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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-07-2305.
 Previous Article | Table of Contents | Next Article 
Blood, 1 February 2003, Vol. 101, No. 3, pp. 807-814
CHEMOKINES
MIP-1 and MIP-1 differentially mediate mucosal and
systemic adaptive immunity
James W. Lillard Jr,
Udai P. Singh,
Prosper N. Boyaka,
Shailesh Singh,
Dennis D. Taub, and
Jerry R. McGhee
From the Department of Microbiology and Immunology,
Morehouse School of Medicine, Atlanta, GA; Department of Microbiology
and The Immunobiology Vaccine Center, University of Alabama at
Birmingham; and Laboratory of Immunology, National Institute on Aging,
Gerontology Research Center, Baltimore, MD.
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Abstract |
Macrophage inflammatory protein-1 (MIP-1) and MIP-1 are
distinct but highly homologous CC chemokines produced by a variety of
host cells in response to various external stimuli and share affinity
for CCR5. To better elucidate the role of these CC chemokines in
adaptive immunity, we have characterized the affects of MIP-1 and
MIP-1 on cellular and humoral immune responses. MIP-1 stimulated strong antigen (Ag)-specific serum immunoglobulin G (IgG) and IgM
responses, while MIP-1 promoted lower IgG and IgM but higher serum
IgA and IgE antibody (Ab) responses. MIP-1 elevated Ag-specific IgG1
and IgG2b followed by IgG2a and IgG3 subclass responses, while MIP-1
only stimulated IgG1 and IgG2b subclasses. Correspondingly, MIP-1
produced higher titers of Ag-specific mucosal secretory IgA Ab levels
when compared with MIP-1. Splenic T cells from MIP-1-
or MIP-1-treated mice displayed higher Ag-specific Th1 (interferon- [IFN-]) as well as selective Th2 (interleukin-5 [IL-5] and IL-6) cytokine responses than did T cells from control groups. Interestingly, mucosally derived T cells from MIP-1-treated mice displayed higher levels of IL-4 and IL-6 compared with
MIP-1-treated mice. However, MIP-1 effectively enhanced
Ag-specific cell-mediated immune responses. In correlation with their
selective effects on humoral and cellular immune responses, these
chemokines also differentially attract CD4+ versus
CD8+ T cells and modulate CD40, CD80, and CD86 expressed by
B220+ cells as well as CD28, 4-1BB, and gp39 expression by
CD4+ and CD8+ T cells in a dose-dependent
fashion. Taken together, these studies suggest that these CC chemokines
differentially enhance mucosal and serum humoral as well as cellular
immune responses.
(Blood. 2003;101:807-814)
© 2003 by The American Society of Hematology.
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Introduction |
Early-acting innate effector molecules are required
to signal the host of potentially lethal agents for protection. In this regard, intestinal epithelial cells produce inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor- (TNF-), IL-6, and IL-8.1,2 Recently, human nasal- and
adenoid-derived epithelial cells have been shown to secrete CC
chemokines such as macrophage inflammatory protein-1 (MIP-1) and
MIP-1.3 Although MIP-1 and MIP-1 are derived from
separate genes, they are highly homologous (about 60%
identity).4 While MIP-1 and MIP-1 bind
CCR5,5,6 MIP-1 also binds CCR1 7,8 as
well as CCR3 in the mouse,9 and MIP-1 is a ligand for
CCR8.10 These chemokines are produced by epithelial
cells,3,11-13 lymphocytes,14 and
platelets15 and act as potent chemoattractants for
monocytes,16,17 natural killer (NK)
cells,18 eosinophils,19 and dendritic
cells.20,21 Several studies have also demonstrated a
selective effect of MIP-1 and MIP-1 on CD8+ and
CD4+ T-cell subset migration22,23 and
microvascular cell adhesion.24 Moreover, MIP-1 was
found to be more efficient at the chemotaxis of B cells when
compared with MIP-1.23 However, it is not certain how
these chemokines affect adaptive immunity.
MIP-1 and MIP-1 have been shown to induce lymphocyte migration
into the nasal mucosa and are highly expressed by influenza virus-infected bronchial and nasal epithelial cells.13
Moreover, Mycobacterium tuberculosis-infected murine
macrophages or Klebsiella pneumoniae challenge results in
the expression MIP-1.17,25 Anti-MIP-1 monoclonal
antibody (mAb) treatment has been shown to block Th1 responses
that are required to clear a Cryptococcus neoformans
infection.26 Correspondingly, MIP-1 has been shown to
enhance interferon-gamma (IFN-) production of in vitro
antigen (Ag)-stimulated T cells27 while decreasing IL-4
production.28 In contrast, CCR5 gene knock-out
(CCR5 /) and MIP-1/ mice displayed
augmented Th1 responses to Leishmania donovani Ag compared
with wild-type mice when challenged with L
donovani.29 These studies have addressed the
expression of chemokines in response to microbial infection and the
important role of MIP-1 and MIP-1 in the outcome of inflammatory
and infectious diseases. Despite these studies, the mechanisms that
these chemokines use for the transition of acute inflammation to host
mucosal immune responses remain elusive.
