| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pathology, Veterans Affairs Medical Center, and
University of Michigan Medical School, Ann Arbor, MI 48105; and
Childrens Hospital, Harvard Medical School, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To help clarify these issues, we performed a detailed analysis of the
expression and function of RANTES in defined models of polarized type 1
(Th1) and type 2 (Th2) cell-mediated pulmonary granuloma formation
elicited with soluble protein Ags of Mycobacterial bovis or
Schistosoma mansoni, respectively (21). The
type 1 granuloma is composed mostly of mononuclear cells and is largely
dependent on IFN-
and TNF-
, whereas the type 2 granuloma is
eosinophil-rich and is largely dependent on IL-4. Our findings clearly
show a differential expression in the two models with RANTES
predominating in the type 1 response at both the site of granuloma
formation and draining lymphoid tissue. In vivo depletion analysis
revealed differing roles for RANTES in the two types of responses.
Specifically, RANTES contributed to type 1 inflammation and limited the
type 2 response analogous to the cross-regulatory effect of IFN-
(22, 23). Thus, our findings suggest that RANTES is an
endogenous regulatory molecule that participates differently in type 1
and 2 inflammatory responses.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female CBA/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). B6 x 129 mice with knockout of the CCR1 receptor gene were prepared as previously described (24). Wild-type control B6 x 129 mice were obtained from The Jackson Laboratory. All mice were maintained under specific pathogen-free conditions and provided with food and water ad libitum.
Sensitization and granuloma induction
Type 1 and 2 granulomas were generated as follows. Briefly, mice were sensitized by s.c. injection with 20 µg purified protein derivative (PPD)3 (Department of Agriculture, Veterinary Division, Ames, IA) of M. bovis incorporated in 0.25 ml CFA (Sigma, St. Louis, MO; product no. F-5881) or 3000 S. mansoni eggs suspended in 0.5 ml PBS. Then, 14–16 days later, PPD and egg-sensitized mice were respectively challenged i.v. with 6000 Sepharose 4B beads (in 0.5 ml PBS) covalently coupled with PPD or to soluble schistosome egg Ags (SEA) obtained from the World Health Organization (Geneva, Switzerland). Nonimmune control (CON) granulomas were generated by injection of Ag-free beads.
Cytokines, Abs, and cytokine assays
All cytokines and chemokines used in this study were obtained as
purified carrier-free recombinant proteins from PeproTech (Rocky Hill,
NJ) and R&D Systems (Minneapolis, MN). Human rRANTES, kindly provided
by Dr. T. Wells (Glaxo Wellcome Research and Development, Research
Triangle Park, NC), was used for in vivo osmotic pump studies that
required large doses of the chemokine. Murine RANTES and IL-2, -4, -5,
-13, and IFN- were measured by standard ELISA using commercially
available reagents (R&D Systems and PharMingen (San Diego, CA)).
Sensitivities ranged from 15 to 50 pg/ml. IL-12 was measured by
sandwich ELISA, as previously described, with sensitivity to 50 pg/ml
(25).
Goat anti-murine RANTES was prepared on contract with Lampire Biological Laboratories (Pipersville, PA) using a 14-aa carboxyl-termial peptide of murine RANTES kindly provided by Dr. B. Daugherty (Merck, Whitehouse Station, NJ). Titer was determined to be >1:1 x 107 by direct ELISA, and neutralization was confirmed by inhibition of RANTES-elicited eosinophil chemotaxis. Specifically, a 1:1000 dilution of antiserum completely neutralized 10 ng of recombinant murine RANTES with no effect on eotaxin-induced chemotaxis.
In vivo cytokine depletion protocol
At the time of bead challenge, mice were given an i.p. injection of 5 mg of protein A-purified rabbit anti-murine RANTES IgG. All polyclonal Ab was depleted of any intrinsic anti-PPD activity by prior passage over a solid-phase PPD immunosorbent column. Nonimmune goat IgG served as a control.
