| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114;
Massachusetts Eye and Ear Infirmary/National Eye Institute Training Program in the Molecular Bases of Eye Diseases, Boston, MA 02114; and
Pulmonary and Critical Care Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The chemokine signals responsible for the directed migration of monocytes, macrophages, and T cells are well known, but the signals controlling NKT cell migration are not. NKT cells are unconventional T cells that express most NK cell markers and the CD1-restricted, canonical TCR. Upon stimulation of their TCR, NKT cells release remarkable amounts of cytokines. Although their biological role is still not entirely understood, NKT cells were originally proposed to bias the outcome of cellular immune responses (e.g., Th1 vs Th2) (6) and are now known to be defective or deficient in several autoimmune diseases (7, 8), implying that they are crucial for the maintenance of peripheral tolerance to self-Ags.
In support of this, Sonoda and colleagues demonstrated that CD1-restricted NKT cells accumulate in the spleen and are required for the induction of T regulatory (Tr)3 cells that convey peripheral tolerance following intraocular exposure to Ag (9). We reasoned that NKT cell accumulation might result either from a local expansion of resident splenic NKT cells or through recruitment of NKT cells from the periphery.
Insights into mechanisms that might induce peripheral tolerance to both self and foreign Ags are gathered from studies of immune-privileged sites. Perhaps the best studied immune-privileged site is the eye, using the anterior chamber-associated immune deviation (ACAID) model, where peripheral tolerance is induced following Ag inoculation in the anterior chamber (a.c.). ACAID is demonstrated experimentally by the inability of a.c.-inoculated mice to mount subsequent Ag-specific delayed-type hypersensitivity (DTH) responses in the periphery (9, 10). In brief, ACAID requires the presence of an intact eye for the first 3 days and an intact spleen the next 4–7 days following a.c. inoculation (10) and is mediated by Ag-specific negative regulatory CD8+ T cells generated within the spleen during the first week of ACAID induction (11). It is believed that an eye-derived F4/80+ monocyte/macrophage appearing in the peripheral blood shortly after a.c. inoculation of Ag travels to the spleen, where it conveys Ag specificity and sets the stage for tolerance-producing mechanisms (12). Evidence is presented in this report to support a unique role for monocyte/macrophage-derived macrophage inflammatory protein (MIP)-2 in the migration of NKT cells to the site of tolerance induction (the spleen) after Ag is presented in the eye.
Traffic of lymphocytes to the spleen is less regulated than traffic through the lymph nodes in that blood-borne cells arriving in the spleen simply "pour out" of the central arterioles into the marginal zones around the periarteriolar lymphoid sheath (PALS) (13). Because the terminal arterioles in the spleen do not end with a network of capillaries, the flow of cells is neither slowed nor regulated. Furthermore, adhesion molecules do not facilitate extravasation of immune cells into the spleen as they do in lymph nodes. It is known that cell clustering is necessary for Ag presentation, the induction of primed T cell responses, and the generation of Ab responses (14). Confocal microscopy studies presented here show that F4/80+ monocytes/macrophages, NKT cells, and conventional CD3+ T cells similarly cluster in the marginal zones of the spleen during the induction of peripheral tolerance. Taken together, our results show that a specific chemokine, MIP-2, mediates the recruitment of NKT cells to a secondary lymphoid organ where they colocalize with newly arrived F4/80+ cells and conventional T cells to induce active suppression.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Female BALB/cAnNTac, C57BL/6, and MHC class II knockout mice (ABBN.5) used in these experiments were obtained from the Schepens Eye Research Institute Vivarium or from Taconic Farms (Taconic, NY). Female Cmkar2tm1Mwm (CXCR2-deficient) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed on a 12/12-h light/dark cycle and provided with food and water ad libitum. All animals were treated humanely and in accordance with the guidelines set forth by the Schepens Eye Research Institute Animal Care and Use Committee and the National Institutes of Health.
Anterior chamber inoculation of Ag
Ags were administered into the a.c. of mice anesthetized with
ketamine/xylazine. Briefly, the cornea was punctured with a 30-gauge
needle, and the aqueous humor (
2 µl) was drained. The anterior
segment of the eye was then re-inflated with 2 µl of air. Using
finely drawn glass needles, 50 µg of OVA (2 µl of a 25-mg/ml
solution in HBSS) was instilled into the a.c., displacing the air. The
OVA solutions used in these studies were passed through DetoxiGel
AffinityPak polymixin B columns (Pierce, Rockford, IL) to remove
contaminating endotoxin/LPS (98% efficiency of endotoxin removal of
concentrations up to 2 mg/ml).
Cell enrichment
PBMCs used in the RNA analyses were isolated from heparinized
venous blood obtained at 1, 3, 5, and 7 days after a.c. inoculation of
Ag. PBMCs were enriched by density gradient centrifugation using
Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada)
according to the manufacturer’s protocol. NKT cells used in the
chemotaxis and RT-PCR assays were enriched from the spleens of naive
MHC class II knockout mice on the B6 background by FACS (Becton
Dickinson, Mountain View, CA) sorting as previously described
(9). The purity of FACS-sorted NKT cells was 94%.
Splenic T cells used for the local adoptive transfer (LAT) assay
(described below) were enriched using IMMULAN T cell enrichment columns
(Biotecx Laboratories, Houston, TX).
Cell lines
Macrophage hybridoma cell lines 59 and 63, originally described by Ishikura et al. (15), were maintained in RPMI 1640, 10% FCS (Life Technologies, Gaithersburg, MD). For all experiments described here, cells were cultured in serum-free medium (SFM) consisting of RPMI 1640, 10 mM HEPES, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies), supplemented with 0.1% BSA and ITS+ culture supplement (1 µg/ml iron-free transferrin, 10 ng/ml linoleic acid, 0.3 ng/ml Na2Se, and 0.2 µg/ml Fe(NO3)3) (Collaborative Biomedical, Medford, MA).
