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Regular Articles |
From the Departments of Pathology and Laboratory
Medicine*
and Pulmonary
Medicine,
Veterans Affairs Medical Center, Ann
Arbor, Michigan; the Department of Pathology,
University of Michigan Hospitals, Ann Arbor, Michigan; and the
Gladstone Institute of Cardiovascular Disease,§
University of California, San Francisco, California
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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mRNA. The latter was possibly related to decreased CD4+ T cell
recruitment. Regionally, draining lymph nodes showed
panlymphoid hyperplasia with impaired production of IFN,
IL-2, and IL-4, but not IL-5, IL-10, or
IL-13. Analysis of procollagen gene expression indicated transient
impairment of procollagen III transcripts on day 4 of granuloma
formation. These findings indicate that agonists of CCR2 contribute to
multiple facets of type-2 hypersensitivity granulomatous
inflammation.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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As a potent chemoattractant for mononuclear phagocytes, MCP-1 may
potentially recruit macrophages during chronic inflammation and indeed
a number of reports seem to support this notion.4-10
We
have previously shown that T-cell-mediated, hypersensitivity-type
granuloma formation can be classified on the basis of cytokine
participation into type-1/Th1 dominant, and type-2/Th2
dominant.11
More recently, we examined type-1 mycobacterial
Ag-elicited granuloma formation in mice with targeted knockout of the
CCR2 receptor and showed a transient defect in local macrophage
recruitment as well as a defect in regional IFN
production.10
That study indicated that CCR2 was involved
at least temporally in local macrophage recruitment and regional
T cell maturational events during a Th1 response. In a previous study
using anti-MCP-1 antibodies, we showed that MCP-1 also appears to
participate in Th2 (type-2) cytokine-mediated granulomas at both local
and regional levels.7
Subsequent studies using MCP-1
knockout mice showed similar results using Schistosoma
mansoni egg challenge.9
In the present study, we
performed a detailed analysis of local, regional, and systemic
parameters during synchronized type-2 granuloma formation in mice
targeted for knockout of the CCR2 receptor. The results indicate a
broad role for CCR2 agonists in hypersensitivity-type granuloma
formation, ranging from chemotaxis to regulation of matrix synthesis.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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CCR2 knockout mice were generated from 129 strain embryonic stem cells using targeting vectors as previously described.10 Control animals consisted of age-matched nonmutant 129 X B6 F1 mice. Mice were maintained in isolator cages under specific pathogen-free conditions and provided with food and water ad libitum.
Sensitization and Granuloma Induction
Pulmonary granulomas with predominantly type-2 cytokine involvement were generated as described.11 Briefly, mice were sensitized by i.p. injection of 3000 S. mansoni eggs suspended in 0.5 ml phosphate-buffered saline (PBS). Fourteen to 16 days later, sensitized mice were challenged by i.v. with 6000 Sepharose 4B beads (in 0.5 ml PBS) covalently coupled with soluble schistosome egg antigens (SEA) obtained from the World Health Organization (Geneva, Switzerland).
Granuloma Dispersal and Draining Lymph Node Culture
Groups of mice were killed at days 1, 2, 4, and 8 of granuloma formation. Following perfusion with cold RPMI, lungs (excluding trachea and major bronchi) were excised. The right upper lung of each mouse was snap-frozen in liquid N2 for mRNA isolation. The left lower lobe was post-inflated and formalin-fixed. The remaining lung lobes were placed in cold RPMI medium; granulomas were then isolated and dispersed as previously described.7,11 For differential counting, duplicate cytospin preparations were prepared from the remaining dispersed granuloma cells and stained with Wright's stain. Blood was also collected from each animal and total white cell count and differential analysis performed.
Mediastinal lymph nodes were collected at the time of lung harvest and teased into single cell suspension. After washing, the cells were cultured in RPMI-1640 medium (JRH Biosciences, Lenexa, KS) containing 10% FBS (Intergen, Purchase, NY), 10 mmol/L glutamine, and 100 mg/ml streptomycin and 100 U/ml penicillin (RPMI-FBS) at 5 x 106/ml in the presence or absence of 5 µg/ml SEA, then cultured as above for 24 hours. Supernates were collected by centrifugation and stored at -45°C.
