| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Induces Significant Recruitment of Neutrophils and Monocytes1
*
Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224;
Catholic University School of Medicine, Seoul, Korea;
Pfizer, Inc., Groton, CT 06340; and
§
Mayo Clinic, Rochester, MN 55905
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
), a member of the CC
chemokine subfamily, has been shown to attract T cells and monocytes in
vitro and to be expressed at sites of inflammation. Although the in
vitro activities of MIP-1 have been well documented, the in vivo
biological activities of MIP-1 in humans have not been studied. To
address this, we challenged human subjects by intradermal injection
with up to 1000 pmol of MIP-1 and performed biopsies 2, 10, and
24 h later. Although no acute cutaneous or systemic reactions were
noted, endothelial cell activation, as indicated by the expression of
E-selectin, was observed. In agreement with its in vitro activity,
monocyte, lymphocyte, and, to a lesser degree, eosinophil infiltration
was observed, peaking at 10–24 h. Surprisingly, in contrast to its
reported lack of in vitro neutrophil-stimulating activity, a rapid
infiltration of neutrophils was observed in vivo. This neutrophil
infiltration occurred as early as 2 h, preceding the appearance of
other cells, and peaked at 10 h. Interestingly, we found that
neutrophils in whole blood, but not after isolation, expressed CCR1 on
their cell surface. This CCR1 was thought to be functional as assessed
by neutrophil CD11b up-regulation following whole-blood MIP-1
stimulation. These studies substantiate the biological
effects of MIP-1 on monocytes and lymphocytes and uncover the
previously unrecognized activity of MIP-1 to induce neutrophil
infiltration and endothelial cell activation, underscoring the need to
evaluate chemokines in vivo in humans. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
)3.
MIP-1 has been shown to be elevated in several inflammatory diseases
including rheumatoid arthritis, idiopathic pulmonary fibrosis,
sarcoidosis, asthma, and the cutaneous disease, lichen planus
(10, 11, 12, 13, 14). In animal model systems, neutralizing Abs to
MIP-1 have been shown to decrease disease intensity and
limit monocyte and lymphocyte recruitment (4, 6, 7, 8). The in vitro profile of activity of MIP-1 in
chemotaxis assays includes effects on B lymphocytes, activated T
lymphocytes (CD8>CD4), NK cells, basophils, dendritic cells, and
eosinophils (15, 16, 17, 18, 19, 20, 21). Many investigators have documented
that MIP-1 has no direct effect on human neutrophil chemotaxis in
vitro. In one report, Schall and colleagues (22, 23)
demonstrated that MIP-1 induced a small but concentration-dependent
intracytoplasmic calcium flux in human neutrophils that was pertussis
toxin sensitive.
Although in vitro studies illustrate the potent chemoattractant
activity of chemokines under static conditions, they cannot absolutely
predict chemokine activity at localized sites in vivo where conditions
of blood flow require several adhesion steps before migration into
tissue sites can occur (24). Furthermore, chemokines have
been shown to bind to proteoglycans and to be modified by surface
peptidases; both of these events can influence their chemotactic
activity in vivo, underscoring the importance of in vivo
experimentation (25, 26, 27, 28, 29). In vivo studies in animals in
which chemokines have been injected into various anatomical sites
indicate significant species differences in the response to some
chemokines, making interpretation of this data difficult
(30, 31, 32, 33, 34). For example, studies in mice engineered to be
deficient in the major MIP-1 receptor CCR1 indicate that
MIP-1/CCR1 are critical for the neutrophilic response to infection
with Aspergillus fumigatus and inflammatory insults such as
acute lung injury following pancreatitis (35, 36). Since
rodents have no CXCR1 homologue (or IL-8 receptor A), but do have
abundant CCR1 on their granulocytes, it has been speculated that
MIP-1 and CCR1 may serve the same function in rodents as CXCR1 and
IL-8 play in humans (35). Thus, studies in rodents and
other animal species have their limitations and make it imperative that
in vivo responses be assessed in humans.