In vivo cytokine treatment by osmotic pump implantation
Miniosmotic pumps were implanted as previously described
(26). Briefly, 2 days before bead challenge, mice were
subjected to ketamine anesthesia, then the abdomen was shaved and
cleaned with 70% alcohol. An 8-mm incision was made in the skin and
peritoneum through which an osmotic pump (Alza, Palo Alto, CA) was
implanted, containing 100 µl of carrier-free human RANTES (hRANTES)
solution. The concentration was adjusted to achieve a dosage of 1
mg/kg/day given as constant infusion (
830 ng/mouse/h). Control pumps
contained the vehicle, PBS. After 2 days of rest, the mice were
challenged i.v. with Ag-coated beads. Then, 3 days after challenge,
lungs and lymph nodes were excised and cultures prepared as described
below. In all experiments, samples of lungs were fixed in 10% buffered
formalin for morphometric analysis, while the remaining lung was used
for granuloma isolation, dispersal, and cell differential analysis.
Morphometry
At 1, 2, 4, or 8 days after challenge, lungs and lymph nodes were excised and prepared as described below. In some experiments, lungs were inflated and fixed with 10% buffered formalin for morphometric analysis. Granuloma area was measured blindly in hematoxylin- and eosin-stained sections of paraffin-embedded lungs using a morphometer and software program (The Morphometer, Woodshole, MA). A minimum of 20 lesions was measured per lung. Only granulomas with full cross-sections of the bead nidus were measured.
Intact granuloma and lymph node cell culture
Intact and dispersed granulomas were prepared as previously described (21). A total of 2 ml of intact granuloma suspension (1500 lesions/ml) was cultured in sterile 100-mm plastic culture dishes in the presence or absence of 5 µg/ml PPD or SEA. Supernatants were collected at 24 h and assayed for RANTES production. A 200 cell differential analysis was performed on duplicate Wright-stained cytospin preparations of dispersed granulomas. Mediastinal lymph nodes were collected aseptically at the time of lung harvest and teased into a single cell suspension. After washing, the cells were cultured in RPMI-FBS at 5 x 106 cells/ml in 100-mm dishes with 5 µg/ml PPD or SEA for 24 h at 37°C in a humidified 5% CO2 atmosphere. In some studies, the cultures were stimulated with 0, 1, 10, or 100 ng/ml murine RANTES and cultured as above. Supernatants were collected by centrifugation and stored at -45°C before performing cytokine assays.
RNA extraction
Perfused lung lobes, excluding major bronchi, were snap frozen with liquid N2, and total cellular RNA was extracted by a modified method of Chirgwin et al. (27) and Jonas et al. (28). The frozen tissues were suspended in extraction buffer (25 mM Tris (pH 8.0), 4.2 M guanidine isothiocyanate, 0.5% Sarkosyl, and 0.1 M 2-ME), homogenized, then added to an equal volume of extraction buffer (100 mM Tris (pH 8.0), 10 mM EDTA, and 1% 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 diethyl pyrocarbonate water, and RNA concentrations were determined spectrophotometrically before storage at -70°C. Yields are routinely >100 µg.
Primers and probes
Primers and probes (18–22 mer) were designed based upon mRNA nucleotide sequences downloaded from the National Center for Biotechnology Information (NCBI) database and using primer design software (Premier Biosoft International, Palo Alto, CA). Designed primer and probe sequences (in 5' to 3' direction) for murine RANTES (NCBI accession no. M77747) and endoplasmic reticulum-specific cyclophilin (NCBI accession no. M60456) mRNAs were as follows: murine RANTES sense primer, GTG-CCC-ACG-TCA-AGG-AGT-ATT-T; antisense primer, A-AGC-GAT-GAC-AGG-GAA-GCG-TAT; probe sequence, TGT-CAC-TCG-AAG-GAA-CCG-CCA-A (spanning bases 231-252); murine cyclophilin sense primer, GTG-GGC-TCC-GTC-GTC-TTC-CTT-T; antisense primer, C-TTT-CCT-CCT-GTG-CCA-TCT-CCC; probe sequence, CAT-CGT-GTC-ATC-AAG-GAC-TTC-A (spanning bases 296-318). Primers and probes were prepared by Genosys Biotechnologies (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.
