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Departments of Pathology and
*
Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
Department of Pathology, Veterans Administration, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 were significantly
reduced in the purified protein derivative Th1-type response, whereas
IL-10 and IL-13 were up-regulated in the schistosome egg Ag Th2-type
response. The regulation of the immune response was further examined by
using the MCP-1 adenovirus at later time points during the elicitation
phase. When MCP-1 was overexpressed during the elicitation phase of the
responses, neither the Th1-type nor the Th2-type granuloma was altered.
Likewise, the cytokine profiles after restimulation of splenocytes ex
vivo were unchanged. Thus, the function of MCP-1 may depend on the
stage and type of immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and C-C (ß). This group of
chemoattractants continues to grow at a staggering pace through
broad-based searches for sequence homology in expressed sequence tag
(EST) databases. However, as this effort continues, the function of all
of these members must be characterized to determine their individual
role in the inflamed and noninflamed environment. In addition to the
continuously growing family of chemokines, their receptors are only now
being identified through the cloning and characterization of numerous
"orphan" receptors. Recent data from a number of laboratories have begun to identify novel mechanisms of immune regulation by CC family chemokines. The recruitment, regulation, and activation of CD4+ T lymphocytes and their cytokine production may be the most critical issue for alteration of immune responses (1, 6, 7, 8, 9, 10, 11, 12, 13). Several studies have begun investigating whether chemokines can direct the immune system toward a specific response. In previous experiments, we have found that monocyte chemoattractant protein (MCP)3-1 can have a regulatory role on the immune system, both by altering an established immune response and by initiation of oral tolerance (14, 15, 16, 17, 18). In the present study, we have further examined the ability of MCP-1 to alter a purified protein derivative (PPD)-induced Th1-type and a schistosome egg Ag (SEA)-induced Th2-type granulomatous reactions (17, 19) by overexpressing the protein during the afferent vs efferent phase of the immune response. Previous studies have demonstrated that MCP-1 had no apparent effect on the developing lesion during the efferent phase of the response, whereas CC chemokine receptor 2 (CCR2) -/- mice (the MCP-1 receptor) had a significantly altered response during the development of both Th1- and Th2-type lesions (20, 21). The results from the present study lend further evidence that MCP-1 can alter immune responses, depending on the time of MCP-1 transgene expression and the type of response, and appear to regulate or enhance the developing response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female CBA/J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained under standard pathogen-free conditions.
Induction of Th1- and Th2-type pulmonary granuloma
Th1- and Th2-type pulmonary granulomas were generated as described elsewhere (17, 19). In brief, mice were immunized i.p. with 20 µg of Mycobacteria bovis (Sigma, St. Louis, MO) for Th1-type or 5000 isolated Schistosome mansoni eggs for Th2-type responses. Immunized mice were challenged 10 days later by an i.v. infusion of 5000 Sepharose 4B beads covalently coupled to PPD or 3000 isolated S. mansoni eggs, respectively. Four days after the challenge, the mice were anesthetized and euthanized. The lungs were inflated and fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and 4-µm-thin sections were stained with hematoxylin and eosin. The granuloma lesions (a minimum of 20 lesions) were randomly chosen by a "blinded" observer, and captured with NIH image (a public domain image processing and analysis program for Macintosh). The area was converted from pixels (2) to µm2 by measuring a known area on a hemocytometer grid and expressed as mean granuloma area/lung.
Overxpression of MCP-1 in the development of pulmonary granuloma
To express increased quantities of MCP-1 in vivo, a recombinant adenovirus coding for murine MCP-1 (AdmMCP-1) was employed. The cDNA for murine MCP-1 was inserted into the E1 region of Ad5 containing human CMV immediate early promoter and the SV40 polyadenylation signal. Transfection of this construct in vitro resulted in the production of biologically active MCP-1 into the culture supernatant (data not shown). To determine the immunomodulating capability of MCP-1 in the development of pulmonary granuloma, the mice were treated i.p. with either 5 x 108 or 5 x 109 PFU of AdmMCP-1 at two different phases: sensitization or elicitation phase. For injecting AdmMCP-1 during the sensitization phase, AdmMCP-1 was given i.p. 1 day before sensitization with PPD or S. mansoni eggs. In case of injecting in the elicitation phase, AdmMCP-1 was given i.p. 1 day before Ag challenge. As a control, the same titer of replication-deficient, E1-deleted Ad5 construct was used.
Spleen cell cytokine production
Spleens were collected at the time of lung harvest and dispersed into single-cell suspensions. RBC were lysed with 0.1 N NH4Cl, and the spleen cells were washed three times with RPMI 1640. The cells (1 x 106/ml) were then cultured in RPMI 1640 containing 10% FCS, 10 mM glutamine, and antibiotics at 37°C in a 5% CO2 incubator. The cells from PPD-immunized mice or S. mansoni egg-immunized mice were restimulated with 10 µg/ml PPD or 5 µg/ml SEA, respectively. Forty-eight hours later, the culture supernatants were collected, centrifuged, and stored at -20°C for cytokine assay.