To address the biological activity of MIP-1 in vivo in humans and
better define its role in disease, we injected MIP-1 into the skin
of normal and atopic volunteers. We designed the present studies to
determine whether the injection of purified human recombinant MIP-1
elicits the selective recruitment of different leukocyte subtypes. We
speculated that by studying the responses to this chemokine in a
complex multicellular tissue environment, we might uncover novel
effects of MIP-1 not predicted by in vitro studies using highly
purified leukocyte subtypes. For example, in our recent analysis of the
dermal response to RANTES challenge, we detected activation of
microvascular endothelial cells (24). Because
allergic subjects have primed or activated eosinophils, T
lymphocytes, and monocytes, we also wanted to determine
whether allergic subjects have a response that was either
quantitatively or qualitatively different from nonallergic subjects
(37, 38, 39, 40). Our studies confirm in vitro results
demonstrating potent effects of MIP-1 on monocytes
and uncover an unexpected and potent effect of MIP-1 on neutrophils
and endothelial cells in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These studies were approved by the Johns Hopkins Bayview Medical
Center Institutional Review Board for human research. Nine otherwise
healthy allergic human subjects between the ages of 23 and 50 years
were enrolled in the phase I study evaluating the effects of escalating
doses of MIP-1 (Table I
). Subjects
were characterized as allergic if they had a history of respiratory
allergies and symptoms referable to these allergies within the last
year, an elevated total IgE and a positive multiallergen RAST screen
(Phadiatop Kabi Pharmacia Diagnostics, Uppsala, Sweden). Use of
antihistamines or systemic glucocorticoids was discontinued for at
least 2 wk or 2 mo before this study, respectively. An initial group of
subjects was divided into three cohorts of three subjects each to
evaluate the safety of intradermal injection of escalating doses of
MIP-1. Successive cohorts were injected with progressively higher
doses of MIP-1 as follows: first cohort, 10, 50, and 100 pmol;
second cohort, 200, 300, and 400 pmol; and third cohort, 600, 800, and
1000 pmol. A sterile saline control was included in all subjects. Each
subject underwent two 6-mm skin biopsies 24 h after injection of
saline and the highest dose of MIP-1.
|
(1000 pmol) injection (Table I
). Subjects were characterized as allergic based on the criteria
listed above and as nonallergic if they had normal total IgE, negative
multiallergen screen, and no history of allergies or symptoms
suggestive of allergic rhinitis, asthma, or atopic dermatitis. All
subjects were injected with saline at one site and 1000 pmol MIP-1
at three sites. A 6-mm skin biopsy was performed at the sites 2, 10,
and 24 h after MIP-1 injections and at one of those time points
for the saline-injected sites. Blood pressure, pulse, body temperature,
and signs and symptoms at the injection sites were monitored over the
course of the study. A complete blood count with differential was done
immediately before the first injection and 24 h later to determine
whether intradermal injections of MIP-1 had any effects on the
number of circulating leukocytes.
The allergic subjects enrolled in phases I and II had significantly
elevated total IgE and multiallergen RAST measurements as compared with
nonallergic subjects (Table I). The allergic subjects enrolled in phase
II had total circulating eosinophil counts which were significantly
elevated compared with those of the nonallergic subjects.
MIP-1
MIP-1 (lot 95351) was purchased from PeproTech (Princeton,
NJ) and was shown to be >99% pure by SDS-PAGE and reversed-phase HPLC
analysis. It contained <1.25 endotoxin units/mg protein as determined
by the Limulus amebocyte lysate assay (BioWhittaker,
Walkersville, MD) and was shown to be free of contaminants by laser
desorption mass spectroscopy. The lyophilized preparation was first
diluted with sterile water to make a stock solution (778.5 µg/ml) and
subsequent dilutions were with sterile saline (no carrier protein).
Stability studies demonstrated that the stock solution of MIP-1
maintained bioactivity for at least 6 mo at -70°C and several weeks
at 4°C. Aliquots of the stock solution were stored at -80°C and
thawed only once just before each injection and then discarded.
Intradermal injections (0.1 ml) of MIP-1 or saline were given at
four separate sites on the volar forearm, and each site was marked with
a surgical pen to provide reference for subsequent clinical evaluations
and skin biopsies.
Routine histology, immunohistochemistry, and immunofluorescent staining
Skin biopsy specimens were fixed in Formalin and embedded in
paraffin. Eosinophil counts were performed on Wright-Giemsa-stained
sections. To further characterize the cellular infiltrate, additional
sections (5 µm) were stained with a variety of cell-specific Abs
(Table II) and the appropriate controls
using the Vectastain ABC-AP kit and the Vector Red substrate kit
(Vector Laboratories, Burlingame, CA) and, where noted, with the
addition of a permeabilization step. Immunofluorescent staining for the
eosinophil granule protein, major basic protein (MBP) was performed to
further quantify both the intact and degranulated eosinophils. The
number of cells/mm2 was determined by counting an
area of 4 x 4 reticules, at x400 magnification using an Olympus
BX60 microscope (Olympus, New Hyde Park, NY) for all markers except
MBP. MBP staining was quantified using a scoring system of 0–3+ based
on the extent and intensity of the fluorescent stain (41).