RT-PCR ELISA detection of cytokine mRNA
The isolated RNA was reverse transcribed to DNA as follows. To 20 µg of RNA (in 25 µl of diethyl pyrocarbonate water) was 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 min in a thermocycler (Perkin-Elmer 9600; Perkin-Elmer, Norwalk, CT). The temperature was then reduced to 43°C, then 69 µl of a first strand buffer (Life Technologies, Grand Island, NY) containing dTT, dNTP, and 1000 U Moloney murine leukemia virus RT was added (29). The mixture was incubated for 2 h, and then the reaction was stopped by heating to 70°C. The DNA was then subjected to PCR (30). Briefly, 5 µl of DNA was added to 95 µl PCR buffer containing unlabeled dNTPs (0.2 mM 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 then performed in a thermocycler as follows: 4 min at 95°C followed by 25 cycles of 1 min at 95°C, 2 min at 57°C, and 1 min at 72°C. After cycling, there was a DNA extension period of 6 min at 72°C, then samples were stored at -20°C before analysis.
Detection of PCR products was performed as follows. Initially, a series of amplification reactions using unlabeled dNTPs was performed, and the products were analyzed by standard agarose gel electrophoresis to confirm that primers yielded predicted products. Once confirmed, labeled products were generated and detected by PCR-ELISA (31). Briefly, 30 µl of amplified RANTES or 5 µl of cyclophilin product was added to a sterile microfuge tube containing 40 µl of denaturing buffer (Boehringer Mannheim) and incubated 10 min. Due to the high degree of assay sensitivity and the relative greater incidence of cyclophilin mRNA species, less cyclophilin product is used to keep the final absorbance signal within readable range. 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 h at 42°C. The plate is then washed, and any bound product is detected with peroxidase-labeled anti-digoxigenin Ab by standard colorimetic reaction using ABTS (2, 2'-azino-bis (3-ethyl benzthiazoline 6-sulfonic acid)) substrate. The amount of color is directly related to the amount of amplification product added. A 96-well plate ELISA reader is used to measure OD at 405 nm at 15 and 30 min. Relative mRNA is then calculated as follows: Relative mRNA ratio = {[(OD target gene)/(OD cyclophilin, challenged lung)]/[(OD target gene)/(OD cyclophilin, unchallenged lung)]}.
Unlike simple gel detection, the PCR-ELISA method employs a hybridiztion step that specifically binds target amplicons. Consequently, it is highly specific and has proven to be 10- to 100-fold more sensitive than gel detection and allows amplification cycles to be kept to a minimum.
Statistics
The paired Student’s t test was used to compare
paired groups. ANOVA was used for multigroup analysis. Values of
p 
0.05 were considered to indicate lack of
significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine the dynamics of RANTES expression during synchronized
type 1 and 2 granuloma formation, we measured levels of spontaneous and
Ag-elicited RANTES in the culture supernatants of intact lesions
isolated at 1, 2, 4, and 8 days of development. This was temporally
related to granuloma size, local RANTES protein secretion, and mRNA
expression. Fig. 1
shows the results of
this multiparameter analysis. Compared with type 2 SEA and non-Ag CON
lesions, the type 1 PPD response was clearly associated with the
greatest overall spontaneous and Ag-elicited RANTES production by
granulomas (F > 1.0 and p < 0.05 for days 1 and
2, as compared with other groups). Levels of RANTES mRNA were likewise
greater for the type 1 response and paralleled the protein expression.
Among the type 1 granuloma cultures, the greatest production occurred
on days 1 and 2 corresponding to the rapid growth period, 0–4 days.