Granuloma Measurement
Granulomas were measured blindly from formalin-inflated lungs that were paraffin-embedded, sectioned, and stained with hematoxylin and eosin. Granuloma area was measured by computerized morphometry. A minimum of 20 lesions was measured per lung.
Cell Identification by Flow Cytometry
Dispersed granulomas and mediastinal lymph node suspensions were subjected to flow cytometry using two- and three-color fluorescent analysis. Anti-CD3, -CD4, -CD8, -CD14, -CD19, and Mac-3 fluorescent-labeled antibodies were obtained commercially (Pharmingen, San Diego, CA). Samples were stained and analyzed immediately using a FACSCAN (Becton-Dickinson, Franklin Lakes, NJ) and WinList analysis program as described previously.12
RNA Extraction
Using a modified method of Chirgwin et al13 and Jonas et al,14 total cellular RNA was extracted from perfused lung lobes excluding major bronchi that had been snap-frozen with liquid N2. The frozen tissues were suspended in extraction buffer (25 mmol/L Tris, pH 8.0, 4.2 mol/L guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 mol/L 2-mercaptoethanol), homogenized, then added to an equal volume of extraction buffer (100 mmol/L Tris, pH 8.0, 10 mmol/L EDTA and 1% sodium dodecyl sulfate (SDS). The mixture was then serially extracted with chloroform-phenol and chloroform-isoamyl alcohol. The RNA is next precipitated at -70°C in ethyl alcohol, washed, and reprecipitated. The pellet was finally dissolved in DEPC water and RNA concentrations determined spectrophotometrically prior to storage at -70°C.
Primers and Probes
Primers and probes (18-22 mer) were designed based upon nucleotide
sequences downloaded from the NCBI data bank and using primer design
software (Premier Biosoft International, Palo Alto, CA). Designed
primer and probe sequences for each of the target RNA species examined
are shown in Table 1
. All primers and
probes were prepared by Genosys Biotechnologies Inc. (The Woodlands,
TX). Probes were biotinylated with biotin-UTP using a standard 3'-end
labeling kit (Boehringer Mannheim, Indianapolis, IN), unincorporated
biotin was removed with QuickSpin columns (Boehringer Mannheim).
Incorporation was confirmed by nitrocellulose blotting followed by
streptavidin-alkaline phosphatase detection.
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The isolated RNA was first reverse transcribed (RT) to DNA as follows. To 25 µg of RNA (in 25 µl of DEPC water) were added 3.6 µl of RNAsin (Boehringer Mannheim) and 10 µl of random hexamer solution (500 mg/ml, Promega, Madison, WI) followed by heating to 70°C for 5 minutes in a thermocycler (9600, Perkin-Elmer Corp., Norwalk, CT). The temperature was then reduced to 43°C and 69 µl of a first-strand buffer (Gibco BRL, Grand Island, NY) containing dTT, dNTPs, and 1000 U murine Moloney leukemia virus reverse transcriptase was added.15 The mixture was incubated for 2 hours, then heated to 95°C to stop the reaction. The DNA was then subjected to PCR.16 Briefly, 5 µl of DNA were added to 95 µl PCR buffer containing unlabeled dNTPs (0.2 mmol/L of each) plus digoxigenin-labeled dUTP, 1 µg sense primer, 1 µg antisense primer, and 5 U Taq polymerase (all from Boehringer Mannheim) in a thin-walled PCR tube. Amplification was performed in a thermocycler as follows: 4 minutes at 95°C, followed by up to 23 cycles of 1 minute at 95°C, 2 minutes at 57°C, and 1 minute at 72°C. After cycling there was a DNA extension period of 6 minutes at 72°C. Samples were stored at -20°C prior to analysis.
Detection of PCR products was performed as follows. Initially a series of amplification reactions using unlabeled dNTPs was performed and the products analyzed by standard agarose gel electrophoresis to confirm that primers yielded predicted products. Once confirmed, la- beled products were generated and detected by PCR-ELISA.17 Briefly, 10–60 µl of amplified product were added to a sterile microfuge tube containing 40 µl of denaturing buffer (Boehringer Mannheim) and incubated for 10 minutes. Next, 500 µl of hybridization buffer containing 4 ng/ml of appropriate (target gene) biotinylated probe was added. Negative controls included tubes with no DNA or DNA with inappropriate probe. The solution was mixed and 200-µl portions were distributed into duplicate wells of a multiwell, streptavidin-coated plate and incubated for 3 hours at 42°C. The plate was then washed and any bound product detected with peroxidase labeled anti-digoxigenin Ab by standard colorimetric reaction using 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate. A 96-well plate ELISA reader was used to measure O.D. at 405 nm. The O.D. was directly proportional to levels of target PCR product, which was normalized to levels of a housekeeping gene, cyclophilin.