The scores for the endothelial adhesion molecule E-selectin were
calculated by dividing the total number of vessels staining for
E-selectin by the total number of von Willebrand factor
(VWF)-positive vessels in an adjacent tissue section and multiplying by
100. All specimens were scored in a blinded fashion by two independent
investigators. The interrater reliability between the two investigators
with regard to cell counts ranged from 63% for
CD68+ cells to 99% for neutrophil
elastase+ cells.
|
Endothelial cells used in this study were from collagenase
digestion of HUVECs or the immortalized human dermal microvascular
endothelial cell line (HMEC, 5A12, a generous gift from Dr. Robert
Swerlick, Emory University, Atlanta, GA) (42, 43).
Endothelial cells were grown to confluence in 6-well plates (Costar,
Cambridge, MA) and either first passage (HUVEC) or passages 25–40
(HMEC) cells were stimulated with either recombinant human MIP-1
(10, 50, or 100 ng/ml), IL-1ß (IL-1ß, 10 ng/ml), or medium alone
for 4 and 24 h (37°C, 5% CO2/95% air).
The expression of endothelial E-selectin, ICAM-1, and VCAM-1 was
analyzed by indirect immunofluorescence with an Epics Profile flow
cytometer (Coulter, Hialeah, FL) using BBIG-E4 (anti-E-selectin;
R&D Systems, Minneapolis, MN), BBA-5 (anti-VCAM-1; R&D Systems),
84H10 (anti-ICAM-1; AMAC, Westbrook, ME) mAb, and an irrelevant
control mAb as described previously (44).
RT-PCR analysis of CCR1 expression
Total RNA was isolated from human neutrophils, eosinophils,
mononuclear cells, and HUVECs using the RNazol B method (Tel-Test,
Friendswood, TX). Human neutrophils were isolated from whole blood of
healthy nonallergic donors, collected into EDTA-containing tubes, and
purified by density centrifugation on 1.079 g/ml Percoll (Pharmacia,
Uppsala, Sweden). Contaminating erythrocytes were removed by hypotonic
lysis and neutrophil purity was enhanced by CD9-negative selection
using the immunomagnetic bead technique (final purity 
98%).
Human eosinophils were purified to 99% purity from allergic donors
using the negative selection immunomagnetic bead technique
(45). Mononuclear cells were obtained from buffy coats of
healthy blood donors using Ficoll-Hypaque (Pharmacia) discontinuous
density gradient centrifugation. These mononuclear cell preparations
were composed of 20% monocytes and 80% lymphocytes.
First-strand cDNA was prepared from the total RNA using oligo(dT) primers (Boehringer Mannheim, Indianapolis, IN) and Superscript RT enzyme (Life Technologies, Gaithersburg, MD) as follows: aliquots of 1 µg of total RNA in 5 µl diethylpyrocarbonate-treated water were mixed with 4 µl of 5x First-Strand buffer (250 mM Tris, 375 mM KCl, and 15 mM MgCl2; Life Technologies), 2 µl of 0.1 M DTT, 4 µl of 20 mM dNTP mix, and 2 µl of 25 µM oligo(dT) and heated at 70°C for 10 min, then chilled on ice. Subsequently, 2 µl of 10 U/µl RNase inhibitor (Life Technologies) and 1 µl of 200 U/µl RT were added to the tube. The reaction was conducted in a Hybaid Omnigene thermocycler (Hybaid) for 50 min at 42°C and then for 5 min at 99°C. Aliquots of cDNA were amplified by PCR using Taq polymerase (Life Technologies) and specific primers for CCR1 and ß-actin in the same tubes according to the method described previously (46). Briefly, 5 µl of the first-strand cDNA was mixed with 3' and 5' primers (20 pmol each), 1 U of Taq DNA polymerase, 5 µl 10x PCR buffer (200 mM Tris and 500 mM KCl, pH 8.4), 2 mM MgCl2, 200 µM dNTP mix, and water to bring the final reaction volume to 50 µl. The samples were sealed with a drop of mineral oil and the PCR reaction was performed as follows: first, a 3-min cycle with a denaturing temperature of 94°C, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min. A final elongation step at 72°C for 7 min was performed. The cDNA from mononuclear cells and HUVECs were used as positive and negative controls, respectively. The nucleotide sequences of CCR1 and ß-actin primers were 5' primer, ACT CCG TGC CAG AAG GTG AA and 3' primer, ATG GCA TCA CCA AAA ACC CA; and 5' primer, TGA CGC GGT CAC CCA CAC TGT GCC CAT CTA and 3' primer, CTA GAA GCA TTG CGC TGG ACG ATG GAG GG, respectively. At the end of the reaction, PCR products were separated by electrophoresis on a 2.5% agarose gel. The amplified products were identified based on the predicted size of 251 bp by comparison with positive control and a DNA ladder of known m.w.