Isolation and analysis of chemokine mRNA
Total cellular RNA was isolated from different cell types using
TRIzol reagent (Life Technologies). RNA isolation was performed
according to the manufacturer’s protocol. Chemokine mRNA was analyzed
with the RiboQuant multiprobe RNase protection assay and the mCK-5
mouse chemokine riboprobe template set (PharMingen-BD Biosciences, San
Diego, CA) encoding lymphotactin (Ltn), RANTES, eotaxin, MIP-1
and
, MIP-2, inflammatory protein 10 (IP-10), monocyte chemoattractant
protein (MCP)-1, TCA-3, L32, and GAPDH. Total RNA (2–5 µg) was used
for the RNase protection assay (RPA), followed by electrophoresis on
polyacrylamide sequencing gels. The gels were dried at 80°C for
1 h. mRNA bands were detected by phosphorimaging with a Bio-Rad
Molecular FX Imaging system (Bio-Rad, Hercules, CA) and quantified with
QuantiOne Molecular Imaging Software (Bio-Rad). To compensate for
loading imperfections and for quantitative purposes, chemokine mRNA
bands were normalized to GAPDH bands. Scanning densitometry results are
expressed as arbitrary OD units
(ODx/ODGAPDH, where "x"
is the OD of the chemokine band of interest).
Chemotaxis assays
To examine the ability of NKT cells to respond to chemokines (i.e., to MIP-2), Boyden chamber chemotaxis assays were performed according to a modified method from Current Protocols in Immunology (16). Briefly, the bottom wells of a 48-well Poretics Boyden chamber (Osmonics, Livermore, CA) were loaded with SFM in the presence or absence of recombinant murine MIP-2 (R&D Systems, Minneapolis, MN) in a total volume of 28–30 µl, while the lower wells contained 2 x 104 NKT cells. The upper and lower segments of the chamber were assembled with an intervening collagen-treated polycarbonate membrane (8-µM pore size; Osmonics) and incubated (37°C, 5% CO2 in air) for 8–12 h. The membranes were air dried and stained with Diff-Quick (VWR Scientific Products, Bridgeport, NJ), and the relative amount of chemotaxis was determined by counting the number of cells that had crossed the membrane within the area of a high-power field containing a 10 x 10 eyepiece grid (five high-power fields per well).
RT-PCR
Total cellular RNA was isolated from murine L-DAP
fibroblasts, sodium caseinate-elicited murine peritoneal neutrophils,
DN32.D3 NKT hybridoma cells, and FACS-sorted NKT and CD8 T cells. One
hundred nanograms of total RNA was reverse transcribed and amplified
using the Access RT-PCR system (Promega, Madison, WI) according to the
manufacturer’s specifications. RT-PCR products were resolved by
electrophoresis in a 1.5% agarose gel containing GelStar nucleic acid
stain (FMC BioProducts, Rockland, ME). The bands were visualized and
the gels were photographed using a Molecular FX Imaging station and
GelDoc (both from Bio-Rad). The primers used were as follows: murine
CXCR2, sense 5'-GTC TAC CTG CTG AAC CTG GCC-3', antisense 5'-GGT
TGT AGG GCA GCC-3'; murine -actin, sense 5'-GTG GGC CGC TCT AGG CAC
CAA-3', antisense 5'-CTC TTT GAT TGC ACG CAC GAT TTC-3'.
Antibodies
The Abs used for flow cytometry were as follows:
biotin-conjugated NK1.1 (PK136); FITC-conjugated CD3
; Cy5-conjugated
TCR -chain (all from PharMingen-BD Biosciences); and
streptavidin-conjugated R-PE (Jackson ImmunoResearch, West Grove, PA).
The primary Abs used for confocal microscopy studies were as follows:
rat anti-mouse F4/80 (Caltag, Burlingame, CA); biotin-conjugated
anti-NK1.1 (hybridoma clone PK136; American Type Culture
Collection, Manassas, VA); and hamster anti-mouse CD3
(PharMingen-BD Biosciences). The secondary reagents used for confocal
microscopy studies were Cy-5-conjugated goat anti-rat
F(ab')2; Rhodamine RedX-conjugated goat
anti-Armenian hamster IgG (H+L) (Jackson ImmunoResearch); and
ExtrAvidin FITC conjugate (Sigma, St. Louis, MO). The Abs used for in
vivo assays were as follows: rat anti-mouse MIP-2 (MAB452, clone
40605.111; R&D Systems) and rat IgG (Sigma). Abs delivered in vivo were
suspended in 100 µl sterile PBS and injected i.p. (50 µg per mouse,
total dose).
Flow cytometric evaluation of NKT cells
Flow cytometric analyses were performed as previously described
(9) on an EPICS XL flow cytometer (Beckman Coulter, Miami,
FL). In brief, splenic NKT cells were examined by first gating
positively on the CD3 intermediate (CD3int)
population and then analyzing within that population for NK1.1 and
TCR- dual-positive cells. The absolute number of NKT cells
determined by flow cytometry was calculated as the percentage of NKT
cells found in the viable cell population, determined by trypan blue
exclusion.
LAT assay
LAT was used to test for the presence or absence of regulatory CD8+ T cells as previously described (9, 17). Briefly, OVA-primed effector T cells were generated by immunizing C57BL/6 mice with OVA in CFA (Sigma). Seven days later, the spleens were collected and enriched for T cells by passage over IMMULAN columns (Biotecx Laboratories). Tr cells were enriched from the spleens of mice that received anti-MIP-2 mAb or control IgG (i.p.) 7 days post-OVA or -HBSS (a.c.). Stimulator cells were obtained by culturing thioglycollate-elicited peritoneal exudate cells (PECs) with OVA (5 ng/ml) overnight. Stimulator, effector, and regulatory cells (5 x 105 each) were resuspended in 10 µl HBSS and injected intradermally into the ear pinnae of completely naive mice. The change in ear thickness was measured at 24 and 48 h after ear challenge using an engineer’s micrometer (Mitutoyo, Paramus, NJ).