Cytokine ELISAs
Immunoreactive murine IFN-, IL-10, IL-12, and IL-13 were
quantitated using a modification of a double ligand method as described
previously (20). Flat-bottom 96-well microtiter plates
(Nunc Immuno Plate I 96-F; Nunc, Denmark, The Netherlands) were coated
with 50 µl/well of anti-murine Abs (1 µg/ml in 0.6 M NaCl, 0.26
M H3BO4, and 0.08 N NaOH,
pH 9.6) for 16 h at 4°C, and then washed with PBS (pH 7.5)
containing 0.05% Tween 20 (wash buffer). Plates were blocked with 2%
BSA in PBS and incubated for 90 min at 37°C, and rinsed four times
with wash buffer. Cell-free culture supernatants (50 µl) were diluted
(undiluted and 1:10) and added in duplicate, followed by incubation for
1 h at 37°C. Plates were washed four times, followed by the
addition of 50 µl/well of biotinylated anti-murine Abs (3.5
µg/ml in PBS, pH 7.5, containing 0.05% Tween 20 and 2% FCS), and
incubated for 30 min at 37°C. Plates were washed four times,
streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added,
and the plates were incubated for 30 min at 37°C. After washing,
chromogen substrate (Bio-Rad) was added and the plates were incubated
at room temperature to the desired extinction. The reaction was
terminated with 50 µl/well of 3 M
H2SO4 solution. Plates were
read at 490 nm in an ELISA reader. Standards were #1/2 log dilution of
murine recombinant cytokines from 1 pg/ml to 100 ng/ml. This ELISA
method consistently detected cytokine levels above 30 pg/ml.
Statistics
Statistical significance was determined by ANOVA with p values <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our initial studies, we attempted to alter the development of
the PPD-specific immune response by giving recombinant MCP-1 and
examining the granuloma response by PPD-bead embolization 10
days later. We found that the presence of exogenously injected MCP-1
(100 ng/mouse i.p.) at the time of Ag sensitization had no effect on
the subsequent pulmonary granuloma formation (data not shown). Because
Ag sensitization is not necessarily a transient event, we surmised that
MCP-1 might need to be produced locally for an extended period of time
and not given as a bolus treatment. To accomplish this, a MCP-1
adenovirus vector was utilized. Two different concentrations of the
virus were used: 5 x 108 and 5 x
109 PFU. The 5 x 108
PFU treatment induced only small amounts of MCP-1 in the peritoneum by
day 1 (<2 ng/ml), whereas the administration of the 5 x
109 dose induced >25 ng/ml when the peritoneal
cavity was washed out (Fig. 1
). A high
level of MCP-1 was also found in the serum after the administration of
5 x 109 PFU MCP-1-adenovirus (Fig. 1).
Increased levels of MCP-1 were only found within the first 24 h
and reduced back to background by day 2 postadenovirus injection. MCP-1
or control adenovirus was injected i.p. 24 h before sensitization
with PPD. The lower concentration had a small and insignificant effect
on the development of subsequent lesions (data not shown), whereas the
higher dose significantly decreased the response to the embolized
PPD-coated beads (Fig. 2). These latter
results with the MCP-1 adenovirus may be explained when examining the
level of production of MCP-1 as described above.
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). Thus, MCP-1 appeared to have a
differential effect on the developing immune responses depending on the
type of response elicited. These results demonstrate differential
effects of MCP-1 on different types of immune responses.
The morphology of the two types of granulomas were also examined.
Histologically, the Th1-type PPD granulomas were noticeably smaller but
major changes in the composition of the granulomas were not evident
(Fig. 3). In contrast, the Th2-type SEA
granuloma was not only larger, but also appeared to have significant
increases in the number and size of multinucleated giant cells (Fig. 3). Thus, it appears that the overexpression of MCP-1 not only altered
the magnitude of the responses, decreasing the Th1-induced lesion and
increasing the Th2-induced lesion, but also in the case of the Th2
response MCP-1 may have altered the composition of the granuloma.
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To determine whether alteration in granuloma development was
accompanied by changes in cytokine profiles, dispersed spleen cells
from immunized and rechallenged mice were cultured and rechallenged
with PPD or SEA in vitro, respectively. The cell-free supernatants were
then assayed for critical Th1- and/or Th2-type cytokines that have
previously been shown to drive the granulomatous lesion development
(19). Administration of MCP-1 adenovirus (5 x
109 PFU) significantly decreased Th1-associated
cytokines in the PPD-immunized mice and increased Th2-type cytokines in
the SEA-immunized mice. Treatment of PPD-immunized mice with the MCP-1
adenovirus significantly lowered IFN- as compared with control (Fig. 4). The IL-12 level, which was present in
control, was not detected after MCP-1 adenovirus treatment (Fig. 4).