Human neutrophil CCR1and CCR5 surface expression
Whole blood was obtained by venipuncture and collected in tubes containing EDTA. Surface expression of CCR1 and CCR5 on neutrophils was examined with indirect immunofluorescence and flow cytometry evaluating granulocytic cells based on scatter characteristics. Concomitant CD16 labeling was used to distinguish eosinophils (CD16-) from neutrophils (CD16+). Whole blood (100 µl) was incubated with saturating concentrations of the unconjugated primary mAb to CCR1 (2D4, a generous gift from LeukoSite, Boston, MA), mAb to CCR5 (R&D Systems), mAb to CD16 (3G8, provided by Dr. Paul Guyre, Dartmouth Medical School, Hanover, NH), or an isotype-matched control. Cells were washed and incubated with saturating dilutions of FITC-conjugated F(ab')2 goat anti-mouse IgG Ab (Tago, Burlingame, CA). RBCs were lysed after the final labeling step using the whole-blood lysing reagent kit (Coulter) following the manufacturer’s instructions. After fixation with 1% paraformaldehyde in PBS, cells were evaluated using an Epics Profile II flow cytometer (Coulter ). Data are expressed as fold control (mean fluorescence intensity(MFI), CCR1 or CCR5, divided by background MFI).
Whole-blood analysis of CD11b up-regulation
The effect of MIP-1 on CD11b expression on monocytes and
neutrophils was determined as described previously (47).
Briefly, human blood was collected in EDTA from 18 healthy donors.
Blood was prewarmed at 37°C for 5 min before adding various
concentrations of MIP-1 (0.01, 0.1, 1.0, 10, 100, and 1000 nM).
After a 15-min stimulation, the tubes were placed into an ice bath and
1 ml of cold PBS containing 2% FCS and 0.2% sodium azide was added.
The tubes were centrifuged at 200 x g for 10 min at
4°C, and cells were stained for the presence of CD11b. CD11b
expression on neutrophils was analyzed by direct immunofluorescence
using the FITC-conjugated anti-CD11b Ab (Caltag, Burlingame, CA)
and a FITC-conjugated human IgG as a control. Monocytes and neutrophils
were identified by their forward and side scatter light profiles. The
data were then expressed as a fold increase over baseline and
calculated as follows: fold increase = (MFI (chemokine) -
MFI (autofluorescence))/(MFI (buffer) - MFI
(autofluorescence)).
Statistics
Statistical analysis was performed using the Student t test. A p value of < 0.05 was considered to be significant. Correlations among cell counts or between cell counts and E-selectin expression were tested using the Spearman rank nonparametric method. Data were expressed as the means ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
No significant side effects were noted after MIP-1 injection in
any of the 27 subjects who participated in this study, and no subjects
dropped out of the study for any reason. Blood pressure, pulse, and
body temperature were monitored over the course of the study (at 0.5,
1, 1.5, 2, 10, and 24 h after challenge in phase I, and at 2, 10,
and 24 h in phase II) and did not change after MIP-1
injections. Additionally, these injections (cumulative dose, 3000 pmol)
did not induce a significant change in peripheral blood neutrophils
(3609 ± 275 cells/mm3 before vs 4283
± 283 cells/mm3 after), lymphocytes (2350
± 150 cells/mm3 before vs 2406 ± 129
cells/mm3 after), monocytes (384 ± 46
cells/mm3 before vs 448 ± 34
cells/mm3 after), or eosinophils (202 ± 39
cells/mm3 before vs 173 ± 33
cells/mm3 after). No immediate cutaneous
reactions were noted in any of the subjects. Several mild reactions
were noted 10 or more h after MIP-1 injection at the injection sites
in 17 subjects (3/9 subjects in phase I and 14/18 subjects in phase
II). These localized reactions occurred at the 1000 pmol MIP-1
injection sites and included tenderness (33%), ecchymosis (33%), and
erythema larger than 1 cm in diameter (33%). No immediate reactions
were noted in any of the subjects.