Interestingly, foreign-body lesions elicited by non-Ag CON beads showed
modestly increased levels of RANTES in response to Ag stimulus (i.e.,
combined PPD and SEA Ags). These lesions, which resemble a primordial
type 1 response, nonspecifically recruit mononuclear cells that could
produce RANTES as an innate response to Ag.
|
, levels
of Ag-elicited RANTES were notably higher during the type 1 PPD
response. Even spontaneous production was significantly higher on days
2 and 4 (F = 11, p = 0.007 and F = 43,
p = 0.0001, respectively). Interestingly, the kinetics
of RANTES production differed from the type 1 granulomas, increasing to
a maximum on day 4 (F > 1 and p < 0.05, as
compared with days 1, 2, and 8). These kinetics paralleled
lymphoproliferation as determined by assessment of lymph node cell
yields (data not shown).
|
To test the in vivo function of RANTES, types 1 and 2 granulomas
were induced in mice treated with a neutralizing preparation of goat
anti-murine RANTES IgG or a corresponding dose of nonimmune goat
IgG. On day 4 of granuloma formation, mice were sacrificed, and lesion
sizes were determined. As shown in Fig. 3, anti-RANTES treatment decreased
type 1 granuloma area by 25% and augmented type 2 granuloma area by
50%. This finding indicated that RANTES functioned differently in the
two systems, supporting type 1 and inhibiting type 2 inflammation.
|
). Ab treatment did
not cause changes in these major cell populations, suggesting that
RANTES was not selectively recruiting mononuclear cells or eosinophils.
However, alterations in specific lymphocyte subpopulations could not be
ruled out.
|
Since RANTES was produced in regional lymphoid tissues, it was of
interest to determine whether its depletion influenced draining lymph
node cytokine-producing capacity. As shown in Fig. 4, typical type 1 and 2 cytokine profiles
were obtained in the two models. The type 1 (PPD) response, dominated
by IFN- and IL-2, was generally resistant to RANTES depletion. In
contrast, the type 2 cytokine profile was broadly augmented with
significant increases in IL-4, IL-5, IL-10, and IL-13. In summary,
endogenous RANTES appeared to temper both type 2 granuloma formation
and its corresponding regional cytokine response. However, RANTES was
not critical to supporting the Th1 profile during the PPD response.
|
To test the direct effect of RANTES on type 1 and 2 inflammation,
granulomas were induced in mice implanted with miniosmotic pumps
providing a constant i.p. infusion (20 µg per mouse per day) of
recombinant hRANTES, which is 85% homologous to murine RANTES at the
amino acid level (32). Because of the high degree of
homology, hRANTES is poorly immunogenic in mice, therefore vehicle
could serve as an appropriate control substance. Fig. 5 shows the effect of the treatment on
granuloma sizes on day 3. Type 1, PPD lesions were statistically
unaffected, although there was a trend to larger lesions, and if values
are extrapolated to volumes, then a significant augmentation could be
detected (data not shown). In contrast, the average type 2, SEA bead
granuloma area was reduced by 27% (40% after subtracting the area
occupied by the bead nidus). Composition analysis of granulomas showed
no change in the type 1 lesions, but there was a significant decrease
(40%) in the percentage of eosinophils in type 2 granuloma,
resulting in a corresponding increase in the proportion of lymphocytes
(Table II). Further analysis of cytokine
profiles in draining lymph node cultures likewise revealed opposing
effects. Fig. 6 shows Ag-elicited levels
of cytokines in lymph node cultures. Compared with day 4, type 1 lymph
node cultures on day 3 tend to show less IFN- and increased IL-12
(compare Figs. 4
and 6), yet the type 1 profile is clearly present. In
these cultures, IFN- and IL-2 were augmented by RANTES infusion with
no change in IL-12. Small increases in IL-5 and IL-13 were also noted,
indicating that like Th1 clones, type 2 cytokines are reduced but not
totally eliminated from the type 1 response. In contrast, during the
type 2 response, there was broad impairment of IL-2, IL-4, IL-5, IL-10,
and IL-13. Thus, RANTES had opposite effects, augmenting the type 1 and
inhibiting the type 2 response. Because the effects were broad, the
bulk of the cytokines generally maintained the same relative
proportions.