Cytokine Measurement
Interleukins 2, 4, 5, 10, and 13 and interferon- were measured
by ELISA using commercially available reagents (Pharmingen; R&D
Systems, Minneapolis, MN); sensitivities ranged from 10 to 50 pg/ml.
Commercially available recombinant murine cytokines served as standards
in all assays (Genzyme, Cambridge, MA; Preprotech Inc., Rocky Hill, NJ;
R&D Systems).
Statistics
Student's t-test (two-tailed) was used to compare control with experimental groups. Values of P > 0.05 were considered to indicate lack of significance.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In order to determine the effect of CCR2 knockout on
leukocyte mobilization, we assessed blood leukocyte differentials and
granuloma size and composition during type-2 granuloma formation. As
shown in Figure 1
, the day 0 baseline
levels of blood leukocyte populations were comparable among groups.
Thereafter, granuloma induction caused peripheral increases in
monocytes, eosinophils, and neutrophils in control mice. Of these
populations, the percentage of circulating monocytes in knockout mice
was reduced by 50–70% during the 8-day study period. This decrease
was absolute and relatively specific to monocytes; total leukocyte
counts were comparable or lower in knockout mice (data not shown).
Despite the persistent decrease in circulating monocytes, granuloma
sizes were only transiently impaired on days 1 and 2 with the mean area
being reduced to near that of the bead diameter on day 1 and reaching
only 30% of controls on day 2 (Figures 2 and 3)
. Analysis of lesion composition
revealed that this impairment was due to a 50% reduction in granuloma
macrophages (Figure 4)
. This effect was
selective in that there was no reduction in lymphocytes, eosinophils,
or neutrophils. By day 4, both granuloma size and composition had
returned to control levels. Although not totally specific for
macrophages, CD14 and Mac-3 markers were evaluated by flow cytometry.
Cells coexpressing these markers were likewise reduced during the early
recruitment stage (Figure 5)
. Thus, CCR2
deficiency was associated with re-duced circulating monocytes and
transient monocyte/macrophage recruitment defect in the early phase of
granuloma formation.
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In order to explore potential compensatory changes in
knockout mice, we assessed granulomatous lungs for mRNA specific for
MCP-1/JE and other CCR2 chemokine agonists, MCP-3 and MCP-5. Message
levels were determined on days 2, 4, and 8 of granuloma formation using
semiquantitative RT-PCR ELISA assay. As shown in Figure 6
, wild-type transcript expression for
MCP-1/JE and MCP-3 was generally 1.5- to 2-fold above granuloma-free
lungs. Message for MCP-5 was less pronounced, ranging from 30–40%
above noninflamed lungs. In all cases, CCR2 knockout caused profound
augmentations. This was especially notable for MCP-1/JE and MCP-3,
which ranged from 3- to 7-fold above levels in unchallenged
lungs on days 4 and 8. Increases were seen on all days for MCP-5
transcripts. These findings suggested that CCR2 participates in a
feedback inhibition of chemokine synthesis.
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In order to determine if CCR2 knockout influenced the local
expression of cytokines, we compared levels of IFN and IL-4
transcripts in lungs with type-2 granulomas. Figure 7
shows the expression of these
transcripts and those for CCR2. As expected, knockout mice showed no
expression of CCR2 mRNA. Interestingly, among the control groups there
was increasing CCR2 transcript expression during the response,
especially on days 4 and 8. With regard to IFN mRNA, wild-type mice
showed about two-fold increases on days 4 and 8. Consistent with the
type-2 response, wild-type mice also showed strong IL-4 expression over
the study period, ranging from two- to three-fold above granuloma-free
control mice. Knockout mice displayed definite alterations in these
patterns. On day 2, IFN message was enhanced in knockout mice,
possibly reflecting the reduced component of macrophages. The latter
may contribute RNA species that dilute IFN mRNA or produce factors
that inhibit IFN synthesis by lymphocytes. By day 4, IFN
transcripts declined by 40%, but recovered to control levels by day 8.
