By
From the * Department of Adult Oncology, Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes,
memory T lymphocytes, and natural killer cells. Because other chemokines have similar target
cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been
uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1-deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and
resident macrophages, MCP-1 Chemokines are low molecular weight secreted proteins that play a variety of roles in intercellular signaling (1). Most chemokines exert their effects on leukocytes
and were first purified on the basis of their ability to attract
specific leukocyte subsets in vitro. For example, monocyte
chemoattractant protein 1 (MCP-1)1 was identified as a
monocyte-specific chemoattractant (2) that was later shown
to attract memory T lymphocytes and NK cells (5). Because of its target cell specificity, MCP-1 was postulated to
play a pathogenetic role in a variety of diseases characterized by mononuclear cell infiltration, including atherosclerosis, rheumatoid arthritis, and multiple sclerosis (8).
Support for MCP-1's importance in the physiology of inflammation comes from demonstrations in transgenic mice
that it functions as a monocyte chemoattractant in vivo
(12).
However, MCP-1's role may be neither essential nor
unique because of the potential for functional redundancy.
Among the known CC chemokines, MCP-1, MCP-2,
MCP-3, MCP-4, MCP-5, macrophage inflammatory protein
(MIP)-1 Targeted Disruption of MCP-1.
Perlmutter
Laboratory, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115; the § Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48105; and
the ¶ Trudeau Institute, Saranac Lake, New York 12983
Abstract
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes
in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni
eggs was blunted in MCP-1
/
mice, as was expression of IL-4, IL-5, and interferon
in splenocytes. In contrast, MCP-1
/
mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for
monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
, MIP-1
, I309, and HCC-1, all have monocyte
chemoattractant activity in vitro. Furthermore, monocytes
express at least three cloned CC chemokine receptors, namely
CCR1, CCR2, and CCR5, and even though MCP-1 binds
only CCR2 with high affinity, CCR2 also binds MCP-3
and MCP-5 (16). With this profusion of monocyte-active chemokines and monocyte-expressed receptors, as well
as ligand-receptor promiscuity in vitro, it can be legitimately asked whether a single chemokine such as MCP-1
could have an essential effect in inflammatory disease. Antibody neutralization experiments indicate that MCP-1 might
play a unique role in models of granuloma formation and pulmonary inflammation (19, 20). However, these studies
suffer from the usual shortcomings of antibody specificity
or secondary effects of exogenously administered antibody.
Therefore, to address the question of whether MCP-1 plays
a unique role in inflammation in vivo, we constructed an
MCP-1-deficient mouse by targeted gene disruption.
Analysis of this mouse indicates that despite functional redundancy with other chemokines in vitro, MCP-1 alone is
responsible for mononuclear cell infiltration in several inflammatory models in vivo.
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
portion of the gene was cloned
into the NotI-XhoI sites of pPNT (23). To modify the 5
portion of the gene, the genomic fragment was digested with NaeI
and PmlI (in the coding region of exon 1) and religated with an
NheI linker to create a small deletion and an in-frame stop
codon. The modified 5
portion was then ligated into the XbaI
site of pPNT to yield the targeting construct pJEKO-9, diagrammed in Fig. 1 A(ii).
View larger version (35K):
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Fig. 1.
(A i) Wild-type SCYA2 locus. The three exons encoding
MCP-1 are shown as hatched boxes; the positions of BamHI and SstI restriction endonuclease sites are indicated in relation to the 5 BamHI site; the NaeI-HpaI fragment used as a probe in Southern blotting is indicated
as the open box. (ii) Targeting construct indicating the transcriptional orientation of the PGK-neo cassette inserted in the second exon of MCP-1.
Asterisk denotes the site of an in-frame stop codon engineered in the first
exon (see Materials and Methods). (iii) Disrupted allele. (B) Southern blot
analysis of wild-type and MCP-1-deficient mice. DNA was extracted
from tails of wild-type mice (+/+), and mice heterozygous (+/
) or
homozygous (
/
) for the disrupted MCP-1 allele. DNA was digested
using SstI and analyzed by Southern blotting using the probe indicated in
A. (C) Chemokine expression in wild-type and MCP-1-deficient mice.
Peritoneal macrophages from wild-type mice (+/+), and mice heterozygous (+/
) or homozygous (
/
) for the disrupted MCP-1 allele were
treated with LPS and radiolabeled using [35S]methionine as described in
Materials and Methods. Conditioned medium was analyzed by immune
precipitation using anti-MCP-1 antiserum and the precipitates were analyzed by SDS-PAGE (left gel). The supernatants from the anti-MCP-1
precipitation were then subjected to immune precipitation using anti-
murine GRO-
/KC, and these precipitates were analyzed by SDS-PAGE (middle gel). In a separate experiment, conditioned medium was
analyzed by immune precipitation using anti-murine MCP-3 antiserum
(right gel). Arrow indicates position of MCP-3. A small amount of cross-reactivity with MCP-1 can be discerned at ~30 kD in supernatants from
wild-type macrophages.