We have previously demonstrated that lymphotactin and RANTES
(regulated on activation normal T cells expressed and secreted) enhance mucosal and systemic immunity when nasally coadministered with
a protein Ag.30,31 These previous studies clearly suggest that chemokines may be major regulatory molecules facilitating the
induction of adaptive mucosal immunity. The current study seeks to
identify the effects of MIP-1 and MIP-1 on mucosal and systemic
immune responses. Our results demonstrate that both of these chemokines
differentially regulate humoral and cellular immunity.
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Materials and methods |
Reagents
Murine MIP-1 and MIP-1 were purchased from PeproTech
(Rocky Hill, NJ). The potential level of endotoxin contamination was quantified by the chromogenic Limulus amebocyte lysate assay
(Associates of Cape Cod, Falmouth, MA) to be less than 5 EU/mg.
Chicken egg albumin (OVA), bovine serum albumin (BSA), and hen egg
white lysozyme were purchased from Sigma Chemical (St Louis, MO).
Cholera toxin was purchased from List Biologicals (Campbell, CA).
Mice and immunizations
Female C57BL/6 and BALB/c mice, aged 5 to 6 weeks, were procured
from The Jackson Laboratory (Bar Harbor, ME). All mice were housed in horizontal laminar flow cabinets free of microbial pathogens. Routine Ab screening for a large panel of pathogens and histologic analysis of organs and tissues were performed to ensure that mice were
pathogen free. C57BL/6 and BALB/c mice used in immunization studies
were 8 to 12 weeks of age. Following anesthesia, mice were nasally
immunized on days 0, 7, and 14 with 75 µg OVA in the presence or
absence of 0.01 to 5 µg MIP-1 or MIP-1 in 10 µL
phosphate-buffered saline (PBS) (5 µL per nare). Control mice received 75 µg OVA and 1 µg cholera toxin in 10 µL PBS, and
negative control mice received 75 µg hen egg lysozyme or PBS alone.
BALB/c mice of similar age were also immunized to confirm the results obtained using C57BL/6 mice. Experimental groups consisted of 5 mice,
and studies were repeated 3 to 5 times. The guidelines proposed by the
National Research Council's committee for the Care of Laboratory
Animal Resources Commission of Life Sciences were followed to minimize
animal pain and distress.
Sample and tissue collection
Fecal samples were weighed and dissolved in PBS containing 0.1%
sodium azide (eg, 1 mL per 100 mg of fecal pellet). Following suspension by vortexing for 10 minutes, fecal samples were centrifuged and supernatants were collected for analysis. Nasal and vaginal cavities were rinsed 3 times with 50 µL PBS. Blood samples were collected by tail vein bleeding, and serum was obtained following centrifugation. Serum and mucosal secretions were collected on days 0, 7, 14, and 21 for OVA-specific Ab analysis by enzyme-linked immunosorbent assay (ELISA). Mice were killed by
CO2 inhalation 1 week after the last immunization to
quantify the OVA-specific Ab-forming cells (AFCs) and T-cell responses
present in immune compartments.
OVA-specific Ab detection by ELISA
Fecal and serum sample levels of OVA-specific Abs were measured
by ELISA, as previously described.30 Briefly, 96-well
Falcon 3912 flexible ELISA plates (Fisher Scientific, Pittsburgh, PA) were coated with 100 µL of 1 mg/mL OVA in PBS overnight at 4°C and
blocked with 1% BSA (Sigma) in PBS (B-PBS) for 3 hours at room
temperature. Individual samples (100 µL) were added and serially diluted in B-PBS. After overnight incubation at 4°C and 3 washes using PBS containing 0.05% Tween 20 (PBS-T), titers of immunoglobulin M (IgM), IgG, or IgA were determined by the addition of a 0.33 µg/mL horseradish peroxidase (HRP)-conjugated goat antimouse µ, , or heavy chain-specific antisera (Southern Biotechnology Associates, Birmingham, AL) in B-PBS-T. Similarly, 100 µL
biotin-conjugated rat antimouse 1 (G1-7.3 at 12.5 ng/mL), 2a
(R19-15 at 125 ng/mL), 2b (R12-3 at 12.5 ng/mL), 3 (R40-82 at 50 ng/mL), and  (G1-7.3 at 1.25 µg/mL) (PharMingen, San Diego, CA)
heavy chain-specific mAbs were used to determine IgG subclass and IgE
isotype titers.30 After incubation and wash steps, 100 µL of 0.5 µg/mL HRP-antibiotin Ab (Vector Laboratories, Burlingame,
CA) in B-PBS-T or 500 ng/mL poly-HRP80 streptavidin (Research
Diagnostics, Flanders, NJ) in Poly-HRP Diluent (Research
Diagnostics) was added to IgG subclass or IgE detection wells,
respectively, and incubated for 3 hours at room temperature. Following
incubation, the plates were washed 6 times and the color reaction for
ELISA was developed by adding 100 µL of 1.1 mM
2,2'-azino-bis(3)-ethylbenzthiazoline-6-sulfonic acid (Sigma)