In contrast, CCR2 deficiency showed greater effect on IL-4 message.
Initially on day 2, IL-4 mRNA was comparable, but then declined by
about 40–50% among knockout mice on days 4 and 8. Thus, CCR2 and
presumably its ligands regulated levels of local cytokine production or
numbers of cytokine-producing cells.
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In order to detect changes in lymphocyte recruitment, flow
cytometric analysis of granuloma lymphocyte populations was performed.
Figure 8
summarizes the gated analysis of
granuloma lymphocytes. A significant component of CD19+ B cells
appeared in granulomas, ranging from 20–30% of lymphocytes (upper
panel). This population was comparable between control and knockout
mice over the study period. The bulk of the remaining lymphocytes were
CD3+ T cells. A subpopulation analysis of CD3+CD4+ and CD3+CD8+ cells
showed a small but significant reduction in the percentage of CD4+ on
day 4 with a corresponding increase in CD8+ cells (middle and lower
panels). It is unclear if this was due to impaired CD4+ T cell
expansion or increased CD8+ T cell recruitment.
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We and others have reported that chemokines can modulate cytokine
production by lymphocytes.18-20
In addition, we
recently demonstrated that draining lymph node cells of CCR2-/- mice
display defective IFN production during the course of type-1 (Th1)
granuloma formation.10
In a similar fashion, we assessed
cytokine profiles in CCR2 knockout during type-2 (Th2) granuloma
formation. Table 2
shows spontaneous and
Ag-elicited levels of IFN and interleukins 2, 4, 5, and 13 in lymph
node cultures. In accord with a Th2 response, the wild-type profile was
dominated by type-2 related cytokines IL-4, IL-5, IL-10, and IL-13,
which were produced in greatest amounts on days 4 and 8. With regard to
IFN- and IL2, the CCR2 knockout mice displayed virtually complete
impairment, especially after day 2, similar to our finding in the
type-1 response.10
Interestingly, partial impairment
also extended to spontaneous and elicited IL-4 production
throughout the study period. Some impairment of spontaneous IL-5 and
IL-13 was also seen on day 4. In contrast, Ag-elicited IL-5 and IL-13
were initially enhanced on day 2 and thereafter were
statistically no different than controls.
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We next examined potential effects of CCR2 deficiency on the
resolution or healing phase of granuloma formation. In view of studies
showing that MCP-1/JE stimulates collagen gene
expression,21
we analyzed procollagens I and III
transcripts by RT-PCR-ELISA. As shown in Figure 10
, wild-type mice
displayed increased mRNA for both procollagen I and III on days 4 and 8
of granuloma formation. In contrast, CCR2-deficient mice did not
display increased expression until day 8, by which time levels were
comparable to those of control animals. The impairment on day 4 was
statistically significant for procollagen III and was observed as a
trend for procollagen I. Thus, the effect of CCR2 deficiency extended
to the resolution phase of granuloma formation by delaying the onset of
maximal collagen synthesis.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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With regard to local cellular mobilization, our synchronized model of granulomatous inflammation revealed that CCR2 knockout mice display impaired mononuclear phagocyte recruitment only in the early phase of granuloma formation suggesting that MCP-1 has a restricted window of chemotactic function. This was likewise observed for the type-1 mycobacterial lesion.10 The eventual recovery of macrophage recruitment by day 4 suggests that other chemotactic factors and receptors must supplant the function of CCR2. Indeed, we demonstrated increased transcripts for MCP-3 that can utilize receptors other than CR2 and potentially compensate for MCP-1.23 We previously reported that treatment with a polyclonal anti-MCP-1 antibody preparation abrogated type-2 granuloma size with the effect persisting until day 4.7 The seemingly greater potency of the Ab was possibly due to nonspecificity, since this preparation has subsequently been shown to cross-react with MCP-3 and its effect would not necessarily be undermined by CCR2 deficiency. However, increases in other promiscuous chemokines could compensate for MCP-1. Because MCP-3 transcripts were readily demonstrable, it could be among a spectrum of chemokines that supplant or compensate for MCP-1 and MCP-5.23,24 Compensatory factors, however, could not overcome the early macrophage recruitment defect. In an analogous fashion, mice with disruption of the eotaxin gene displayed only early phase impairment of eosinophil recruitment, whereas later secondary recruitment was unaffected.25
Interestingly, the apparently compensated lesion on day 4 did undergo changes in T cell subpopulations resulting in a decreased CD4/CD8 T cell ratio. Because MCP-1/JE is reportedly chemotactic for Th cells, this effect may have been caused by impaired CD4+ T cell recruitment.26 Alternatively, the early macrophage recruitment defect may have resulted in downstream changes in T cell populations. Local macrophages may be required to support proliferation or produce secondary waves of T cell chemotactic signals. Based upon our observations to date, we hypothesize that local recruitment during the initiation, mobilization, sustenance, and resolution stages of chronic inflammation may be mediated by successive waves of chemotactic mediators. Efforts are presently underway in our laboratory to test this hypothesis.