Confocal microscopy
To examine the anatomical distribution of NKT cells in the
spleen relative to the F4/80+ APCs and
conventional T cells, OVA- (or HBSS)-inoculated (a.c.) mice were
euthanized at various times after intraocular injection, and their
spleens were removed and snap-frozen in Tissue-Tek OCT compound (Sakura
Finetek USA, Torrance, CA). The frozen spleens were sectioned at 4-µm
increments and stored at -20°C until ready for use. Following
ice-cold acetone fixation, the tissue samples were immunostained with
anti-F4/80- and Cy5-conjugated goat anti-rat IgG,
anti-CD3-, and Rhodamine-conjugated goat anti-Armenian
hamster IgG, and biotinylated anti-NK1.1- and FITC-conjugated
ExtrAvidin. After a final wash, the tissue sections were mounted with
ProLong-AntiFade mounting media (Molecular Probes, Eugene, OR),
protected with coverslips and stored at 4°C until analyzed by
confocal microscopy. The immunofluorescence staining resulted in the
appearance of F4/80+ cells as bright pink,
CD3+ cells as red/orange, conventional NK
cells as green, and NKT cells as yellow (resulting from cancellation of
the red/orange and green wavelengths).
Immunostained tissue sections were analyzed using a TCS 4D Confocal Laser Scanning Microscope (Leica, Deerfield, IL) fitted with a krypton-argon laser and three-color detection capability. The tissue sections were scanned at 15–25 sections/µm, averaging 16 or 20 passes per section to obtain ultra-high-resolution images. Digital imaging and overlay of single and multicolor image stacks were done with Adobe Photoshop version 5.0 (Adobe Systems, Mountain View, CA).
Statistical analyses
All statistical analyses were performed by ANOVA with
Neuman-Keuls post-hoc analyses. Significance was determined at
p 
0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous reports by Wilbanks and Streilein 12 showed that a presumably eye-derived ACAID-conferring monocyte/macrophage could be isolated from the bloodstream during the first week after a.c. inoculation of Ag and could transfer ACAID. We postulated that the macrophages that encounter Ag within the a.c. of the eye (a compartment rich in immunomodulatory factors) expressed a unique chemokine profile and that these chemokine-producing cells increased to detectable numbers in the bloodstream after inoculation of Ag (a.c.). In studies by Wilbanks and Streilein, ACAID-conferring cells (PBMCs) were more easily detected in 500 µl of blood from a.c.-inoculated mice if the mice were splenectomized, presumably to prevent the exit of the cells to the spleen, thus enriching the circulating eye-derived F4/80 cell(s). In this report, instead of performing splenectomy, we collected and pooled the blood of five a.c.-inoculated mice to obtain detectable numbers of ACAID-conferring monocytes/macrophages from which RNA could be isolated.
Using a multiprobe RPA system, we observed that selective up-regulation
of MIP-2 mRNA occurred in vivo among blood monocytes/macrophages after
Ag (OVA) inoculation via the eye (a.c.), peaking at day 3 after Ag
inoculation (Fig. 1
A). It
should be noted that while MIP-2 increased in blood monocytes from
a.c.-inoculated mice, other chemokines on the template either remained
constant throughout the time course (i.e., MIP-1, MIP-1, and
RANTES) or were not expressed (Ltn, eotaxin, MCP-1, and TCA-3). To
compensate for slight variations in RNA loading from lane to lane, the
bands were analyzed by scanning densitometry, and the chemokine gene
expression was normalized to GAPDH levels. Densitometric analyses (Fig. 1B) confirmed that MIP-2 mRNA expression among PBMCs from
a.c.-inoculated mice increased more than 2-fold at day 3 and returned
to baseline by day 5 after a.c. inoculation. The appearance and
disappearance of the MIP-2-producing cells in the blood coincides with
the trafficking of ACAID-conferring PBMCs previously described by
Wilbanks and Streilein (12). Additionally, the selective
induction of MIP-2 in PBMCs 3 days after a.c. inoculation of Ag did not
occur following s.c. inoculation (data not shown). We also observed a
slight increase in IP-10, which followed the same profile as MIP-2.
Experiments are currently in progress to determine a role for IP-10 in
ACAID.
|
2 (to mimic the high levels of intraocular TGF-2), whereas
the 63 macrophage hybridoma cells were only pulsed with Ag because they
synthesize TGF- (18). In examining the chemokine
profiles of these cell lines, we found that, similar to the PBMCs from
OVA-inoculated (a.c.) mice, ACAID-conferring 59 and 63 macrophages also
selectively expressed increased amounts of MIP-2 mRNA, whereas
non-ACAID-conferring macrophages did not (Fig. 2). Together, these data suggest that
increased expression of MIP-2 is a function of the specialized APCs
associated with tolerance induction to eye-derived Ags.
|
Because the kinetics of MIP-2 mRNA expression and trafficking of
ACAID-conferring APCs to the spleen seemed to herald the NKT cell
accumulation in the spleen after OVA inoculation (a.c.), we postulated
that MIP-2 was a chemoattractant signal for NKT cells. Results from
Boyden chamber chemotaxis assays confirmed that freshly isolated
FACS-sorted NKT cells, but not CD8+ T cells,
migrated along a concentration gradient of MIP-2 (Fig. 3B). Evaluation of the cells
on the Boyden chamber membranes by light microscopy showed that NKT
cells incubated with recombinant murine (rm)MIP-2 exhibited marked
morphologic changes. Many of the NKT cells found on the underside of
the membrane were spindle-shaped, with prominent lamellipodia,
morphology consistent with (but not restricted to) active migration or
movement of the cells (Fig. 3A).