Leukocyte recruitment following MIP-1 injection
The cellular infiltrates observed in biopsies taken 24 h
after injection of MIP-1 at 100-, 400-, and 1000-pmol doses were
assessed by Giemsa and immunohistochemical staining (Fig. 1). A parabolic dose-response curve was
observed for CD68+ and CD3+
cell recruitment, with maximal recruitment noted at 400 pmol (Fig. 1, A and C). Unexpectedly, neutrophils were
recruited at all three doses, although there was no apparent
dose-response relationship for the MIP-1 doses tested (Fig. 1B). Eosinophil migration was quite modest, reaching maximal
migration at 1000 pmol (Fig. 1D). Despite this modest
eosinophil infiltration, the presence of extracellular MBP staining
suggested that the eosinophils were activated as demonstrated by their
degranulation (Fig. 2).
|
|
-induced cellular recruitment, we
injected 1000 pmol of MIP-1 at three sites and performed skin
biopsies 2, 10, and 24 h later. Results of the cellular
phenotyping from this study are presented in Fig. 3. Recruitment of neutrophils was
apparent as early as 2 h after MIP-1 injection (Fig. 3A). Recruitment of monocytes (CD68+;
Fig. 3B), neutrophils, cutaneous lymphocyte Ag
(CLA)+ cells (Fig. 3C), and T
lymphocytes (CD3+; Fig. 3D) plateaued
by 10 h after challenge, whereas recruitment of eosinophils had
not plateaued by 24 h (Fig. 3E). The cellular MBP
semiquantitative staining score correlated with eosinophil counts from
Giemsa-stained sections (r = 0.64, p =
0.0001), and eosinophil degranulation only reached statistical
significance at 24 h. Interestingly, the magnitude and kinetics of
leukocyte recruitment did not differ between allergic and nonallergic
subjects (data not shown). Therefore, all of the cell counts reported
in Fig. 3 represent the combined data of 18 (9 allergic and 9
nonallergic) subjects. The order of magnitude of leukocyte recruitment
in response to the 1000-pmol dose listed in order of absolute cell
counts was: neutrophils > monocytes > lymphocytes >
eosinophils. Importantly, the MIP-1 lot used in this study failed to
induce chemotaxis of purified human neutrophils in vitro (data not
shown). Representative immunohistochemical and Giemsa-stained sections
from skin biopsies taken 24 h after MIP-1 injection are
illustrated in Fig. 4, A–E.
To better characterize the selectivity of the leukocyte migration in
response to intradermal MIP-1 challenge, we compared the
differential of the tissue-infiltrating leukocytes 10 h after
MIP-1 injection to that seen in the peripheral blood of the same
subjects before MIP-1 challenge, as shown in Table III. When the migration was evaluated in
the context of the circulating pool of leukocytes, the recruitment of
monocytes was the most selective; monocytes represented 5 ± 1%
of the circulating leukocytes and 41 ± 3% of the tissue
leukocytes.
|
|
|
Skin biopsies were also analyzed for the expression of the
endothelial adhesion molecule E-selectin. We noted a dramatic induction
of E-selectin expression in dermal endothelial cells as early as 2
h after MIP-1 (1000 pmol) injection, reaching maximal expression
10 h after challenge (Figs. 4F and
5). There was no significant difference
in the endothelial E-selectin expression between allergic and
nonallergic groups and therefore their values are combined (data not
shown). The extent of endothelial E-selectin expression at 24 h
correlated with tissue infiltration by monocytes
(CD68+), T lymphocytes
(CD3+), and neutrophils (r
0.49, p = 0.0001). The tissue migration of
CLA+ cells did not correlate with E-selectin
expression, which is surprising since CLA is thought to be an important
ligand for E-selectin (48, 49).
Lack of direct effects of MIP-1 on endothelial cells
To determine whether MIP-1 was capable of directly inducing
endothelial adhesion molecule expression, we incubated HUVECs and HMECs
with MIP-1 (10, 50, and 100 ng/ml) in vitro for 4 or 24 h and
performed flow cytometry with Abs to the endothelial adhesion molecules
E-selectin, VCAM-1, and ICAM-1 (n = 2). MIP-1 had no
effect on the expression of any of these endothelial adhesion molecules
at any of the concentrations or stimulation times tested (data not
shown). IL-1ß (10 ng/ml x 4 h) stimulation was used as a
positive control and induced expression of all three adhesion molecules
(data not shown).