in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01%
H2O2 (ABTS solution). End-point titers were
expressed as the reciprocal log2 of the highest dilution,
which indicated an optical density that was 415 nm (OD415)
of at least 0.1 OD unit above the OD415 of negative
controls after a 20-minute incubation.30
Cell isolation
Single-cell suspensions of spleen, Peyer patches, nasal tract,
and cervical lymph nodes were prepared by aseptically removing tissues
and then passing them through a sterile wire screen. Lower respiratory
tract tissues were excised, minced, and further disrupted by stirring
in collagenase type IV (Sigma) in RPMI 1640 (collagenase solution)
during incubation at 37°C.30 After removal of Peyer patches, the small intestine was cut into 1 cm strips and stirred in
PBS containing 1 mM EDTA (ethylenediaminetetraacetic acid) at
37°C for 30 minutes. Next, intestinal lamina propria lymphocytes were
isolated by digesting tissue in the collagenase solution for about 45 minutes with stirring at 37°C. Lymphocytes were further purified
using a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient,
collecting at the 40% to 75% interface.30 Cell
suspensions were washed twice in RPMI 1640. Lymphocytes were maintained
in complete medium, which consisted of RPMI 1640 supplemented with 10 mL/L nonessential amino acids (Mediatech, Washington, DC), 1 mM sodium pyruvate (Sigma), 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (Mediatech), 100 U/mL penicillin, 100 µg/mL
streptomycin, 40 µg/mL gentamycin (Elkins-Sinn, Cherry Hill, NJ), 50 µM mercaptoethanol (Sigma), and 10% fetal calf serum (FCS) (Atlanta
Biologicals, Norcross, GA). T-cell fractions were obtained by passing
single-cell suspensions over nylon wool for 1 hour at 37°C (more than
95% purity).
IgA, IgM, and IgG enzyme-linked immunospot (ELISPOT)
analysis
An ELISPOT assay was used to detect total or OVA-specific
AFCs.30 In brief, 96-well Millititer HA
nitrocellulose-based plates (Millipore, Bedford, MA) were coated with
100 µL of 1 mg/mL OVA in PBS, PBS only (negative control), or 0.5 µg/mL goat antimouse Ig (H+L) polyclonal Ab (positive control)
(Southern Biotechnology Associates) and incubated overnight (12 hours)
at 4°C. Subsequently, wells were blocked with B-PBS for 2 hours and
washed with complete media. Whole cells were added to wells in
duplicate at 1 × 106/mL, 5 × 105/mL, and
1 × 105/mL concentrations in complete medium and
incubated for 6 hours at 37°C in 5% CO2. After washing
with PBS-T, individual AFCs were detected with HRP-labeled goat
antimouse µ, , or chain-specific Abs (1 µg/mL) (Southern
Biotechnology Associates), visualized by adding
3-amino-9-ethylcarbazole (AEC) buffer (Moss, Pasadena, MD), and counted
using a dissecting microscope (SZH Zoom Stereo Microscope System;
Olympus, Lake Success, NY).
Ag-specific T-helper cell responses
Purified T cells were cultured at a density of
5 × 106/mL with 1 × 106/mL T
cell-depleted and irradiated 30 Gy (3000 rad) splenic feeder cells in complete medium containing OVA (1 mg/mL) at 37°C in 5% CO2. T cells were cultured in 96-well round-bottom plates
(Corning Glass Works, Corning, NY) to determine Ag-specific
proliferative responses. After 3 days of culture, cells were pulsed
with 0.5 µCi (0.0185 MBq) methyl-3H-thymidine
(Amersham Life Sciences, Arlington Heights, IL) per well for 18 hours. Cells were harvested on glass microfiber filter paper
(Whatman, Clifton, NJ), and radioactivity levels were obtained by liquid scintillation counting.
Effects of MIP-1 and MIP-1 on resting and OVA-stimulated
DO11.10 primary lymphocytes
Splenocytes from DO11.10 mice were isolated and added at a
density of 1 × 106 cells per milliliter in complete
medium containing 0, 1, 10, 100, or 1000 ng/mL MIP-1 and MIP-1. A
class II-restricted OVA peptide containing amino acids 323 to 339 (50 µg/mL) was used to activate primary OVA-specific T-cell
receptor (TCR) transgenic CD4+ T cells from
DO11.10 mice.32 Lymphocytes were also cultured with
optimal doses of concanavalin A (ConA; 5 µg/mL) or antimouse CD3 mAb (10 µg/mL-coated plates) as positive controls or alone as
negative controls. After incubation for 2 days, cells were stained with
phycoerythrin (PE)-conjugated rat antimouse CD28, CD30, CD37, CD80,
CD86, CD154, or CD153 (PharMingen) and Cy5-conjugated CD4, and/or B220,
fluorescein isothiocyanate (FITC)-conjugated MAC-1 and/or CD8 mAbs
(PharMingen) for 30 minutes with agitation. Lymphocytes were
then washed with fluorescence-activated cell sorter (FACS)
buffer (PBS with 1% BSA) and fixed in 2% paraformaldehyde in PBS and
analyzed by flow cytometry using a FACSCaliber (Becton Dickinson,
San Jose, CA).
