was found in spleens (Fig. 5A), but it was readily
detectable in BM (Fig. 5B). Immunofluorescence double
staining of BM cytospin preparations further demonstrated that MIP-2
protein colocalized with granulocytesGr-1+ (Fig. 5C). In addition, morphological analysis revealed MIP-2
staining in two cell forms with either ring-shaped nuclei, namely,
PMN-like ring cells (Fig. 5E) or PMN/neutrophils (Fig. 5D). The data indicate that MIP-2 gene expression occurs
constitutively in BM and further imply that MIP-2 can exist in
peripheral granulocytesGr-1+ as a prestored
chemokine in the absence of detectable MIP-2 mRNA transcription.
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Because PMN are normally short-lived, the fate of
granulocytesGr-1+/MIP-2+ was analyzed in response
to infection with Y. enterocolitica. In control
mice, high numbers of
granulocytesGr-1+/MIP-2+ were found in BM,
concomitant with low numbers in peripheral blood and spleens. During
infection, however, the amount of
granulocytesGr-1+/MIP-2+ markedly decreased in BM
(Fig. 6A). The loss of
granulocytesGr-1+/MIP-2+ in BM of infected mice
was specific in that no significant decrease was observed in the total
number of granulocytesGr-1+ (Fig. 6B).
The data indicate that mobilization of leukocytes during infection
yields a selective depletion of
granulocytesGr-1+/MIP-2+ from BM. In
addition, data summarized in Table I show that Yersinia infection
produced a change in the proportion of
granulocytesGr-1+/MIP-2+ in BM such that the
relative amount of
PMN/neutrophilsMIP-2+ increased, while there was
a decrease of PMN-like ring cellsMIP-2+. The data
argue in favor of a subset of granulocytes committed for MIP-2
production.
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To gain insights into the regulatory mechanisms of MIP-2
production, primary cultures of BM were stimulated with IL-1 and TNF.
For each stimulus, one set of cultures was analyzed for MIP-2 mRNA
expression by PCR and a second set was used for analysis of MIP-2
protein synthesis by immunostaining. Both cytokines were found to
strongly increase the relative amounts of MIP-2 mRNA as compared with
untreated cultures (Fig. 7A).
However, none of the two cytokines was able to significantly change the
numbers of cells with detectable MIP-2 protein after 2 or 24 h of
exposition (Fig. 7B), suggesting that transcription and
translation of MIP-2 are independently regulated. Alternatively, it is
possible that MIP-2 mRNA is increased in cells already committed for
MIP-2 production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B) and
synthesis of MIP-2 protein in BM was associated with
granulocytesGr-1+ (Fig. 5C). Second,
in the absence of MIP-2 mRNA (Fig. 5A), the MIP-2 protein
was found in splenic granulocytesGr-1+ of
uninfected mice (Fig. 4E) as well as in circulating blood
leukocytes (data not shown). Similar to our results with MIP-2, IL-6
protein was previously found in circulating murine PMN devoid of IL-6
mRNA, while at the same time expression of IL-6 mRNA was readily
detectable in BM cells (37). In addition, murine
neutrophils have recently been shown to contain intracellular stores of
IL-12 (38). The data point to a common regulatory
mechanism through which intracellular cytokines are acquired as part of
the normal developmental program of neutrophils.
As determined by immunofluorescence microscopy, detection of MIP-2
strictly correlated with the cell surface marker Gr-1, which is
primarily expressed on mature granulocytes (39). In BM,
45% of all cells were recognized by the Gr-1 Ab, whereas 15% of
the cells stained positive for both Gr-1 and MIP-2. This suggests that
within the population of granulocytesGr-1+ only a
subset of cells is committed to produce MIP-2. In addition, a
morphological classification based on the criteria provided by Biermann
et al. (40) indicates that
granulocytesGr-1+/MIP-2+ can be subdivided into
two subsets, namely, PMN-like ring cells and PMN/neutrophils (Table I).
One characteristic of PMN maturation includes the acquisition of
granules and granule products (41). Hence, a likely
explanation for the existence of subsets of
granulocytesGr-1+/MIP-2+ are differences in
timing of granule protein synthesis during maturation of PMN
precursors. In cell culture experiments as well as in animals, other
cell types such as macrophages, epithelial cells (23), and
mast cells (27) have been described as a source of MIP-2.
In control experiments, we observed also that anti-MIP-2 antisera
recognized MIP-2 in LPS-stimulated macrophages (data not shown),
consistent with the detection of MIP-2 mRNA expression (Fig. 1).
However, throughout the entire study, immunostaining of tissue
specimens from infected mice as well as control mice failed to
specifically identify MIP-2-synthesizing cells other than
granulocytesGr-1+. It cannot be excluded,
however, that the levels of MIP-2 protein are increased in
granulocytesGr-1+ due to its storage in granules,
which in turn may facilitate immunorecognition of neutrophil-associated
MIP-2.