|
|
|
The above studies indicated that RANTES plays a differential
regulatory role in cytokine production during type 1 and 2 granuloma
formation. To begin to explore potential mechanisms, PPD- or
SEA-sensitized lymphoid cultures from mice at day 4 of granuloma
formation were exposed to specific Ags and graded doses of exogenous
murine RANTES in vitro. Representative type 1 and 2 cytokines (IL-2,
IFN-, IL-4, and IL-5) were then measured after 24 h of culture.
As shown in Fig. 7, type 1 (PPD) and type
2 (SEA) responses had the expected Th1- and Th2-related cytokine
profiles with IL-2 and IFN- dominating the former and IL-4 and IL-5
dominating in the latter. In response to RANTES treatment, the
SEA-sensitive cultures displayed a specific dose-dependent inhibition
of IL-4, dropping to nearly 50% of control levels at 100 ng/ml (ANOVA,
0 ng/ml vs 1 ng/ml, p = 0.6; 1 ng/ml vs 10 ng/ml,
p = 0.016; 10 ng/ml vs 100 ng/ml, p =
0.008). Interestingly, IL-5 was unaffected. In the type 1 (PPD)
cultures, RANTES had no consistent effect. Thus, a selective inhibitory
effect of mRANTES on IL-4 could be demonstrated in vitro. However, this
was unlike the broad effects on type 2 cytokines that we observed when
RANTES was administered in vivo over several days, suggesting the
presence of intermediary events leading to in vivo changes.
|
RANTES is known to interact with a number of known chemokine
receptors including CCR1, -3, -4, and -5 (9). As an
initial effort to determine which of these receptors was involved in
IL-4 regulation, we compared the effects of RANTES treatment on day 4,
type 2-responsive draining lymph node cultures from wild-type and CCR1
receptor knockout mice. This was likewise compared with the in vitro
effect of eotaxin, a chemokine thought to selectively bind to the CCR3
receptor. Fig. 8 shows the results of
these studies. The first observation that became apparent upon
inspecting the baseline response was that CCR1 knockout mice produced
higher levels of IL-2 and IL-4 than wild-type mice. This effect did not
extend to IFN-, which tended to be lower than controls. Ag-elicited
IL-5 levels were comparable. However, we did note that spontaneous
levels of IL-5 and -13 were greater in CCR1 knockout cultures (data not
shown). With regard to RANTES treatment, the B6 x 129 wild-type
mice responded similarly to the CBA strain, showing a selective
dose-dependent inhibition of IL-4 production. In CCR1 knockout
cultures, RANTES failed to demonstrate statistically significant
effects. The CCR3 agonist, eotaxin, did not effect IL-4 in either
wild-type or knockout strains. However, as we previously reported
(33), 100 ng/ml eotaxin augmented IFN- levels (ANOVA,
p < 0.001 and p < 0.0001 for control
and knockout mice, as compared with 0 ng/ml). Surprisingly, eotaxin at
100 ng/ml inhibited IL-5 in CCR1 knockout, but not in wild-type,
cultures, suggesting that CCR1 deficiency enhanced sensitivity to the
CCR3 agonist.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF- augment RANTES, whereas
type 2 cytokines IL-4 and IL-13 reduce expression (7, 41, 42).