Human neutrophil CCR1 and CCR5 expression
The influx of neutrophils in response to intradermal MIP-1
challenge (Figs. 1B, 3A, and 4B)
suggested that these leukocytes might express one of the MIP-1
receptors CCR1 or CCR5 (15, 50). To investigate this
possibility, we analyzed neutrophils for the expression of CCR1 mRNA
using RT-PCR and for surface expression of CCR1 and CCR5 using flow
cytometry. Fig. 6A shows that
highly purified (99%) neutrophils from four donors constitutively
express CCR1 mRNA (lanes 4–7). Eosinophils (>99%
purity, lane 1) are shown as a positive control, and HUVECs
and water (lanes 3 and 8, respectively) as
negative controls. To demonstrate that the small number of
contaminating eosinophils in the neutrophil samples did not explain our
ability to amplify CCR1 mRNA, we simultaneously analyzed mRNA isolated
from a CCR1-negative HUVEC sample (lane 3, HUVEC
alone) to which we added a number of eosinophils equal to those
contaminating the neutrophil preparation (lane 2,
HUVEC and 1% eosinophils). Because we did not detect CCR1 mRNA
expression under these conditions, we concluded from these studies that
neutrophils themselves express CCR1 mRNA.
|
stimulation (e.g.,
chemotaxis or up-regulation of CD11b surface expression). Subsequent
studies were performed on whole-blood preparations from healthy
subjects (n = 6). In these studies, we observed an
average MFI of 2.1 ± 0.3-fold control, with 31% positivity for
CCR1 and no expression of CCR5 (1.0 ± 0.1-fold control). A
representative CCR1 histogram is shown in Fig. 6B. The cells
within the granulocyte scatter region were almost entirely neutrophils
as determined by their CD16 staining (99.5 ± 0.4% positivity,
Fig. 6C). Therefore, the CCR1 surface expression could not
be attributed to eosinophils. Interestingly, the neutrophil CCR1
expression was greater than that seen on the cells found within the
lymphocyte scatter, which were at the limit of detection (MFI, 1.1
± 0.1-fold control; 9% positive), but considerably less than that
seen on cells within the monocyte scatter (MFI, 14.4 ± 2.9-fold
control; 88% positive; data not shown). One interpretation of these
findings is that neutrophil CCR1 expression is lost during cell
isolation. Next, we determined the effects of MIP-1 on human
neutrophils in vitro.
In initial studies, we were unable to demonstrate chemotaxis or
up-regulation of surface expression of the integrin subunit CD11b with
MIP-1 on isolated neutrophils, in agreement with the work of other
investigators (22, 23). In contrast, when freshly
collected whole blood was stimulated for 15 min with MIP-1 (10 nM),
CD11b up-regulation was observed (n = 18 donors) on
both neutrophils (MFI-fold increase, 1.4 ± 0.1) and monocytes
(MFI-fold increase, 2.1 ± 0.1) as shown in Fig. 7, A and B,
respectively. Although the magnitude of this increase was small, the
EC50 for this response, as shown in Fig. 7C, was comparable to that achieved using leukotriene
(LT)B4 as the agonist
(EC50, 3.8 nM for MIP-1 vs 4.0 nM for
LTC4). Whole-blood chemotaxis assays were
attempted but were futile, since erythrocytes prevented any leukocyte
migration because they plugged the filter in the Boyden microchemotaxis
chamber (Neuroprobe, Cabin John, MD).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
is a chemokine thought to be important in several
diseases characterized by the recruitment and activation of mononuclear
cells. In vitro studies to date have led to the belief that it is not
active as a neutrophil chemoattractant (3). However, our
studies indicate that MIP-1, either by direct or indirect
effects, is a neutrophil chemoattractant in vivo in humans.
We found that intradermal challenge with even low doses of MIP-1
induced a marked influx of neutrophils. We also observed that
intradermal injection of MIP-1 led to the activation of
microvascular endothelial cells. Both of these findings could have
important, unrealized implications for the role of MIP-1 in
disease.
The infiltration of lymphocytes and monocytes into intradermal sites
following MIP-1 injection is in agreement with in vitro chemotaxis
studies. Memory T lymphocyte migration into skin is believed to rely on
expression of the CLA Ag (51). However,
characterization of the lymphocytes infiltrating the skin after
MIP-1 challenge revealed that only 50% of these cells expressed
CLA. Furthermore, although CLA+ cells bind to
endothelial E-selectin, which is believed to be responsible for their
migration to skin (52), in our study there was no
correlation between the percentage of vessels staining for E-selectin
and the number of CLA+ cells following MIP-1
challenge. It is also interesting to note that when comparing the
percentage of infiltrating cells with the number of cells in the
periphery of the same individual, lymphocyte recruitment in response to
MIP-1 was the least selective as compared with the other cell types
(Table III). In contrast, MIP-1 induced the most selective
infiltration of monocytes. This supports the hypothesis that MIP-1
may be a critical cytokine in chronic inflammatory conditions
characterized by large numbers of tissue monocytes/macrophages, such as
multiple sclerosis and rheumatoid arthritis (6, 8).