Immune Precipitation.
To activate peritoneal macrophages, mice were administered 1.5 ml of 4% thioglycollate broth intraperitoneally. After 72 h, cells were harvested by peritoneal lavage with 5 ml cold HBSS with 10 U/ml heparin, and plated in RPMI 1640 medium supplemented with 10% bovine calf serum. After 2 h, nonadherent cells were removed by washing and adherent cells were stimulated with 10 µg/ml LPS (Sigma Chemical Co., St. Louis, MO) for 4 h. Cells were radiolabeled with 2 mCi/ml [35S]methionine (NEN-DuPont, Boston, MA) in the presence of 10 µg/ml LPS for an additional 4 h. Medium was collected, diluted with an equal volume of RIPA buffer, and precleared with normal rabbit serum and protein A-Sepharose beads (Bio-Rad Laboratories, Hercules, CA). Immune precipitations were then performed sequentially with anti-murine MCP-1 (21) and anti-KC (27). Anti-MCP-3 (28) (a gift from R. Bravo, Bristol-Myers Squibb, Princeton, NJ) was used in a separate experiment. Similar results were obtained without thioglycollate treatment, although the levels of chemokine synthesis were much lower in wild-type and MCP-1Thioglycollate Challenge.
8-9-wk-old male mice were administered 1 ml of 4% thioglycollate broth intraperitoneally, and 72 h later were killed by CO2 asphyxiation. Cells were recovered by peritoneal lavage and counted using a hemocytometer. Cells were applied to microscope slides using a cytospin centrifuge, stained with Diff-Quik (Baxter Healthcare Corp., McGaw Park, IL), and differential counts were obtained by morphological analysis.Contact Hypersensitivity.
8-12-wk-old wild-type and MCP-1Tuberculin-type Hypersensitivity.
8-12-wk-old wild-type and MCP-1Schistosoma mansoni Egg-induced Pulmonary Granulomata.
Mice were sensitized by intraperitoneal injection of 3,000 viable Schistosoma mansoni eggs. 2 wk later, synchronous pulmonary granulomata were induced by intravenous injection of 3,000 viable eggs as previously described (20). At 2, 4, and 8 d after embolization, lungs were inflated with formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin, and granuloma area was measured by an investigator blinded to treatment group using a computerized morphometer (The Morphometer; Woods Hole Educational Associates, Woods Hole, MA).Cytokine Production by Splenocytes.
Mice were sensitized by injection with Schistosome egg antigen (SEA). 2 wk later, splenocytes were isolated and cultured in RPMI 1640 with 10% fetal calf serum alone or with 10 µg/ml SEA. 24 h later medium was collected and concentrations of IL-2, IFN-Infection with Mycobacterium tuberculosis.
Mice were injected intravenously with 105 CFU of the H37Rv strain of Mycobacterium tuberculosis. Four wild-type and four MCP-1 ![]() |
Results and Discussion |
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Fig. 1 A describes the gene targeting strategy for MCP-1. The frequency of homologous recombination in embryonic stem (ES) cells was ~3% and resulted in an MCP-1 allele that was disrupted by a neomycin resistance cassette in the second exon (in a transcriptional orientation opposite to that of MCP-1) and a linker with an in-frame stop codon in the first exon. Two targeted ES clones were injected into blastocysts, but only one resulted in a live birth after transfer to foster mothers. This chimera passed the disrupted allele to its progeny which were intercrossed to produce mice homozygous for the disrupted allele (Fig. 1 B) in the expected Mendelian proportion.
To test whether MCP-1 expression had been disrupted,
peritoneal macrophages were isolated from wild-type mice
and from mice heterozygous or homozygous for the disrupted
allele. Fig. 1 C shows that LPS-stimulated macrophages
from wild-type mice secreted MCP-1 protein, a 25-35 kD
microheterogeneous glycoprotein (21, 29), while macrophages from homozygous mice secreted no detectable MCP-1. Macrophages from heterozygous mice secreted intermediate amounts. The absence of MCP-1 secretion by
macrophages from homozygous mice was not due to absence of peritoneal macrophages since the resident macrophage population was similar in number to wild-type mice (see below). In addition, stimulated macrophages from
homozygous mice secreted wild-type amounts of other
chemokines such as GRO-/KC and MCP-3 (Fig. 1 C),
as well as MIP-1
(data not shown).
Litter size
and sex distribution of MCP-1/
mice were indistinguishable from wild-type mice. MCP-1
/
mice developed normally and had the same life span as wild-type mice.