|
C). The modest expression of CXCR2 on NKT cells,
compared with expression of CXCR2 mRNA in the neutrophil, may reflect a
resting level or a response to manipulation and may not reflect the
true level of receptor expression that occurs in stimulated NKT cells
in vivo. MIP-2 directs the migration of NKT cells to the spleen after a.c. inoculation of Ag
In comparing routes of Ag inoculation (a.c., i.v.,
or s.c.), we previously observed that only the a.c. route was
associated with accumulation of NKT cells in the spleen
(9). Because MIP-2 was a chemoattractant for NKT cells and
its expression increased selectively in the monocyte/macrophage
population, presumably in transit from the eye to the spleen via the
blood, we reasoned that blocking MIP-2 systemically following OVA
inoculation (a.c.) would prevent the NKT cell accumulation in the
spleen associated with ACAID. MIP-2 was neutralized with specific Abs
in vivo. Mice were given OVA or HBSS (a.c.) and cotreated with either
anti-MIP-2 mAb or rat IgG (i.p.) on days 0 and 4 after inoculation.
Seven days after the initial a.c. inoculation and Ab treatment, splenic
NKT cell frequency was determined by flow cytometry of whole
splenocytes, as previously described (9). Mice that
received OVA (a.c.) and were cotreated with rat IgG displayed a 3-fold
increase in the percentage and a 2-fold increase in the absolute number
of splenic NKT cells, whereas a.c.-inoculated mice that were cotreated
with anti-MIP-2 mAb showed no increase in NKT cells (Fig. 4). Additionally, we noted that MIP-2
increased the percentage of NKT cells in the spleen, but not the
percentage of either the CD3 or NK1.1 single-positive populations
between experimental and control groups (Table I). Administration of anti-MIP-2 mAb
to HBSS-inoculated (a.c.) mice showed no effect on the baseline number
of NKT cells present in the spleen (Fig. 4). These data are in
agreement with our previous observations that a small increase (from
3% in naive mice to 6% in a.c.-inoculated mice) in splenic NKT
cells occurred after inoculation of OVA (a.c.) and was required to
generate ACAID-regulatory T cells.
|
|
Because NKT cells are crucial for the generation of
CD8+ Tr cells and the suppression of the effector
phase of the DTH response, we determined whether blocking the
recruitment of NKT cells to the spleen also blocked the generation of
the Tr cell. Single-cell suspensions of spleen cells prepared from mice
that received either OVA or HBSS (a.c.) in conjunction with
anti-MIP-2 mAb or rat IgG (i.p.) were enriched for T lymphocytes
(containing NKT cells) and used as regulatory cells in a LAT assay
(9, 17). In the LAT assay, all of the cells necessary for
a localized DTH reaction (stimulator, effector) are injected into the
ear pinna of a naive recipient mouse. The presence of Ag-pulsed APCs
and Ag-primed effector T cells initiates the efferent arm of a local
cellular immune response that can be quantified 24 or 48 h later
by measuring the ear swelling. The ear of the recipient mouse acts, in
effect, as a test tube, allowing the local DTH response to occur
regardless of the recipient’s immunologic status. The addition of
regulatory cells to the inoculum tests their ability to suppress the
local DTH response. Here, regulatory cells (enriched splenic T
lymphocytes from experimental mice) were mixed with OVA-pulsed APCs
(stimulators) and T cells enriched from spleens of OVA-primed mice
(effector cells) and cotransferred into the ear pinnae of naive mice.
Previously, we showed that Tr cells from OVA-inoculated (a.c.) mice,
cotransferred with OVA-pulsed PECs and OVA-primed T cells, suppressed
the local DTH response (9). In contrast to regulatory
cells obtained from mice inoculated with OVA (a.c.) and treated with
rat IgG, T cells from spleens of mice inoculated with OVA (a.c.) and
treated with anti-MIP-2 were unable to down-regulate the adoptively
transferred local DTH response (Fig. 5).
The inability to down-regulate a primed T cell response in the
recipient’s ear correlated positively with a statistically significant
decrease in the number of NKT cells in the spleens of mice given OVA
(a.c.) and treated with anti-MIP-2 mAb. Thus, in the absence of
MIP-2, NKT cells were not recruited to the spleen, and
CD8+ Tr cells were not generated. These data
extend our previous reports (9) that an increase in
splenic NKT cells was associated with Ag inoculation into the eye by
showing that NKT cell recruitment is mediated by MIP-2.
|
).
Untreated CXCR2-deficient mice also have fewer numbers of splenic NKT
cells compared with WT mice, suggesting that MIP-2-CXCR2 interactions
also may be important in either the basal trafficking of NKT cells to
lymphoid organs or their development.
|
Traffic patterns of ACAID-conferring macrophages (F4/80+) and NKT cells suggested to us that, similar to the induction of conventional immune responses, a novel group of cells formed clusters in secondary lymphoid organs (i.e., the spleen) for the sole purpose of inducing peripheral tolerance. To examine the anatomical distribution and localization of NKT cells in relation to F4/80+ macrophages and T cells during the induction of ACAID, we visualized F4/80+ cells, conventional T cells, and NKT cells in frozen tissue sections from the spleens of mice at various days post-a.c. inoculation of OVA or vehicle (HBSS) with confocal microscopy.
Tissue sections from mouse spleens 1 day after a.c. inoculation showed
a typical splenic architecture comprised of T cell-rich areas (white
pulp or PALS), red pulp, marginal zones (transition from red pulp to
PALS, the site of Ag presentation), and reticular elements of the
spleen (Fig. 7A). In the
frozen sections harvested 1 day post-a.c. inoculation, NK cells (green)
were randomly scattered throughout the red pulp, whereas NKT cells
(yellow) were rarely observed near the PALS.