To help clarify the function of RANTES in vivo, we performed a detailed
multiparameter analysis of RANTES expression and function in defined
models of polarized type 1 and 2 pulmonary granuloma formation. Both
protein and mRNA measurements revealed strong expression of RANTES
during the type 1 (Th1) response to M. bovis PPD with weaker
expression in the type 2 (Th2) response to the S. mansoni
egg Ags. This was in complete accord with studies of human cells
reported by Schrum et al. (20) and noted above. Our
results demonstrated differential production at both the local sites of
inflammation and in draining lymphoid tissues. The association with the
type 1 response fits well with reports showing that IFN- and TNF-
promote RANTES production (41, 42), since these cytokines
characterize the type 1 granuloma (43). This notion is
also supported by our previously reported observation that RANTES and
TNF- mRNA are reduced in IFN- knockout mice during the type 1
granulomatous response (44).
Because of the synchronized nature of our models, we were able to define the time course of local and regional RANTES production. Maximal local production during the type 1 response occurred during the cellular recruitment phase, 1–4 days. In addition, RANTES neutralization resulted in decreased granuloma size. These findings are fully consistent with RANTES chemotactic role and concur with studies showing that Th1 cells are preferentially attracted to RANTES (45). However, in view of RANTES reported effects on T cells (10, 11), other interpretations must be considered. The chemotactic function of RANTES may be secondary to immunoregulatory effects. If RANTES is promoting local Th1 cell activation, then neutralization might similarly cause decreased inflammation, independent of chemotactic function. Alternatively, or in addition, if RANTES is inhibiting the cross-regulatory influence of type 2 cytokines, like IL-4 and IL-13 (which are present but at low levels in type 1 lesions), then its neutralization might lead to lesion impairment by enhancing the effect of these opposing cytokines. Further studies will be needed to determine which mechanisms are participating.
In lymph nodes, RANTES production paralleled the lymphoproliferative response maximizing on day 4 and suggesting the expansion of RANTES producing cells, possibly Th1 clones as the studies of Schrum et al. (20) would suggest. Interestingly, Ag-stimulated cultures produced significant levels of RANTES within 24 h. Based on in vitro studies, this seems rapid since Ag-stimulated T cells reportedly produce RANTES primarily as a late transcription (3–5 days) product (46). This would suggest that production occurs more rapidly after in vivo priming or that other RANTES-producing cells are present.
Our depletion studies provided compelling and novel findings regarding
the role of RANTES in the type 1 and 2 responses. Although RANTES may
act as an eosinophil chemotactic agent in acute allergy
(37), it did not seem to recruit eosinophils in the type 2
granuloma. In fact, endogenous RANTES supported the type 1 and
inhibited the type 2 granuloma. This result was analogous to the
cross-regulatory effect reported with IFN- depletion (22, 23). Since maximal RANTES production is in part dependent on
IFN-, it would suggest that RANTES is a downstream cross-regulatory
agent. Furthermore, it is known that IL-4 and IL-13 down-regulate
RANTES (7, 41, 42), thus defining a cross-regulatory
circuit analogous to that involving IFN- and IL-10
(47). In this scenario, RANTES would be another element in
defining the balance of Th1 and Th2 cell expression.
This regulatory role was further revealed by observed effects on lymphoid tissue during the type 2 response, where RANTES depletion broadly augmented and exogenous administration broadly abrogated type 2 cytokines. Although it might be argued that the observed effects on granulomas were due to complex modifications of chemotactic gradients, it is difficult to conceive how this would apply to the changes in regional lymph node cytokine profiles. In view of RANTES role in T cell activation (10, 11), it seems more reasonable to propose that our findings reflect regulation of Th2 lymphocyte function or maturation. Although not providing a detailed mechanism, our in vitro studies would support this explanation. During the in vitro Ag-elicited response, RANTES inhibited IL-4 production, but not IL-5. Superficially, this seems inconsistent with the broad effects on type 2 cytokines observed following in vivo treatments, but based on current paradigms of T cell maturation, there is likely no contradiction. Since IL-4 is an upstream promoter of Th2 development, its abrogation by RANTES would eventually broadly degrade the type 2 profile, as we observed in over 5 days of in vivo RANTES treatment. However, in vitro cultures treated for just 24 h may only show effects on the sensitive target cell population. Downstream changes in other Th2 cytokines would require longer exposure. Although it is possible that RANTES mediates specific IL-4 gene regulation, we would rather offer that RANTES acts on a precursor IL-4-producing stage of Th2 cell development with mature Th2 cells being resistant. This would explain why RANTES treatment achieved only 50% inhibition of IL-4 in vitro. This explanation is purely speculative and many basic mechanistic questions have yet to be resolved, such as characterization of the specific source(s) and target(s) of RANTES action within lymphoid tissues.