Their hematologic profiles were also similar. In addition, MCP-1-deficient mice had normal numbers of Küpffer
cells and alveolar macrophages as determined by immunohistochemical staining with F4/80 (data not shown).
In response to intraperitoneal thioglycollate instillation, wild-type mice develop peritonitis that, after 72 h, consists primarily of monocytes and macrophages. Fig. 2 shows that before challenge, peritoneal lavage of wild-type and MCP-1- deficient mice recovered ~5 × 106 cells/mouse, of which ~95% were macrophages. This indicates that, similar to resident macrophage populations in liver and lung, MCP-1 is not required for the establishment of resident peritoneal cells.
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72 h after thioglycollate administration, wild-type mice
were observed to have a sixfold increase in the number of
cells in their peritoneal cavities (Fig. 2). These cells consisted of a small number of neutrophils and eosinophils elicited over days 1-3, but most of the increase was due to
monocytes and macrophages. In contrast, MCP-1/
mice
experienced only a doubling of total intraperitoneal cell number due to an increase in neutrophils and eosinophils
that was statistically similar to the increase seen in wild-type
mice. However, the MCP-1
/
mice showed essentially
no recruitment of monocytes or macrophages to their peritonea.
In this model of peritonitis, both neutrophil and monocyte accumulation depend on the expression of selectins
(30, 31), 2 integrins (32), LFA-1 (33, 34), intracellular adhesion molecule-1 (ICAM-1) (35), and platelet-endothelial
cell adhesion molecule-1 (PECAM-1) (36). Although the
absence of elicited monocytes in MCP-1
/
mice could be
due to deficient adhesion molecule expression, elicitation of
neutrophils and eosinophils in numbers identical to those in
wild-type mice suggest that adhesion molecule expression was intact. We did not test for monocyte accumulation after 72 h, and it is possible that other chemokines might compensate for MCP-1 at later time points. However, at 72 h,
when maximal monocyte recruitment normally occurs,
monocyte accumulation clearly depends on MCP-1.
To test the role
of MCP-1 in a contact hypersensitivity response, mice
were sensitized with DNFB, then challenged by application of the hapten on the skin of the ear. Fig. 3 A shows
that sensitized wild-type and MCP-1/
mice experienced
the same increase in ear swelling compared to nonsensitized
mice of matched genotype. Similarly, in a tuberculin-type delayed-type hypersensitivity (DTH) model, sensitized mice
were challenged by footpad injection of NP-O-Su. Again,
as shown in Fig. 3 B, sensitized wild-type and MCP-1
/
mice had the same amount of footpad swelling compared
to nonsensitized mice. These results indicate that MCP-1 is
not required for the component of the DTH response that
produces edema.
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However, examination of infiltrates in contact hypersensitivity responses showed that the proportion of F4/80+
cells in the lesions of MCP-1/
mice was decreased threefold compared to wild-type mice (Fig. 3 C). Furthermore,
while the total number of elicited cells per unit area appeared to be lower in MCP-1
/
mice, the number of
neutrophils appeared to be the same (data not shown).
Thus MCP-1 is required for eliciting a full complement of
mononuclear cells in a DTH response but not for generating edema, suggesting that other cell types, such as neutrophils, may be responsible for this manifestation of DTH
(37). This may be particularly relevant in the mouse where
neutrophils comprise a much larger proportion of the infiltrate compared to humans. A similar dissociation between
cell recruitment and vascular leak has been observed in
P-selectin-deficient mice (38).
The presence of DTH-associated swelling in MCP-1/
mice indicates that sensitization occurred without MCP-1.
This may mean that MCP-1 is not necessary for activation
of dendritic cells or for their migration into draining lymph
nodes, which is a notable finding because of the evidence
from some laboratories that MCP-1 can attract dendritic
cells (15, 39). Our data suggest that this property of MCP-1
may not be relevant in the two models of DTH used in this
study.
To examine MCP-1's
role in a different hypersensitivity system, we challenged
sensitized mice by intravenous administration of Schistosoma
mansoni eggs to elicit the synchronous formation of secondary granulomata around eggs embolized to the pulmonary vasculature. It had been previously demonstrated that administration of anti-MCP-1 antibodies reduced the size of
secondary pulmonary granulomata in this model by ~40%
(20). Consistent with that finding, Fig. 4 shows that the size
of secondary granulomata 8 d after challenge in MCP-1/
mice was also ~40% smaller than granulomata in wild-type
mice, thereby genetically confirming the importance of
MCP-1 in this process.