F4/80+ cells were observed at a very low
frequency in splenic tissue obtained from both naive B6 mice and
sensitized mice 1 day after a.c. OVA. Also,
F4/80+ cells that were visualized in control
sections were not associated with T cell areas or the marginal zone,
but instead, as previously noted (19), were found as
isolated cells in the red pulp or in the subcapsular space, tightly
associated with splenic reticular elements. Three days after OVA
inoculation (a.c.), F4/80+ cells appeared within
marginal zones of the spleen (data not shown). However, 5–7 days
post-OVA inoculation (a.c.), there was a marked increase in the number
of F4/80+ cells (bright pink) and NKT cells
(yellow) within the marginal zones (Fig. 7, B and
C). As predicted from flow cytometric studies above
correlating increases of NKT and F4/80+ cells to
the spleen after a.c. inoculation, NKT cells colocalized in the
marginal zones with the F4/80+ cells (bright
pink) and conventional marginal zone T cells (orange) (Fig. 7, B–D). Analyses of tissue sections 7 days
post-a.c. inoculation were similar to day 5 in terms of cluster
quantity and heterogeneity.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Moreover, the data showed that MIP-2 mRNA was uniquely up-regulated in mononuclear cells that appeared in the blood following inoculation of Ag via the a.c. and in macrophage cell lines that exhibited the phenotype of eye-derived macrophages. Together, these data imply that eye-derived macrophages, in part, contribute to the production of MIP-2. Furthermore, because neutrophils were excluded from the density gradient preparations for PBMCs, and T cells do not generally produce MIP-2, we concluded that the cells of the monocyte/macrophage population found in the blood were those that produced MIP-2. Moreover, we found that the patterns of MIP-2 up-regulation coincided precisely with the trafficking of the ACAID-conferring cells from the blood to the spleen, as previously described by Wilbanks and Streilein 12 . As our results show, the level of MIP-2 mRNA expression rose nearly 3-fold among blood monocytes at day 3 and returned to baseline by day 7 after a.c. inoculation of Ag. In additional experiments, we observed that as MIP-2 disappeared from the blood monocyte population, its expression increased by 23.2% in the total splenic adherent cell population at day 7 (data not shown).
In studies extending work reported here, we have since identified the cellular source of MIP-2 mRNA as being F4/80+ cells and observed by RT-PCR that F4/80+ cells enriched from PBMCs and harvested from a.c.-inoculated mice expressed comparatively higher levels of MIP-2 mRNA than did enriched cells from i.v.-inoculated mice (manuscript in preparation). Furthermore, such data exclude the possibility of artifactual MIP-2 production due to endotoxin contamination of the Ag, OVA.
Because our previous data showed that NKT cells and F4/80+ cells accumulated in the spleen after OVA was inoculated a.c. and were needed for the generation of the Tr cells (9), we predicted that NKT cells would interact not only with the APCs in the spleen, but also with the Tr cell precursor for the induction of peripheral tolerance and the development of the Tr cell. Indeed, histologic evidence showed the colocalization of at least three cells types (F4/80+ APCs, NKT, and T cells) subsequent to the a.c. inoculation of Ag. It is well known that T cells, APCs, and B cells are in close association in the spleen and other lymphoid organs during the generation of primary Ab responses. This is the first evidence that the set of cells that is required to interact for tolerance induction is different from the set required for immune responses. Conventional wisdom previously suggested that the cells involved in tolerance might be the same as those for an immune response, but that the signals (soluble, cellular) might be different.
Although unknown, we predicted chemokines to be responsible for
directing the trafficking of NKT cells to and from secondary lymphoid
organs. Qin and colleagues reported several years ago that aside from
its expression on neutrophils, the IL-8 receptor (CXCR2) was present
only on a very small subpopulation of CD3+
lymphocytes that coexpressed the NK marker, CD56 (22). In
a subsequent report, Wang and colleagues definitively showed that
neither murine B220+, CD4+,
nor CD8+ cells stained positively with an
anti-mouse IL-8 receptor homologue Ab, whereas a small
subpopulation of NK1.1+ cells did
(23). Although these reports suggested that NKT cells
express the CXCR2 receptor, the data presented here definitively show
that NKT cells respond and migrate preferentially along a chemotactic
gradient of MIP-2, a murine functional IL-8 homologue. Additionally, we
confirmed the mRNA expression of the high-affinity murine IL-8 receptor
homologue, CXCR2, on NKT cells by RT-PCR (Fig. 3C). In
contrast to other chemokine receptors in both the CCR and CXCR
families, murine CXCR2 exhibits little, if any, redundancy in its
ligand binding in that it binds the only two known murine functional
IL-8 homologues, MIP-2 and KC (24). This suggests that in
the mouse, only neutrophils and NKT cells respond to MIP-2. However,
the data presented do not address the role of neutrophils in
tolerance.
The origin of the F4/80+ cells that accumulate with NKT cells in the splenic marginal zones subsequent to a.c. inoculation of Ag is presumed to be the eye, primarily by circumstantial evidence. Wilbanks and Streilein described a population of F4/80+ cells that were indigenous to the iris and ciliary body (12) and also showed that OVA-pulsed PECs were capable of inducing ACAID if injected into the a.c. (12). Given that F4/80 is a marker associated exclusively with professional APCs such as macrophages and dendritic cells (19, 25), and that the Ag-bearing ACAID-conferring signal observed in the blood after a.c. inoculation is F4/80+ (12), it was proposed that the APCs we observe in the marginal zone after a.c. inoculation of Ag included eye-derived APCs. It could be concluded from the confocal data that more F4/80+ APCs were detected in the spleen 7 days post-a.c. inoculation than could have emigrated from the eye. Therefore, the potentially small numbers of eye-derived APCs may also influence the recruitment of additional F4/80+ APCs and their ability to either induce or sustain tolerance. The exact origin of the NKT cells that accumulate in the spleen also remains undefined. It is possible that the NKT cells are sequestered in the spleen from the bloodstream after they emerge from NKT precursor cells in the bone marrow (26) or thymus (27).