Cytokine analysis by ELISA
For the assessment of cytokine production, 800 µL of culture
supernatants from 24-well flat bottom plates (Corning Glass Works) was
harvested after 2 days of incubation. The T-helper (Th) cytokines, IL-2, IL-4, IL-5, IL-6, IL-10, TNF-, IFN-, and
granulocyte-macrophage colony-stimulating factor (GM-CSF), in
cell culture supernatants were determined by ELISA following the
manufacturer's instructions (E-Biosciences, San Diego, CA). The IL-5
cytokines in culture supernanatants were determined as described
previously by Lillard et al.30 Briefly, Falcon 3912 Microtest plates (Fischer Scientific) were coated with 100 µL of 2.5 µg/mL rat antimouse IL-5 (PharMingen) in 0.1 M
bicarbonate buffer (pH 9.5) overnight at 4°C and blocked with 3% BSA
in PBS at room temperature for 3 hours. Next, serially diluted
recombinant murine cytokines as standards (PharMingen) or cultured
supernatant samples were added in duplicate and incubated overnight at
4°C. The plates were washed with PBS-T and incubated with 0.2 µg/mL
biotinylated secondary murine cytokine detection Abs (PharMingen) in
B-PBS-T at room temperature for 3 hours. After washing with PBS-T and
PBS, wells were incubated for 2 hours in 100 µL of 0.5 µg/mL
peroxidase-conjugated antibiotin Ab (Vector Laboratories) and developed
with ABTS solution, as described above. The cytokine ELISA assays were
capable of detecting 15 pg/mL IFN-; 5 pg/mL IL-2, IL-4, and IL-5;
100 pg/mL IL-6; and 200 pg/mL IL-10.
In vitro T-cell proliferation assay
Proliferating T lymphocytes were labeled with 5-bromo-2'-deoxy
uridine (BrdU, Roche Diagnostics, Dusseldorf, Germany);
subsequently, BrdU incorporation was detected using a scanning
multiwell spectrophotometer (SpectraMax 250 ELISA reader, Molecular
Devices, Sunnyvale, CA). In brief, after 2 days of culture, cells at a
density of 1 × 106/mL were transferred in polystyrene
96-well plate (Corning Glass Works). An aliquot of 10 µL BrdU
labeling solution (10 µm/mL) per well was added and incubated for 18 hours at 37°C in a humidified atmosphere containing 5%
CO2. The cells were then fixed and incubated with 100 µL
nuclease in each well for 30 minutes at 37°C. The cells were washed
with complete media and again incubated with BrdU solution for 30 minutes at 37°C. The incorporation was developed in ABTS solution,
and the optical density was read at 450 nm.
Cytotoxic T lymphocyte (CTL) analysis
The CD8+ T-cell subset from the cervical lymph
nodes, Peyer patches, and spleen were isolated and purified as
described above and restimulated ex vivo with the class I-restricted
peptide of OVA257-264 (SIINFEKL) for 5 days with syngeneic
EL4 feeder cells.33 The cytotoxic T lymphocyte (CTL)
response was determined by a 4-hour 51Cr release assay
using E.G7.OVA target cells.34
Statistics
The data are expressed as the mean ± 1 SEM and compared using
a 2-tailed Student t test or an unpaired Mann-Whitney
U test. The results were analyzed using the Statview II
statistical program (Abacus Concepts, Berkeley, CA) for Macintosh
computers and were considered statistically significant if P
values were less than .05. When cytokine levels were below the
detection limit (BD), they were recorded as one half the lower
detection limit (eg, 50 pg/mL for IL-6) for statistical analysis.
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Results |
MIP-1 and MIP-1 stimulate OVA-specific systemic Ab
responses
We first determined the optimal dose of MIP-1 and MIP-1 that
would affect Ag-specific serum Ab responses. For this purpose, mice
were nasally administered 3 times at weekly intervals with OVA (75 µg) in the presence of increasing concentrations of the 2 chemokines
(eg, 0.0, 0.01, 0.1, 1.0, and 5.0 µg). Accordingly, we analyzed
OVA-specific Ab isotypes and IgG subclasses in sera and mucosal
secretions. Significant titers of OVA-specific Ab responses were
elicited when mice were given a 1.0 µg dose of MIP-1 or MIP-1,
and the Ab responses were not further increased when chemokine doses
were increased (data not shown). Therefore, a dose of 1 µg MIP-1
or MIP-1 was used for subsequent in vivo experiments. Mice nasally
immunized 3 times with OVA plus 1 µg MIP-1 or MIP-1 displayed
significantly increased Ag-specific serum IgG Ab levels (Figure
1). Serum IgE and IgA Ab responses were
noted only in mice that received MIP-1 but not MIP-1 (Figure 1).