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Secondary granulomatous responses have been suggested
to be predominantly controlled by Th2 cells (40). To
examine the effect of MCP-1 on the development of T
helper cells in this model, splenocytes from sensitized mice
were tested for cytokine secretion in response to SEA in
vitro. As shown in Table 1, IL-4 and IL-5 production were
significantly reduced in MCP-1/
splenocytes compared
to wild-type splenocytes. However, IFN-
secretion was
also 59% lower in MCP-1
/
mice (but with a P value of
only 0.08), whereas IL-2 and IL-10 expression were unchanged (as were the proliferative responses of splenocytes
to SEA [data not shown]). Thus the absence of MCP-1 altered patterns of cytokine expression in sensitized mice, although the defect was not restricted to Th2 cytokines.
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It remains to be determined whether this phenotype reflects a direct influence of MCP-1 on Th development (43)
or an indirect influence of other abnormalities that may be
present in MCP-1/
mice. For example, NK cells are a
likely source of IFN-
in splenocytes of sensitized mice (44),
and there may be fewer NK cells in MCP-1
/
spleens. It
is not inconceivable that MCP-1 has important influences on the basal population of lymphoid tissues.
The preceding experiments demonstrated that lack of MCP-1 causes deficiencies in cellular recruitment to DTH and granulomatous lesions. This raised the question of whether MCP-1 also
plays a role in systemic inflammatory challenges. To test
this idea, we intravenously inoculated mice with 105 CFU
of the virulent Mycobacterium tuberculosis strain H37Rv. At
various times after challenge, CFU in lung, liver, and spleen were counted. Fig. 5 shows that MCP-1/
mice cleared
organisms from spleen and lung slightly less efficiently than
wild-type mice at early time points, but by 80 days after inoculation the two genotypes were indistinguishable. Thus
macrophages in these organs are capable of suppressing infection by this systemically administered pathogen even in
the absence of MCP-1.
|
It is possible that this resistance to mycobacteria reflects
intact Th1-like responses in MCP-1/
mice. This interpretation would be consistent with results from an experimental model in which intravenously injected SEA-coupled beads, but not purified protein derivative (PPD)-coupled
beads, elicited MCP-1 expression in pulmonary granulomata,
and anti-MCP-1 treatment decreased the size of the SEA-induced, but not the PPD-induced, granulomata (45). These
results differ from responses in transgenic mice expressing
MCP-1 under the control of the mouse mammary tumor virus LTR (46). Those mice had high serum levels of
MCP-1 and were deficient in clearing intravenously administered mycobacteria. Thus, persistent ambient MCP-1 may
cause defects in macrophage function that are distinct from
those caused by absence of MCP-1. For example, high serum levels of MCP-1 may downregulate another receptor
on monocytes in addition to CCR2.
However, in vitro ligand binding experiments suggest
that MCP-1's sole cloned receptor is CCR2 (an early assignment of MCP-1 to CCR4 [47] has not been reliably
reproduced [48]). Recently, two models of CCR2-deficient mice constructed by gene targeting have been described, and both share several features with the MCP-1-
deficient model reported here, including a selective defect in macrophage elicitation in response to intraperitoneal thioglycollate (49, 50). This suggests that CCR2 is probably
MCP-1's sole receptor in vivo as well. Interestingly, one of
these models (49) demonstrated a relatively selective defect
in Th1 responses, which differs from suggestions using antibody neutralization or TCR transgenic T cells in vitro that
MCP-1 influences Th2 responses (43, 45). However, those
CCR2-deficient mice also showed defects in IL-4 and IL-5
secretion as well as IFN- secretion, similar to our mice.
This indicates, again, that MCP-1's role as an immunoregulatory molecule is complex and not restricted to type 1 or
type 2 helper T cells. When examined in vivo, MCP-1's
influence on acquired immunity may be due to its attraction of specific leukocyte subsets to secondary lymphatic
organs rather than a direct effect on T lymphocyte differentiation.
In summary, the major finding from our work concerns
chemokine redundancy. In spite of the existence of many
CC chemokines that attract monocytes in vitro, our data
show that loss of MCP-1 alone is sufficient to impair monocyte trafficking in several different models at the times we
examined. In addition, absence of MCP-1 results in profound alterations in cytokine secretion by splenocytes sensitized to SEA. This suggests that chemokine redundancy as
defined by receptor binding in vitro may not be relevant in
vivo, where specificity is achieved by timing and levels of
expression. For example, while many CC chemokines can attract monocytes, only MCP-1 appears to be expressed at
high levels in the peritoneum in response to thioglycollate.
Thus, strategies designed to disrupt a single chemokine-
receptor pair may be beneficial. This will depend on demonstrating an essential role for a specific chemokine in disease models, an approach that is now feasible using MCP-1/
mice.
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Footnotes |
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Received for publication Received for publication 14 October 1997 and in revised form 2 December 1997..
The authors thank Dr. Arlene Sharpe for blastocyst injections; Dr. Abul Abbas for helpful comments and encouragement; Drs. Rolf Freter and Charles Stiles for the 5 ![]() |
References |
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