Although CCR2 was transiently required for local macrophage recruitment, its absence resulted in a chronic partial monocytopenia in the peripheral blood. As previously reported, CCR2 deficiency does not cause myelosuppression,10 so MCP-1 presumably promotes monocyte production or the release of monocytes from the marrow during an inflammatory stress. It should be emphasized that the observed impairment was consistently about 50% and ultimately did not prevent granulomas from attaining maximal cellularity. The potential role of MCP-1 in regulating peripheral blood monocytosis merits further investigation.
Our finding that CCR2 knockout mice display altered cytokine production
in draining lymph nodes and granulomas provides further support for the
notion that chemokines influence lymphoid maturational or activation
events. As reported for the mycobacterial response,10
IFN and IL-2 were notably abrogated in lymph node cultures of
knockout mice and this effect extended to IL-4 in the type-2 response.
The latter is consistent with the effect of anti-MCP-1 Ab
treatment7
and MCP-1 knockout.9
The partial
IL-4 abrogation caused by CCR2 deficiency did not impair type-2
granuloma formation after day 2 nor the production of other Ag-elicited
Th2-related cytokines such as IL-5 and IL-13. However, it was noted
that spontaneous production of IL-5 and IL-13 was reduced on day 4,
suggesting a subtle but ineffectual defect. This is not surprising,
because alternative sources of IL-4 or compensatory mechanisms can
support the type-2 responses.24,27
In addition, it is known
that endogenous IFN limits S. mansoni egg Ag-induced
inflammation and cytokines production by means of cross-regulatory
inhibition.28,29
Therefore, any concurrent abrogation of
IFN might be predicted to offset impairment conferred by decreased
levels of IL-4. The overall increased cell proliferation in knockout
lymph nodes likely reflects the absence of IFN-mediated
cross-regulation of the Th2 response.28
It was of interest
that overall reductions of IFN mRNA in granulomatous lungs were less
pronounced than protein levels in lymph node cultures. At present, we
have no definitive explanation. The relationship between mRNA levels
and protein secretion remains unclear and it is possible that cytokine
producing cells are enriched at sites of granuloma formation.
In our previous study,7
anti-MCP-1 Ab treatment caused
greater abrogation of Th2 cytokines than we observed in CCR2 knockout
mice. This observation has subsequently been repeated with new, highly
specific anti-murine MCP-1/JE Abs (data not shown). Therefore, the
disparity likely reflects different experimental conditions. This is an
especially important consideration because congenital CCR2 deficiency
likely influences both induction and elicitation phases of immunity.
Notably, our findings in CCR2 knockout mice agree well with in
vitro studies showing that MCP-1 promotes both IL-4 and IFN
during primary and secondary lymphocyte stimulation in
vitro.19
Thus, CCR2 potentially contributes to both
sensitization and elicitation phases of immunity and its absence could
impair both IFN and IL-4. However, as we observed, compensatory
mechanisms ultimately established a CCR2/MCP-1-independent response.
Our previous Ab-mediated depletion study examined the elicitation phase
of the type-2 response which, not having been deprived of CCR2-mediated
stimulation during induction, likely will have a greater MCP-1/JE
dependence and sensitivity to anti-MCP-1/JE treatment. In any case,
both MCP-1 depletion and CCR2 knockout studies point to a role for
MCP-1/JE in the generation or activity of IFN- and IL-4-producing
lymphoid cells.