Both molecules clearly exerted different effects on serum IgG subclass
responses. The humoral adjuvant activity of MIP-1 induced
significant increases in anti-OVA IgG1 and IgG2b Ab titers followed by
IgG2a and IgG3, while MIP-1 only induced IgG1 and IgG2b Ab responses
(Figure 2).
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| Figure 1.
OVA-specific serum IgA, IgM, IgG, and IgE Ab responses
following nasal immunization with MIP-1 or MIP-1.
Groups of 5 C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA and 0.0 ( ) or 1.0 µg MIP-1 or MIP-1 ( )
in PBS. The data presented are the mean Ab titers ± SEMs of 3 separate experiments. ELISA determined the distribution of OVA-specific
serum and fecal Ab titers on day 21. Asterisks indicate statistically
significant differences (P < .05) from Ab titers of mice
immunized with OVA alone.
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| Figure 2.
OVA-specific serum IgG subclass Ab responses following
nasal immunization with MIP-1 or MIP-1.
Groups of 5 C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA and 0.0 () or 1.0 µg MIP-1 or MIP-1 ()
in PBS. The data presented are the mean IgG subclass Ab titers ± SEMs of 3 separate experiments. ELISA determined the distribution of
OVA-specific serum and fecal Ab titers on day 21. Asterisks indicate
statistically significant differences (P < .05) from Ab
titers of mice immunized with OVA alone.
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MIP-1 and MIP-1 effects on mucosal IgA Ab
responses
We next asked whether the adjuvant activity of nasally
coadministered MIP-1 or MIP-1 could promote mucosal secretory IgA Ab responses. Analysis of OVA-specific S-IgA Ab responses in mucosal secretions revealed significant S-IgA Ab titers in fecal extracts as
well as vaginal and nasal washes of mice nasally immunized with OVA
plus MIP-1 or MIP-1 (Table 1).
These results were further confirmed by analysis of OVA-specific AFCs
in cells isolated from mucosal and systemic tissues. Mice that received
OVA alone did not display substantial Ag-specific AFCs in any of the
tissues analyzed (Figure 3). Both
MIP-1 and MIP-1 increased OVA-specific IgM and IgG AFCs in
splenic and respiratory tract cell isolates but not in intestinal
lamina propria-derived cells (Figure 3). We noted the presence of
significant IgA AFCs in tissues from mice immunized with MIP-1 but
limited IgA AFC generation by MIP-1 (Figure 3). These results
suggest that the nasal delivery of MIP-1 or MIP-1 enhanced
OVA-specific peripheral IgG Ab responses, while MIP-1 significantly
stimulated S-IgA Abs.
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| Figure 3.
OVA-specific AFCs in spleen, nasal tract, lung, and
intestinal lamina propria.
Groups of 5 C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA and 0.0 ()or 1.0 µg MIP-1 ( ) or MIP-1
() in PBS. Levels of OVA-specific AFCs present in spleen, intestinal
lamina propria, and respiratory tracts, including associated lymphoid
tissue, were determined by ELISPOT analysis 7 days after the last
immunization. AFCs below detectable levels (fewer than 10 AFCs per
106 lymphocytes) are designated BD. The data presented are
the mean AFCs ± SEMs, in duplicate cultures, of 3 separate
experiments. Asterisks indicate statistically significant differences
(P < .05) from AFCs of mice immunized with OVA
alone.
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Proliferation and cytokine secretion by MIP-1- and
MIP-1-induced OVA-specific T cells
Because MIP-1 and MIP-1 differentially regulated mucosal and
systemic Ab responses, we next examined whether the differences in
their effects could be attributed to their ability to differentially promote Th1- versus Th2-type cytokine responses. The CD4+ T
cells isolated from the lower respiratory tract, Peyer patches, cervical lymph nodes (CLNs), or spleens of mice immunized with OVA in
the presence of MIP-1 or MIP-1 exhibited marked increases in
OVA-specific proliferative responses in comparison with
CD4+ T cells derived from control mice that received OVA
alone (Figure 4). In addition, these
chemokines enhanced the splenic-, Peyer patch-, lower respiratory
tract-, and CLN-derived Th cell-derived responses to in vitro
restimulation with OVA. In fact, CD4+ T cells from these
inductive lymphoid tissues of mice immunized with either MIP-1 or
MIP-1 exhibited greater IFN- secretion when compared with
CD4+ T cells from control mice given OVA only (Figure
5). IFN- responses were always higher
in groups that received MIP-1. On the other hand, MIP-1 promoted
higher levels of Th2 cytokine responses (ie, IL-4, IL-5, and IL-6) by
ex vivo-restimulated OVA-specific T cells (Figure 5). Thus, MIP-1
and, to a lesser degree, MIP-1 promote IFN- and Th1-type
responses, while IL-4 and Th2-type responses were especially induced
by MIP-1.
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| Figure 4.
Proliferation responses by OVA-specific CD4+
T cells from mice immunized with OVA plus MIP-1 or MIP-1.