Th2-type cytokines were not detectable in either PPD group (data not
shown). In contrast, both IL-10 and IL-13 were significantly increased
in the SEA-immunized mice treated with the MCP-1 adenovirus, as
compared with the control adenovirus treated group, with no detectable
Th1-type cytokines, IFN- and IL-12 (Fig. 5). These cytokine profiles correlate
well with the altered pulmonary granuloma formation observed in both
groups of sensitized animals.
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We were next interested in examining the ability to alter
the pulmonary granuloma responses by overexpressing MCP-1 during the
elicitation phase (efferent) by administrating the adenovirus (5
x 109 PFU) i.p. on day 9 after sensitization.
PPD-coated beads or schistosome eggs were then injected i.v. on day 10
and granulomas were morphometrically measured on day 14 (4 days after
PPD-bead or schistosome egg embolization). The data in Fig. 6 illustrate that overexpression of MCP-1
during the efferent phase of the Th1- or Th2-type response had no
altered effect on granuloma development (Fig. 6). Subsequently, when
spleen cells were stimulated ex vivo, no alteration of cytokine
production was detected (Fig. 7). These
data suggest that MCP-1 regulation during the efferent phase of the
response had no effect on granuloma development and cytokine
production. Thus, the stage of the response that MCP-1 was administered
during development of the immune responses may determine its
function.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12 (14, 15).
Most convincingly, MCP-1 was found to be involved in the development of
oral tolerance through the regulation of IL-12 production in the
subsequent responses (16). The data in the present study
help to extend these findings by demonstrating that the overexpression
of MCP-1 at specific phases of the developing responses appears to
differentially alter the outcome of the immune responses. When MCP-1
was introduced at the beginning of the immune response at a time when T
lymphocytes would first be in contact with Ag, a decreased immune
response was observed with Th1-type granuloma while increasing the
Th2-type granuloma. Interestingly, Ab isotypes IgG2a and IgG1 were not
altered during these T cell-mediated responses (data not shown). In
contrast, when MCP-1 was overexpressed during the efferent phase of the
immune response, at a time when the system was already sensitized, no
effect on the developing Th1 and Th2 granulomatous response was
observed.
The direct role of MCP-1 is unclear in these studies. However, MCP-1
has been shown to promote the activation of several molecules that may
participate in altering the immune response. As mentioned above and in
these studies, MCP-1 can alter the production of IL-12 and IFN- in
vivo (16, 17, 25, 26). The direct regulation of IL-12 by
MCP-1 has previously been observed using isolated macrophages in vitro
(17) and may be the primary mechanism of PPD (Th1) lesion
regulation. Alternatively, MCP-1 can induce the production of
suppressive cytokines that may directly impact on the production of
Th1-type responses. Fibroblast-derived TGF-ß can be induced by MCP-1
activation of structural cells and may contribute to the alteration of
responses observed in these studies (27). MCP-1 has also
been shown to be a potent inducer of mast cell degranulation in vivo
and may alter the response through the release of factors during the
developing response (28). Finally, MCP-1 may alter the
expression of specific costimulatory molecules during Ag presentation
either directly or indirectly by induction of suppressive factors, such
as IL-10, which was up-regulated in the Th2-response model.
Interestingly, MCP-1-transgenic mice appear to have problems clearing
bacterial infections, possibly reflecting an altered ability to
generate the proper immune response (18). Likewise,
MCP-1-deficient mice appear to have altered development of Th2-type
granulomatous responses (29). These previous results would
be reflective of the data presented in the present studies. A final
possible explanation is that the overexpression of MCP-1 either altered
the specific trafficking of cells into their proper compartment for
activation or desensitized the cells for specific movement. We
attempted to address this by examining the in vitro chemotactic
responses of peripheral cells from adenoviral-treated animals and found
no difference in movement of cells from MCP-1 compared with control
virus-treated animals (data not shown). However, alteration of
trafficking may still be a significant event in vivo during these
analyses. A number of conflicting studies have suggested that MCP-1 and
its receptor CCR2 may be involved in the enhancement of Th1-type
responses (30, 31, 32). These data may, in fact, underscore
the complexity of the chemokine regulation during development of immune
responses.
Altogether, these data further support the contention that chemokines have multiple effects on a developing immune response. The fact that MCP-1, and other chemokines, can influence the direction of an immune response and aid in determining the intensity of the reaction further indicates the complexity and importance of these molecules. Future studies will examine the in vivo mechanism of MCP-1 in the differentiation of the immune response.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; PPD, purified protein derivative; SEA, schistosome egg Ag; CCR CC chemokine receptor; AdmMCP-1, adenovirus coding for murine MCP-1.
Received for publication September 9, 1999. Accepted for publication November 24, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks. J. Immunol. 159:3565.[Abstract]
and ß chemokines induce NK cell migration and enhance NK- mediated cytolysis. J. Immunol. 155:3877.[Abstract]
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