As mentioned above, MCP-1/JE is a known lymphocyte chemoattractant26,30 and it is possible that local cytokine defects of CCR2 knockout mice are related to this chemotactic function. Studies by Qin et al suggest that CCR2 is more densely expressed on CD26+ (activated/memory) than naive T cells.31,32 If this is the case, then our findings may reflect memory T cells with suboptimal homing to granulomas. In addition, because chemokines positively regulate T cell adhesion molecules33,34 then CCR2 deficiency could result in impaired T cell migration events. Adhesion defects might also compromise T cell stimulation. Specifically, MCP-1 is reported to enhance ß1 integrin binding to fibronectin,35 which not only promotes T cell migration but is also known to be a potent costimulatory factor.36,37 Reduced integrin-mediated stimulation could result in partial impairments of T cell activation, maturation, and cytokine production.
Finally, our finding that granulomatous lungs of CCR2 knockout mice have delayed onset of procollagen transcripts suggested that agonists of this receptor participate in the healing or resolution phase of granuloma formation. The eventual recovery of procollagen transcripts on day 8 suggests that the CCR2 defect is overcome or supplanted by other mediators. This may be a simple indirect effect of the early impairment of macrophage and lymphocyte recruitment slowing the efficiency of a variety of downstream events that support collagen synthesis. However, our finding is also consistent with the report of Gharee-Kermani et al demonstrating that MCP-1 is a direct mediator of collagen gene expression.21 We have consistently noted more severe fibrosis in resolving type-2 lesions, which express greater levels of MCP-1/JE than type-1 lesions.7 Studies by Hogaboam et al indicate that fibroblasts of type-2 lesions express CCR2, MCP-1/JE, and procollagen mRNA in response to IL-4 (manuscript submitted). Such a clustered expression implies that IL-4 may induce an autocrine feedback circuit among type-2 fibroblasts that promotes collagen synthesis.
In conclusion, analysis of type-2, T-cell-mediated hypersensitivity granuloma formation in CCR2 knockout mice suggests that MCP-1/JE participates at multiple stages and facets of this chronic immunoinflammatory response. Specifically, we have demonstrated contribution to monocyte mobilization to peripheral blood, early phase macrophage recruitment, cytokine production, and procollagen gene expression. Thus, chemokines appear to have a broad role in T-cell-mediated inflammation.
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Footnotes |
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Dr. Curtis is a career investigator of the American Lung Association of Michigan.
Address to Stephen W. Chensue, M.D., Ph.D., Pathology and Laboratory Medicine 113, Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105. E-mail: schensue@umich.edu.
Accepted for publication January 21, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and interleukin-4 knockout mice: Analysis of local and regional cytokine and chemokine networks. J Immunol 1997, 159:3565–3573
-IFN, IL-4, and IL-10 in schistosome egg granuloma formation: in vivo regulation of Th activity and inflammation. Clin Exp Immunol 1994, 98:395-400[Medline]
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T. R. Traynor, A. C. Herring, M. E. Dorf, W. A. Kuziel, G. B. Toews, and G. B. Huffnagle Differential Roles of CC Chemokine Ligand 2/Monocyte Chemotactic Protein-1 and CCR2 in the Development of T1 Immunity J. Immunol., May 1, 2002; 168(9): 4659 - 4666. [Abstract] [Full Text] [PDF]
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Z. Zhu, B. Ma, T. Zheng, R. J. Homer, C. G. Lee, I. F. Charo, P. Noble, and J. A. Elias IL-13-Induced Chemokine Responses in the Lung: Role of CCR2 in the Pathogenesis of IL-13-Induced Inflammation and Remodeling J. Immunol., March 15, 2002; 168(6): 2953 - 2962. [Abstract] [Full Text] [PDF]
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M. K. Park, K. F. Hoffmann, A. W. Cheever, D. Amichay, T. A. Wynn, and J. M. Farber Patterns of Chemokine Expression in Models of Schistosoma mansoni Inflammation and Infection Reveal Relationships between Type 1 and Type 2 Responses and Chemokines In Vivo Infect. Immun., November 1, 2001; 69(11): 6755 - 6768. [Abstract] [Full Text] [PDF]
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B. P. Chen, W. A. Kuziel, and T. E. Lane |