Groups of 5 C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA and 0.0 ()or 1.0 µg MIP-1 () or MIP-1
() in PBS. One week after the last immunization, lower respiratory
tract (lung and mediastinal lymph nodes)-, Peyer patch-, spleen-, and
CLN-derived T cells were purified and cultured at a density of
5 × 106/mL with 500 µg/mL OVA for 3 days with T
cell-depleted, irradiated splenic feeder cells
(1 × 106/mL) in complete medium. Experimental groups
consisted of 5 mice, and studies were repeated 3 times. Proliferation
was measured by 3H-thymidine incorporation. The
stimulation index corresponds to counts per minute (cpm) of cell
cultures containing OVA divided by the cpm of cultures without OVA. The
data presented are the mean stimulation indexes ± SEMs of
quadruplicate cultures. Asterisks indicate statistically significant
differences (P < .05) from the stimulation index of mice
immunized with OVA alone.
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| Figure 5.
MIP-1 and MIP-1 differentially regulate
Ag-specific Th cytokine responses.
Groups of 5 C57BL/6 mice were intranasally immunized on days 0, 7, and
14 with 75 µg OVA and 0.0 () or 1.0 µg MIP-1 () or
MIP-1 () in PBS. One week after the last immunization, spleen-
(A), Peyer patch- (B), lower respiratory tract (lung and mediastinal
lymph nodes)- (C), and CLN-derived (D) T cells were purified and
cultured at a density of 5 × 106/mL with 500 µg/mL OVA
for 5 days with T cell-depleted, irradiated splenic feeder cells
(1 × 106/mL) in complete medium. Experimental groups
consisted of 5 mice, and studies were repeated 3 times. Cytokine
production of cultured supernatants was determined by ELISA. Th1- and
Th2-type cytokine profiles are presented as the mean cytokine levels
(picograms per milliliter) ± SEMs of duplicate cultures from each
group. Asterisks indicate statistically significant differences
(P < .05) from cytokine levels of mice immunized with OVA
alone, while cytokines below detectable levels are designated
BD.
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MIP-1, but not MIP-1, promotes mucosal and systemic
CD8+ CTL responses
It was important to establish whether MIP-1 and/or MIP-
support CTL responses. For this purpose, CLN, Peyer patch, and spleen CD8+ T cells were assessed for their cytotoxic T-cell
activity. No specific CTL response was noted in CLN, Peyer patch, or
spleen cells from control mice immunized with OVA only or MIP-1 plus OVA. In contrast, cells from mice that received the MIP-1 plus OVA
showed dramatic increases in the percent cell lysis of the H-2b-restricted EG7.OVA tumor cell line34;
interestingly, increased CTL responses were seen in cells isolated from
both mucosal and systemic immune compartments (Figure
6).
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| Figure 6.
MIP-1- and MIP-1-mediated OVA-specific CTL
responses.
Groups of 5 C57BL/6 mice were nasally immunized on days 0, 7, and 14 with 75 µg OVA and 0.0 () or 1.0 µg MIP-1 ( ) or MIP-1
( ) in PBS. One week after the last immunization, spleen-, Peyer
patch-, and CLN-derived CD8+ lymphocytes were purified and
restimulated ex vivo with OVA peptide 257-264 (SIINFEKL) for 5 days
with syngeneic EL4 cells in complete medium. Experimental groups
consisted of 5 mice, and studies were repeated 3 times. The cytotoxic
response was determined by 4-hour 51Cr release against
E.G7.OVA or OVA peptide 257-264 (SIINFEKL) pulsed EL4 cells. Data shown
are mean values of triplicates obtained at varying effector-target
ratios as indicated.
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Effects of MIP-1 and MIP-1 differentially regulate
costimulatory molecule expression by Ag-presenting cells in
vitro
The adjuvant effects of cholera toxin (CT) have been attributed to
the ability of this enterotoxin to up-regulate CD86
expression.35 Earlier studies with RANTES showed that it
enhances CD80 but not CD86 expression.33 To better
elucidate the mechanisms underlying the adjuvant effects of MIP-1
and MIP-1, we assessed their potential to modulate the expression of
costimulatory molecules (ie, CD80, CD86, and CD40) on B cells and that
of corresponding receptors (ie, CD28 and CD154/gp39) on T cells.
Results depicted in Figure 7 clearly show
that MIP-1 and MIP-1 differentially regulate costimulatory
molecule expression. MIP-1 but not MIP-1 enhanced CD80 expression
by both resting and Ag-stimulated B cells from DO11.10 mice (Figure 7).
The stimulatory effect of MIP-1 was also seen on CD86 expression by
resting B cells while MIP-1 did not significantly affect CD86
expression by resting or OVA-stimulated B cells (Figure 7). CD40
expression on resting and OVA-stimulated B cells was also up-regulated
by MIP-1, but only OVA-stimulated B cells showed increased CD40 expression after treatment with MIP-1 (Figure 7). The same pattern of effects was seen on macrophages (CD11b+) where MIP-1
but not MIP-1 up-regulated CD80 and CD86 expression (data not
shown).