Whatever the underlying mechanism, our findings indicate that the action of RANTES on the type 2 lymphoid response involves ligation of the CCR1 receptor. This was unexpected since CCR1 is only one of several potential RANTES receptors (9). The importance of this receptor was suggested by the baseline augmentation of IL-4 production in CCR1 knockout mice, but this was directly demonstrated by their failure to respond to RANTES-mediated IL-4 inhibition. Of course, future studies designed to reintroduce the CCR1 receptor will help to confirm its regulatory role. Interestingly, IL-4 inhibition could not be replicated by the CCR3 agonist, eotaxin. In view of reports showing that CCR3 is selectively expressed on Th2 cells (48), the participation of the CCR1 receptor in regulating the type 2 response suggests an additional level of complexity. If mature Th2 memory cells cannot directly respond to RANTES via CCR1, then indirect effects must be considered, such as intermediary cells or, as proposed above, stage-specific regulation. Recent evidence suggests the existence of a dynamic T cell subset selective expression of chemokine receptors that is under cytokine-mediated control (49, 50). Therefore, lymphocyte regulation via transient, stage-specific chemokine receptor expression is a reasonable hypothesis. It should be noted that a detailed analysis of type 1 and 2 granuloma formation in CCR1 knockout mice is underway and will be reported later.
Although RANTES clearly appears to temper the type 2 response, its role
in type 1 lymphoid response is unclear. A seeming inconsistent finding
was that during the type 1 (PPD) response, RANTES depletion did not
reduce Th1 cytokines, yet parenteral RANTES administration augmented
production. A simple explanation is that due to chemokine redundancy,
RANTES is not by itself essential in promoting the Th1 response. We
have reported that both monocyte chemoattractant protein-1/JE and
macrophage-inflammatory protein (MIP)-1 can promote IFN-
production in lymphoid cultures, and, like RANTES, MIP1 inhibits
IL-4 production (51). Thus, there is definite potential
for redundancy. The administration of exogenous RANTES may simply
augment a mixed pool of endogenous agonists that promote the type 1
response, yet elimination of the endogenous RANTES component may not be
sufficient to impair the response.
In summary, this is the first comprehensive in vivo investigation of RANTES expression and function in polarized type 1 and 2, T cell-mediated granuloma formation. Our studies clarify a number of confusing observations and provide novel evidence for RANTES as a cross-regulatory molecule that tempers type 2, hypersensitivity inflammation. Finally, we demonstrate the potential use of exogenous RANTES to temper established type 2 hypersensitivity states.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Stephen W. Chensue, Pathology and Laboratory Medicine, 113 Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105