The magnitude of monocyte and lymphocyte infiltration at 24 h
after challenge increased from the 100–400 pmol dose, but was less at
the 1000 pM dose. The reason for this parabolic dose-response curve is
unclear. One possible explanation is that at higher concentrations,
diffusion of MIP-1 into the systemic circulation desensitizes (and
internalizes) MIP-1 receptors on circulating leukocytes, resulting
in less migration. However, since neutrophil and eosinophil migration
was not decreased at higher dose levels, such a systemic receptor
desensitization seems unlikely. Alternatively, one could speculate that
at higher dose levels, MIP-1 is also activating cells as they
infiltrate the skin sites, releasing factors that may feedback and
dampen further cell infiltration. Further studies will be necessary to
address these possibilities.
The robust infiltration of neutrophils occurring following MIP-1
injection was unexpected based on in vitro results (23, 50, 53, 54). In a similar study with RANTES, another CC chemokine which
shares CCR1 and CCR5 with MIP-1, no neutrophils were observed.
Possible explanations for these paradoxical results include the
presence of a unique chemokine receptor for MIP-1 on neutrophils,
the possibility that RANTES does not stimulate sufficient signaling
through their shared receptors (CCR1 or CCR5), or that the neutrophil
infiltration was secondary to the release of other factors. Since the
kinetics of neutrophil infiltration was rapid, especially as compared
with other cell types, and the neutrophil infiltration response was
near maximal at a dose of MIP-1 (100 pmol) that was too low to
recruit other cell types (Fig. 1), an indirect effect involving other
infiltrating cells seems less likely. We have recently made the
observation that epithelial cells express CCR1 and CCR5 (S.
Shahabuddin, unpublished data). Therefore, we cannot rule out the
possibility that MIP-1 may act on other resident cells, such as
epithelial cells, to cause the release of inflammatory mediators which
in turn recruit neutrophils. Of note, no neutrophil chemotactic
activity was observed in supernatants from a resident tissue cell
(HUVEC) stimulated with MIP-1 despite the fact that these cells have
been shown to express relevant receptors (CCR4 and CCR5)
(55).
Although most investigators have found that MIP-1 is not chemotactic
for purified human neutrophils, we (and others) have shown that these
cells express mRNA for the MIP-1 receptor CCR1, and we demonstrate
here the constitutive surface expression of CCR1 by flow cytometry
(Fig. 6) (53, 54). Interestingly, we could only detect
CCR1 surface expression on neutrophils from whole-blood preparations
and could not detect surface expression on cells that had undergone
isolation procedures, including density gradient centrifugation and
hypotonic RBC lysis (data not shown). In agreement with these results,
we detected MIP-1-dependent CD11b up-regulation on neutrophils from
whole blood but not in purified neutrophils. This suggests
that the standard isolation techniques may affect neutrophil chemokine
receptor surface expression and might help explain the lack of MIP-1
chemotactic activity on purified neutrophil preparations. A possible
explanation for this is the release of RANTES or MIP-1 from
platelets during the isolation procedure, since platelets are a rich
source of these CC chemokines (56). RANTES release could
desensitize and internalize CCR1 and thereby prevent responses to CCR1
ligands in vitro (57). Interestingly, Bonecchi et al.
(53) have recently demonstrated that IFN-
up-regulates
CCR1 mRNA and surface expression on purified neutrophils, and that
these stimulated neutrophils now undergo chemotaxis to MIP-1 and
RANTES in vitro. Collectively, this work suggests that MIP-1 may
directly or indirectly play a role in the development of tissue
neutrophilia, in sharp contrast to other members of the CC chemokine
subfamily. In support of this notion, Murch et al. (58)
have shown that infants who develop bronchopulmonary dysplasia have
increased numbers of neutrophils and elevated MIP-1 in their
bronchoalveolar lavage fluid.