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| Figure 7.
Regulation of CD80, CD86, and CD40 expression by
B220+ cells by MIP-1 and MIP-1.
Splenocytes from DO11.10 mice were incubated with 0, 1, 10, 100, or
1000 ng/mL MIP-1 (triangles) or MIP-1 (squares) in plates
containing 0 (open symbols) or 500 (filled symbols) µg/mL OVA. The
percent increase (or decrease) of the costimulatory molecule expression
by resting (open symbols) or OVA-activated (filled symbols) lymphocytes
was calculated as the percent of double-positive B220+ and
CD80+, CD86+, or CD40+ cells in
cultures containing MIP-1 or MIP-1 minus the percent gated of
double-positive cells in cultures without these chemokines, divided by
the latter. Studies were repeated 3 times, and the data presented are
the mean percent changes ± SEMs of these experiments.
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MIP-1 and MIP-1 differentially regulate costimulatory
molecules on T cells
To further establish the differences of MIP-1 and MIP-1
effects on distinct regulation of costimulatory signals, we
next assessed their potential to modulate the expression of receptors for costimulatory molecules expressed by T cells. MIP-1
significantly increased the expression of CD28 and CD154 (ie, CD40L and
gp39) by OVA-stimulated CD4+ T cells; on the other hand,
MIP-1 promoted only modest increases in CD154 expression by
OVA-stimulated CD4+ T cells (Figure
8). MIP-1 did not affect CD28
expression by either resting or OVA-stimulated CD4+ T
cells. The same pattern of regulatory effects by MIP-1 and MIP-1
were observed by CD8+ T cells. In contrast with the effect
on CD4+ T cells, MIP-1 failed to stimulate CD40L
expression by Ag-stimulated CD8+ T cells (data not
shown).
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| Figure 8.
Regulation of CD28 and CD154 (CD40L) expression on
CD4+ cells by MIP-1 and MIP-1.
Spleen cells from DO11.10 mice were incubated with 0, 1, 10, 100, or
1000 ng/mL MIP-1 (triangles) or MIP-1 (squares) in plates
containing 0 (open symbols) or 500 (filled symbols) µg/mL OVA. The
percent increase (or decrease) of the costimulatory molecule expression
by resting (open symbols) or OVA-activated (filled symbols) T cells was
calculated as the percent of double-positive CD4+ and
CD28+ or CD154+ cells in cultures containing
MIP-1 or MIP-1 minus the percent gated of double-positive cells
in cultures without these chemokines, divided by the latter. Studies
were repeated 3 times, and the data presented are the mean percent
changes ± SEMs of these experiments.
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We finally investigated the effect of MIP-1 and MIP-1 on the
expression of CD137 (4-1BB) by CD4+ and CD8+ T
cells, because this molecule has been associated with the preferential development of CTL and Th1-type responses.36-40 MIP-1
dramatically increased the expression of 4-1BB on both OVA-activated
CD4+ and CD8+ T cells (Figure
9). On the other hand, MIP-1 did not
enhance CD137 expression by CD4+ T cells and only modestly
up-regulated the expression of this molecule on CD8+ T
cells. Taken together, our findings suggest that the differential adjuvant activity of MIP-1 and MIP-1 could result from their distinct effects on the expression of T- and B-cell costimulatory molecules.
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| Figure 9.
Regulation of CD137 (4-1BB) expression on
CD4+ and CD8+ cells by MIP-1 and MIP-1.
Spleen cells from DO11.10 mice were incubated with 0, 1, 10, 100, or
1000 ng/mL MIP-1 (triangles) or MIP-1 (squares) in plates
containing 0 (open symbols) or 500 (filled symbols) µg/mL OVA. The
percent increase (or decrease) of the costimulatory molecule expression
by resting (open symbols) or OVA-activated (filled symbols) T cells was
calculated as the percent of double-positive CD4+ or
CD8+ and 4-1BB+ cells in cultures containing
MIP-1 or MIP-1 minus the percent gated of double-positive cells
in cultures without these chemokines, divided by the latter. Studies
were repeated 3 times, and the data presented are the mean percent
changes ± SEMs of these experiments.
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Discussion |
The spectrum of mucosal cells expressing MIP-1- and
MIP-1-specific receptors as well as the ability of these chemokines to attract lymphocytes and affect the outcome of Th1- and Th2-mediated disease pathologies provided the rationale to test the effects of
MIP-1 and MIP-1 on acquired mucosal immunity. The results reported here support our hypothesis that MIP-1 and MIP-1 are able to enhance the development of both humoral as well as
cellular mucosal and systemic immunity. For the first time, we have
also shown that MIP-1- and MIP-1-mediated immunity is fostered
by differential regulation of costimulatory molecule expression for support of humoral and cell-mediated immune responses.