Although our studies focused on the interaction of NKT,
F4/80+, and CD3+ T cells
during ACAID induction in the spleen, the results do not exclude the
possibility of other cells being involved in the tolerance-inducing
clusters. Both B cells (28) and perhaps 
cells
(29) are required for ACAID induction, and it is known
that CD4+ and CD8+ T cells
differentiate into afferent and efferent regulatory cells as a result
of these complex cellular interactions. Thus, the question is raised as
to why so many cells are required to interact in ACAID-induced
tolerance. The F4/80+ cell and the B cell are
potential APCs and could present Ag to different T cell populations,
i.e., one may present to CD4+ and the other to
CD8+ T cells. The NKT cell binds to the CD1d
molecule that could be expressed by both conventional APCs and marginal
zone B cells and could deliver amplifying signals (i.e., TGF- and
IL-10) to impose an environment that is regulatory cell promoting.
Alternatively, the two APC populations could present to the same T cell
populations but signal differently. Thus, unlike in the a.c., where
cells are bathed in immunosuppressive aqueous humor, the observations
reported here present evidence for a cell:cell-induced
immunosuppressive microenvironment in the spleen.
Several reports in both mice and humans show that defective or
deficient NKT cells correlate with the emergence of autoimmune disease
(30, 31, 32), implying that NKT cells are crucial for
maintaining peripheral self-tolerance. The precise effector mechanisms
used by NKT cells to stave off autoimmunity are likely to involve
soluble mediators such as IL-4, IL-10, and TGF-. Interestingly, this
exclusive group of cytokines is involved in the induction of tolerance
that is induced via immune-privileged sites, such as the eye, brain,
and testis (10). Our data support the possibility that
certain forms of autoimmunity emerge because of a defect in the
recruitment of CD1-reactive NKT cells to secondary lymphoid organs
where active tolerance is induced. This defect could be at the level of
the local APC that fails to recruit the appropriate cells to the
secondary lymphoid organ. Bendelac and colleagues previously suggested
that CD1 expression might serve to recruit NKT cells and activate them
to release substantial amounts of immunomodulatory cytokines
(33). We propose that "tolerogenic" cytokines, such as
TGF-, induce APCs to produce a selective set of chemokine(s) (as
shown above) necessary for attraction of the CD1-restricted NKT cell to
the site of tolerance induction. Interaction of NKT cells with CD1 on
APCs leads to the release of an immunomodulatory cytokine that
initiates the generation of CD8+ Tr cells.
The unique data reported here show definitively that a specialized chemokine is associated with the movement of cells into secondary lymphoid organs during the activation and differentiation of negative regulatory cells required for peripheral tolerance and active suppression of DTH. In contrast to the cells that interact during immune activation (34), this novel tolerance-inducing cell cluster in the marginal zone is, at the very least, dependent on MIP-2 secreted by tolerance-inducing APCs and on the recruitment of NKT cells for the generation of Tr cells. Although the magnitude and heterogeneity of cells within the cluster are yet to be defined, we can conclude that peripheral tolerance induced via an immune-privileged site is dependent on MIP-2-mediated recruitment of NKT cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Joan Stein-Streilein, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114.
3 Abbreviations used in this paper: Tr, T regulatory; a.c., anterior chamber; ACAID, a.c.-associated immune deviation; DTH, delayed-type hypersensitivity; IP-10, inflammatory protein-10; LAT, local adoptive transfer; SFM, serum-free medium; MIP, macrophage inflammatory protein; PALS, periarteriolar lymphoid sheath; PEC, peritoneal exudate cells; RPA, RNase protection assay; WT, wild type; int, intermediate; rm, recombinant murine; MCP, monocyte chemoattractant protein; Ltn, lymphotactin.
Received for publication August 16, 2000. Accepted for publication September 28, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
upon activation by anti-CD3 or CD1. J. Immunol. 159:2240.24JQ antigen receptor in patients with systemic sclerosis. J. Exp. Med. 182:1163. T cells are critical for the induction of ACAID. Invest. Ophthalmol. Vis. Sci. 41:S117.