3 Abbreviations used in this paper: PPD, purified protein derivative; SEA, schistosome egg Ags; CON, control; h, human.
Received for publication February 3, 1999. Accepted for publication April 14, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induces differential upregulation of and ß chemokine secretion in colonic epithelial cell lines. Gut 42:208. in bronchoalveolar lavage fluid of allergic asthmatic patients. Am. J. Respir. Crit. Care Med. 153:1398.[Abstract]
, MIP-1ß, and RANTES is associated with a type-1 immune response. J. Immunol. 157:3598.[Abstract]
, IL-10, and TNF. J. Immunol. 154:5969.[Abstract]
(IFN-), IL-4 and IL-10 in schistosome egg granuloma formation: in vivo regulation of Th activity and inflammation. Clin. Exp. Immunol. 98:395.[Medline]
plus TNF- and inhibition by IL-4 and IL-13. J. Immunol. 154:1870.[Abstract]
and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks. J. Immunol. 159:3565.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  W. Hansen, A. M. Westendorf, S. Reinwald, D. Bruder, S. Deppenmeier, L. Groebe, M. Probst-Kepper, A. D. Gruber, R. Geffers, and J. Buer Chronic Antigen Stimulation In Vivo Induces a Distinct Population of Antigen-Specific Foxp3 CD25 Regulatory T Cells J. Immunol., December 15, 2007; 179(12): 8059 - 8068. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Chen, P. Keshavan, G. D. Gregory, and D. J. Klumpp RANTES mediates TNF-dependent lamina propria mast cell accumulation and barrier dysfunction in neurogenic cystitis Am J Physiol Renal Physiol, May 1, 2007; 292(5): F1372 - F1379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Koya, K. Takeda, T. Kodama, N. Miyahara, S. Matsubara, A. Balhorn, A. Joetham, A. Dakhama, and E. W. Gelfand RANTES (CCL5) Regulates Airway Responsiveness after Repeated Allergen Challenge Am. J. Respir. Cell Mol. Biol., August 1, 2006; 35(2): 147 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tjarnlund, A. Rodriguez, P.-J. Cardona, E. Guirado, J. Ivanyi, M. Singh, M. Troye-Blomberg, and C. Fernandez Polymeric IgR knockout mice are more susceptible to mycobacterial infections in the respiratory tract than wild-type mice Int. Immunol., May 1, 2006; 18(5): 807 - 816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Palaniappan, S. Singh, U. P. Singh, R. Singh, E. W. Ades, D. E. Briles, S. K. Hollingshead, W. Royal III, J. S. Sampson, J. K. Stiles, et al. CCL5 Modulates Pneumococcal Immunity and Carriage J. Immunol., February 15, 2006; 176(4): 2346 - 2356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. S. Souza, E. Roffe, V. Pinho, D. G. Souza, A. F. Silva, R. C. Russo, R. Guabiraba, C. A. J. Pereira, F. M. Carvalho, M. M. Barsante, et al. Potential Role of the Chemokine Macrophage Inflammatory Protein 1{alpha} in Human and Experimental Schistosomiasis Infect. Immun., April 1, 2005; 73(4): 2515 - 2523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Olleros, R. Guler, D. Vesin, R. Parapanov, G. Marchal, E. Martinez-Soria, N. Corazza, J.-C. Pache, C. Mueller, and I. Garcia Contribution of Transmembrane Tumor Necrosis Factor to Host Defense against Mycobacterium bovis Bacillus Calmette-Guerin and Mycobacterium tuberculosis Infections Am. J. Pathol., April 1, 2005; 166(4): 1109 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Wickremasinghe, L. H. Thomas, C. M. O'Kane, J. Uddin, and J. S. Friedland Transcriptional Mechanisms Regulating Alveolar Epithelial Cell-specific CCL5 Secretion in Pulmonary Tuberculosis J. Biol. Chem., June 25, 2004; 279(26): 27199 - 27210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Morimoto, M. Inobe, C. Kimura, S. Kon, H. Diao, M. Aoki, T. Miyazaki, D. T. Denhardt, S. Rittling, and T. Uede Osteopontin affects the persistence of {beta}-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms Int. Immunol., March 1, 2004; 16(3): 477 - 488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Booth, J. K. Mwatha, S. Joseph, F. M. Jones, H. Kadzo, E. Ireri, F. Kazibwe, J. Kemijumbi, C. Kariuki, G. Kimani, et al. Periportal Fibrosis in Human Schistosoma mansoni Infection Is Associated with Low IL-10, Low IFN-{gamma}, High TNF-{alpha}, or Low RANTES, Depending on Age and Gender J. Immunol., January 15, 2004; 172(2): 1295 - 1303. [Abstract] [Full Text] [PDF]
|
|
|