Immunization of mice with OVA and MIP-1 or MIP-1 enhanced
Ag-specific serum and mucosal Ab and CD4+ T-cell
proliferative and cytokine responses in systemic and mucosal compartments. Previous studies in our laboratory have shown that the
classical mucosal adjuvant, CT, can induce Ag-specific IgE and IgG1 Ab
titers.41 However, when RANTES was used as an adjuvant, Ag-specific serum IgG subclass Ab responses were predominantly IgG2a,
followed by IgG2b, IgG3, and IgG1 OVA-specific titers.31 However, nasal immunization regimens that use soluble protein Ag often
result in higher IgG1 Ag-specific Ab titers.42,43 In this
regard, both MIP-1 and MIP-1 supported OVA-specific IgG1 and
IgG2b responses. The cytokine IL-4 supports IgG1 and IgE Ab generation
and production44-46; hence, the levels of anti-OVA IgG1
Abs were consistent with the observed cytokine secretion by
OVA-restimulated CD4+ T cells of MIP-1- and MIP-1-
treated mice. Interestingly, the Ag-induced secretion of IL-4 was most
pronounced in CD4+ T lymphocytes from mice immunized with
MIP-1 plus OVA.
IFN- production is often associated with IgG2a and IgG3 Ab
production47 and may account for the MIP-1- and
MIP-1-mediated anti-OVA IgG2a and IgG3 Ab responses. Even low doses
of IFN- (1500 units) have been shown to increase IgG2a production in
vivo, while considerably higher doses of IFN- (12 500 units) are
required to induce decreases in IgG1 and IgE responses.48
In fact, the Ag-specific IFN- responses were greatest in mice
immunized with MIP-1 plus OVA, which correlates with the
MIP-1-induced increases in OVA-specific IgG2a and IgG3 Ab
responses. Taken together, the analysis of the OVA-specific humoral and
T-helper responses revealed that MIP-1 yields a mixed Th1- and
Th2-type response with strong IFN- and lower IL-4, while MIP-1
generates a predominate Th2 response.
The precise cytokine signals required for S-IgA production are not
completely understood, and studies support that both Th1- and Th2-type
cell-derived cytokines are important for S-IgA.49-51 We
have previously shown that chemokines like lymphotactin and RANTES also
induce S-IgA.30,31 Clearly, serum and mucosal Ag-specific Ab responses were enhanced by MIP-1 and MIP-1. However, MIP-1 yielded higher increases in S-IgA responses in fecal, nasal, and vaginal secretions. In confirmation, anti-OVA AFCs observed in the
spleen, intestinal lamina propria, nasal tract, and lung confirmed the
Ag-specific IgA Abs detected in the corresponding mucosal secretions.
The heightened IgA Ab response generated by MIP-1 also correlates
with the predominant Th2 > Th1 cytokine response induced by
this chemokine.
We have also shown that the cytokine responses promoted by these
chemokines, particularly MIP-1, can induce significant Ag-specific CTLs in both systemic and mucosal tissues. No doubt, the Th1 > Th2
profile of CLN, Peyer patch, and splenic lymphocytes from the
MIP-1-treated mice also supported the observed Ag-specific CTL
responses. This response was mediated by CTLs from both mucosal (CLNs
and Peyer patches) and systemic (splenic) immune compartments. Our
results are clearly consistent with the pattern of cytokine and IgG
subclass responses. Most important, our findings demonstrate that CTL
responses can be induced in mucosal and systemic tissues by a
protein-based vaccine harboring MIP-1.
Cytokines produced by CD4+ T cells after mucosal
administration of MIP-1 or MIP-1 only partially explain the
increases in OVA-specific Ab and T-cell responses. While these
chemotactic molecules directly aid in the accumulation of lymphocytes
at infection or immunization sites, lymphocyte recruitment alone does
not ensure the initiation of an adaptive immune response, particularly
not a mucosal immune response.52 We have previously shown
that MIP-1 and RANTES can increase CD80 expression as well as
augment T-cell and Ag-presenting cell (APC)
functions.31,33 To address the potential mechanisms that
are used by these chemokines to initiate adaptive immune responses, we
investigated how MIP-1 and MIP-1 would affect the expression of
costimulatory molecules on B cells and corresponding receptors on T
cells. Indeed, the mucosal adjuvanticity of CT involves the selective
up-regulation of CD86 expression.35 Our previous studies
with RANTES have shown that this chemokine positively regulates the
expression of CD80, but not CD86, by Ag-presenting cells and CD28,
CD40L, CD30, but not 4-1BB, expression by T cells.31,33
MIP-1 significantly up-regulates expression of CD80, like
RANTES,33 but also increases CD86 surface levels on
resting B cells. In addition to B7 ligands, CD40 is another receptor
important for B-cell activation and differentiation.53 We
have observed significant changes in CD40L expression by activated T
cells cocultured with RANTES.31 A modest yet
dose-dependent increase (and subsequent decrease) in CD40 expression
was observed following MIP-1 and MIP-1 incubation with
Ag-stimulated or resting DO11.10 CD11b+ or
B220+ lymphocytes. In general, our data suggest that
MIP-1 is more effective at modulating surface expression of
costimulatory molecules necessary |
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Abstract
Introduction