24JQ T cells in type 1 diabetes: [Published erratum appears in 1999 Nature 399:84.]. Nature. 391:177.[Medline]
14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1831.This article has been cited by other articles:
|
|
 |
|
  R. E. Cone, S. Chattopadhyay, R. Sharafieh, Y. Lemire, J. O'Rourke, R. A. Flavell, and R. B. Clark T cell sensitivity to TGF-{beta} is required for the effector function but not the generation of splenic CD8+ regulatory T cells induced via the injection of antigen into the anterior chamber Int. Immunol., May 1, 2009; 21(5): 567 - 574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Qiao, K. Lucas, and J. Stein-Streilein Retinal Laser Burn Disrupts Immune Privilege in the Eye Am. J. Pathol., February 1, 2009; 174(2): 414 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Fraccaroli, J. Alfieri, L. Larocca, M. Calafat, G. Mor, C. P. Leiros, and R. Ramhorst A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation Hum. Reprod., January 1, 2009; 24(1): 166 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Grajewski, A. M. Hansen, R. K. Agarwal, M. Kronenberg, S. Sidobre, S. B. Su, P. B. Silver, M. Tsuji, R. W. Franck, A. P. Lawton, et al. Activation of Invariant NKT Cells Ameliorates Experimental Ocular Autoimmunity by A Mechanism Involving Innate IFN-{gamma} Production and Dampening of the Adaptive Th1 and Th17 Responses J. Immunol., October 1, 2008; 181(7): 4791 - 4797. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Watte, T. Nakamura, C. H. Lau, J. R. Ortaldo, and J. Stein-Streilein Ly49 C/I-dependent NKT cell-derived IL-10 is required for corneal graft survival and peripheral tolerance J. Leukoc. Biol., April 1, 2008; 83(4): 928 - 935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chattopadhyay, J. O'Rourke, and R. E. Cone Implication for the CD94/NKG2A-Qa-1 system in the generation and function of ocular-induced splenic CD8+ regulatory T cells Int. Immunol., April 1, 2008; 20(4): 509 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Sonoda, T. Nakamura, H. A. Young, D. Hart, P. Carmeliet, and J. Stein-Streilein NKT Cell-Derived Urokinase-Type Plasminogen Activator Promotes Peripheral Tolerance Associated with Eye J. Immunol., August 15, 2007; 179(4): 2215 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Stein-Streilein and A. W. Taylor An eye's view of T regulatory cells J. Leukoc. Biol., March 1, 2007; 81(3): 593 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Ashour and J. Y. Niederkorn {gamma}{delta} T Cells Promote Anterior Chamber-Associated Immune Deviation and Immune Privilege through Their Production of IL-10 J. Immunol., December 15, 2006; 177(12): 8331 - 8337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Meng, P. Yang, B. Li, H. Zhou, X. Huang, L. Zhu, Y. Ren, and A. Kijlstra CD4+PD-1+ T Cells Acting as Regulatory Cells during the Induction of Anterior Chamber-Associated Immune Deviation. Invest. Ophthalmol. Vis. Sci., October 1, 2006; 47(10): 4444 - 4452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Palmer, J. M. Tulley, E. J. Kovacs, R. L. Gamelli, M. Taniguchi, and D. E. Faunce Injury-Induced Suppression of Effector T Cell Immunity Requires CD1d-Positive APCs and CD1d-Restricted NKT Cells J. Immunol., July 1, 2006; 177(1): 92 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Ashour and J. Y. Niederkorn Peripheral Tolerance Via the Anterior Chamber of the Eye: Role of B Cells in MHC Class I and II Antigen Presentation J. Immunol., May 15, 2006; 176(10): 5950 - 5957. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Masli, B. Turpie, and J W. Streilein Thrombospondin orchestrates the tolerance-promoting properties of TGF{beta}-treated antigen-presenting cells Int. Immunol., May 1, 2006; 18(5): 689 - 699. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nowak, F. Kopp, K. Roelofs-Haarhuis, X. Wu, and E. Gleichmann Oral nickel tolerance: fas ligand-expressing invariant NK T cells promote tolerance induction by eliciting apoptotic death of antigen-carrying, effete B cells. J. Immunol., April 15, 2006; 176(8): 4581 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. M. Vissers, B. C. A. M. van Esch, G. A. Hofman, and A. J. M. van Oosterhout Macrophages induce an allergen-specific and long-term suppression in a mouse asthma model Eur. Respir. J., December 1, 2005; 26(6): 1040 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang-Hoover, P. Finn, and J. Stein-Streilein Modulation of Ovalbumin-Induced Airway Inflammation and Hyperreactivity by Tolerogenic APC J. Immunol., December 1, 2005; 175(11): 7117 - 7124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Faunce, J. L. Palmer, K. K. Paskowicz, P. L. Witte, and E. J. Kovacs CD1d-Restricted NKT Cells Contribute to the Age-Associated Decline of T Cell Immunity J. Immunol., September 1, 2005; 175(5): 3102 - 3109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakamura, A. Terajewicz, and J. Stein-Streilein Mechanisms of Peripheral Tolerance following Intracameral Inoculation Are Independent of IL-13 or STAT6 J. Immunol., August 15, 2005; 175(4): 2643 - 2646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-H. Lin, D. E. Faunce, M. Stacey, A. Terajewicz, T. Nakamura, J. Zhang-Hoover, M. Kerley, M. L. Mucenski, S. Gordon, and J. Stein-Streilein The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance J. Exp. Med., May 16, 2005; 201(10): 1615 - 1625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Oh, S. Kim, S.-H. Park, H. Gu, D. Roopenian, D. H. Chung, Y. S. Kim, and D.-S. Lee Direct Regulatory Role of NKT Cells in Allogeneic Graft Survival Is Dependent on the Quantitative Strength of Antigenicity J. Immunol., February 15, 2005; 174(4): 2030 - 2036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Matzer, F. Rodel, R. M. Strieter, M. Rollinghoff, and H. U. Beuscher Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes Int. Immunol., November 1, 2004; 16(11): 1675 - 1683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Furlan MBP-Specific Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice J. Immunol., July 1, 2004; 173(1): 5 - 5. [Full Text] [PDF]