Infection of mice with Y. enterocolitica caused a
preferential accumulation of MIP-2 mRNA in infected tissue, as compared
with mRNA levels of other chemokines such as MIP-1, MIP-1,
RANTES, MCP-1, and IP-10 (Fig. 1). In contrast, no such preferential
induction of MIP-2 mRNA was observed when macrophage cultures were
infected with Y. enterocolitica. Instead, each of the
macrophage-derived chemokines was induced equally well by either LPS or
the bacteria. This suggests that expression of MIP-2 in
Yersinia infection is selective. Underlying regulatory
mechanisms may be provided by the release of proinflammatory mediators
which like TNF have the potential to increase MIP-2 mRNA expression
(27), whereas IL-10, one of the most potent
anti-inflammatory agents, has been shown to suppress chemokine gene
expression (21, 42). Alternatively, we propose that
accumulation of MIP-2 mRNA at sites of infection is largely regulated
through the influx of newly recruited neutrophils retaining the ability
to express MIP-2 mRNA. Experimental support for this comes from kinetic
experiments showing a temporal correlation between appearance of MIP-2
mRNA and neutrophil recruitment. Moreover, evidence that neutrophils
display the ability to up-regulate MIP-2 expression in response to
proinflammatory mediators comes from experiments showing that purified
BM-derived granulocytes can be stimulated with TNF to increase MIP-2
mRNA (Fig. 7C). The constitutive expression of MIP-2 mRNA
and protein found in BM-granulocytes (Figs. 7 and Fig. 5C)
does not seem to be due to constitutive TNF expression in the BM,
because granulocytesGr-1+/MIP-2+ were found in BM
of TNF-deficient mice (S. P. Matzer and H. U. Beuscher,
unpublished data).
In conclusion, the data of this study identify neutrophils as a major source of the chemokine MIP-2. They contribute to the concept of neutrophils as rapidly mobilized regulators of antimicrobial host responses (1) through preformed inflammatory as well as anti-inflammatory cytokines. Synthesis and release of preformed MIP-2 by neutrophils may have several biological implications: 1) peripheral neutrophils containing the preformed MIP-2 may act as alarm cells and regulate the early influx of PMNs to sites of infection and 2) in the setting of an ongoing acute inflammatory process neutrophil-derived MIP-2 may serve to amplify and perpetuate the recruitment of leukocytes from the circulation.
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Acknowledgments |
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Footnotes |
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2 S.P.M. and T.B. contributed equally to this study.
3 Address correspondence and reprint requests to Prof. Dr. H. Ulrich Beuscher, Institute for Clinical Microbiology, Immunology, and Hygiene, University of Erlangen, Wasserturmstrasse 3, 91054 Erlangen, Germany. E-mail address: beuscher{at}mikrobio.med.uni-erlangen.de
4 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; MIP, macrophage-inflammatory protein; BM, bone marrow; MOI, multiplicity of infection; DAPI, 4-diamidino-2-phenylindol-di-hypochloride; MCP-1, monocyte chemoattractant protein 1; IP-10, IFN--inducible protein 10; TCA-3, T cell activation gene 3; PBL, peripheral blood leukocyte.
Received for publication April 27, 2001. Accepted for publication August 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 176:593.. J. Immunol. 161:1204.. J. Immunol. 159:3595.[Abstract]
(IL-1) to IL-1 production during maturation of inflammatory macrophages in vivo. J. Exp. Med. 175:1793.This article has been cited by other articles:
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  S. A. Handley, P. H. Dube, and V. L. Miller From the Cover: Histamine signaling through the H2 receptor in the Peyer's patch is important for controlling Yersinia enterocolitica infection PNAS, June 13, 2006; 103(24): 9268 - 9273. [Abstract] [Full Text] [PDF]
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 S. Bennouna and E. Y. Denkers Microbial Antigen Triggers Rapid Mobilization of TNF-{alpha} to the Surface of Mouse Neutrophils Transforming Them into Inducers of High-Level Dendritic Cell TNF-{alpha} Production J. Immunol., April 15, 2005; 174(8): 4845 - 4851. [Abstract] [Full Text] [PDF]
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 S. P. Matzer, F. Rodel, R. M. Strieter, M. Rollinghoff, and H. U. Beuscher Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes Int. Immunol., November 1, 2004; 16(11): 1675 - 1683. [Abstract] [Full Text] [PDF]
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), PBLs (
), and spleen cells (SC; 
) were
subjected to immunofluorescence double staining using anti-MIP-2 in
combination with anti-Gr-1 Abs. To determine the total cell count,
slides were counterstained with DAPI. Results show the number of
Gr-1-expressing cells (B) and MIP-2-positive
granulocytesGr-1+ (A) in percentage of the
number of all cells counted. At least 400 cells were counted for each
specimen; two separate experiments were performed. Numbers are
mean ± SEM.