|
|
|
|
|
|
D. E. Faunce and J. Stein-Streilein The Authors Respond J. Immunol., July 1, 2004; 173(1): 5 - 6. [Full Text] [PDF]
|
|
|
|
|
|
S. Camelo, A. Shanley, A. S. P. Voon, and P. G. McMenamin The Distribution of Antigen in Lymphoid Tissues following Its Injection into the Anterior Chamber of the Rat Eye J. Immunol., May 1, 2004; 172(9): 5388 - 5395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Faunce, A. Terajewicz, and J. Stein-Streilein Cutting Edge: In Vitro-Generated Tolerogenic APC Induce CD8+ T Regulatory Cells That Can Suppress Ongoing Experimental Autoimmune Encephalomyelitis J. Immunol., February 15, 2004; 172(4): 1991 - 1995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. F. H. M. Boonman, G. J. D. van Mierlo, M. F. Fransen, K. L. M. C. Franken, R. Offringa, C. J. M. Melief, M. J. Jager, and R. E. M. Toes Intraocular Tumor Antigen Drains Specifically to Submandibular Lymph Nodes, Resulting in an Abortive Cytotoxic T Cell Reaction J. Immunol., February 1, 2004; 172(3): 1567 - 1574. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang-Hoover and J. Stein-Streilein Tolerogenic APC Generate CD8+ T Regulatory Cells That Modulate Pulmonary Interstitial Fibrosis J. Immunol., January 1, 2004; 172(1): 178 - 185. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Stein-Streilein Invariant NKT Cells as Initiators, Licensors, and Facilitators of the Adaptive Immune Response J. Exp. Med., December 15, 2003; 198(12): 1779 - 1783. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Muller, S. Bischof, F. Sommer, M. Lohoff, W. Solbach, and T. Laskay Differential Production of Macrophage Inflammatory Protein 1{gamma} (MIP-1{gamma}), Lymphotactin, and MIP-2 by CD4+ Th Subsets Polarized In Vitro and In Vivo Infect. Immun., November 1, 2003; 71(11): 6178 - 6183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Johnston, C. H. Kim, D. Soler, M. Emoto, and E. C. Butcher Differential Chemokine Responses and Homing Patterns of Murine TCR{alpha}{beta} NKT Cell Subsets J. Immunol., September 15, 2003; 171(6): 2960 - 2969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Thomas, R. Hou, J. E. Boyson, T. K. Means, C. Hess, D. P. Olson, J. L. Strominger, M. B. Brenner, J. E. Gumperz, S. B. Wilson, et al. CD1d-Restricted NKT Cells Express a Chemokine Receptor Profile Indicative of Th1-Type Inflammatory Homing Cells J. Immunol., September 1, 2003; 171(5): 2571 - 2580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Dieli, M. Taniguchi, M. Kronenberg, S. Sidobre, J. Ivanyi, L. Fattorini, E. Iona, G. Orefici, G. De Leo, D. Russo, et al. An Anti-Inflammatory Role for V{alpha}14 NK T cells in Mycobacterium bovis Bacillus Calmette-Guerin-Infected Mice J. Immunol., August 15, 2003; 171(4): 1961 - 1968. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakamura, K.-H. Sonoda, D. E. Faunce, J. Gumperz, T. Yamamura, S. Miyake, and J. Stein-Streilein CD4+ NKT Cells, But Not Conventional CD4+ T Cells, Are Required to Generate Efferent CD8+ T Regulatory Cells Following Antigen Inoculation in an Immune-Privileged Site J. Immunol., August 1, 2003; 171(3): 1266 - 1271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gillessen, Y. N. Naumov, E. E. S. Nieuwenhuis, M. A. Exley, F. S. Lee, N. Mach, A. D. Luster, R. S. Blumberg, M. Taniguchi, S. P. Balk, et al. CD1d-restricted T cells regulate dendritic cell function and antitumor immunity in a granulocyte-macrophage colony-stimulating factor-dependent fashion PNAS, July 22, 2003; 100(15): 8874 - 8879. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Faunce, R. L. Gamelli, M. A. Choudhry, and E. J. Kovacs A role for CD1d-restricted NKT cells in injury-associated T cell suppression J. Leukoc. Biol., June 1, 2003; 73(6): 747 - 755. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kuwata, H. Watanabe, S.-Y. Jiang, T. Yamamoto, C. Tomiyama-Miyaji, T. Abo, T. Miyazaki, and M. Naito AIM Inhibits Apoptosis of T Cells and NKT Cells in Corynebacterium-Induced Granuloma Formation in Mice Am. J. Pathol., March 1, 2003; 162(3): 837 - 847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xu and J. A. Kapp {gamma}{delta} T Cells in Anterior Chamber-Induced Tolerance in CD8+ CTL Responses Invest. Ophthalmol. Vis. Sci., November 1, 2002; 43(11): 3473 - 3479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. E. Faunce and J. Stein-Streilein NKT Cell-Derived RANTES Recruits APCs and CD8+ T Cells to the Spleen During the Generation of Regulatory T Cells in Tolerance J. Immunol., July 1, 2002; 169(1): 31 - 38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Masli, B. Turpie, K. H. Hecker, and J. W. Streilein Expression of Thrombospondin in TGF{beta}-Treated APCs and Its Relevance to Their Immune Deviation-Promoting Properties J. Immunol., March 1, 2002; 168(5): 2264 - 2273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kawakami, Y. Kinjo, K. Uezu, S. Yara, K. Miyagi, Y. Koguchi, T. Nakayama, M. Taniguchi, and A. Saito Monocyte Chemoattractant Protein-1-Dependent Increase of V{alpha}14 NKT Cells in Lungs and Their Roles in Th1 Response and Host Defense in Cryptococcal Infection J. Immunol., December 1, 2001; 167(11): 6525 - 6532. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yang, J. Luan, Y. Yu, C. Li, R. A. DePinho, L. Chin, and A. Richmond Induction of Melanoma in Murine Macrophage Inflammatory Protein 2 Transgenic Mice Heterozygous for Inhibitor of Kinase/Alternate Reading Frame Cancer Res., November 1, 2001; 61(22): 8150 - 8157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Matzer, T. Baumann, N. W. Lukacs, M. Rollinghoff, and H. U. Beuscher Constitutive Expression of Macrophage-Inflammatory Protein 2 (MIP-2) mRNA in Bone Marrow Gives Rise to Peripheral Neutrophils with Preformed MIP-2 Protein J. Immunol., October 15, 2001; 167(8): 4635 - 4643. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