In addition to causing leukocyte recruitment, we also observed an
effect of MIP-1 on endothelial cell activation in vivo. This
suggests that chemokines may recruit leukocytes in vivo in part by
inducing endothelial adhesion molecule expression. The up-regulation of
selectins on endothelial cells would be expected to facilitate
leukocyte rolling under flow and thus prepare them for subsequent steps
of integrin-induced firm adherence and chemotaxis into tissue. There
was a significant correlation between endothelial E-selectin expression
and the influx of all leukocytes, suggesting that the endothelial
activation resulting from MIP-1 challenge may have contributed to
cell migration. Since we could not detect enhanced expression of
ICAM-1, E-selectin, or VCAM-1 on HUVECs and HMECs stimulated with
MIP-1 in vitro, it is likely that endothelial activation in vivo
occurred indirectly, possibly due to the release of
endothelial-activating cytokines (e.g., IL-1 or TNF) by other cell
types. Although monocytes have been shown to release TNF- in
response to MIP-1, a close view of the kinetics of monocyte
recruitment and E-selectin expression suggests that the endothelial
activation preceded significant monocyte influx. Alternatively,
MIP-1 may induce IL-1 or TNF release from other cell types or the
ability of MIP-1 to activate endothelium in vivo may require factors
not provided in our tissue culture systems (59). The
E-selectin expression is not due to endotoxin because the levels
detected in the MIP-1 preparation were <1.25 endotoxin units/mg
protein (60).
Strikingly different results were observed following the intradermal
challenge of RANTES as compared with MIP-1. Intradermal RANTES
challenge led to a selective migration of eosinophils and lymphocytes,
whereas MIP-1 recruited neutrophils and monocytes as well as
lymphocytes and eosinophils. The recruitment of eosinophils by 500 pmol
of RANTES was substantially greater than that seen with 1000 pmol of
MIP-1 (112 ± 23 cells/mm2 vs 26 ±
6 cells/mm2, respectively). There were no clear
differences in the kinetics of eosinophil tissue infiltration between
allergic and nonallergic subjects following MIP-1 challenge, in
contrast to the accelerated migration of eosinophils in allergic
subjects that occurred with RANTES. Furthermore, the allergic phenotype
of enrolled subjects had no influence on the kinetics or magnitude of
the monocyte, neutrophil, or lymphocyte response to MIP-1,
suggesting that allergic status does not affect the in vivo response to
MIP-1. A likely explanation for the greater eosinophil response
observed in the RANTES study may relate to the ability of RANTES, but
not MIP1, to interact with the eosinophil receptor CCR3.
Although MIP-1 is known best for its effects on cell migration, it
is thought to have several other important biological functions which
include cell activation, mast cell and/or basophil histamine release,
and PG-independent pyrogenic activity (30, 61, 62). Our
results suggest that MIP-1 skin challenge can induce eosinophil
activation as measured by interstitial MBP staining. We were unable to
assess monocyte or neutrophil activation in this model. MIP-1 had no
effect on cutaneous mast cell histamine release since no acute
wheal-and-flare reactions were seen in any of the 27 subjects injected
with MIP-1 in doses ranging from 100 to 1000 pmol. This is in
contrast to studies by Alam et al. (63) demonstrating that
human MIP-1 induced mouse footpad swelling, which was maximal 30 min
after injection and was felt to represent mast cell degranulation based
on electron microscopic examination. These differences may be due to
differences in chemokine and chemokine receptor specificity between
mouse and human. Our study was not designed to test the effects of
MIP-1 on basophil mediator release, although it is possible that the
erythema seen 10 h after 1000 pmol MIP-1 in approximately
one-third of the subjects may have been secondary to basophil
recruitment and activation. Even with a cumulative dose of 3000 pmol
(24 µg) per subject, we were unable to detect any elevations in body
temperature during monitoring of subjects for up to 24 h after
their last intradermal injection. These findings suggest either that
MIP-1 does not have pyrogenic effects in vivo in humans or that
these localized cutaneous injections were not sufficient to develop
systemic levels of MIP-1.
In summary, we demonstrate that MIP-1 is a potent in vivo leukocyte
recruiting agent in humans. Relative to the corresponding peripheral
cell numbers, MIP-1 is most selective for monocytes. However, we
also identified previously unrecognized effects of MIP-1 on
endothelial cells and neutrophils in vivo. We have shown that the
tissue neutrophilia could be due to a direct effect of MIP-1 on
neutrophils but we cannot exclude the possibility that this
neutrophilia was due to secondary mediators released by other cells
within the tissue. The potent in vivo effect of MIP-1 on tissue
neutrophilia raises important questions about the role of MIP-1 and
its receptor, CCR1, in neutrophil-mediated diseases. Our results also
provide further support for the contention that the human challenge
model we have developed will yield new insights into the role of
chemokines in human diseases.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Lisa A. Beck, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. E-mail address:
3 Abbreviations used in this paper: MIP-1, macrophage inflammatory protein-1; MBP, major basic protein; CLA, cutaneous lymphocyte Ag; VWF, von Willebrand factor; HMEC, human microvascular dermal endothelial cell; MFI, mean fluorescence intensity.
Received for publication August 16, 1999. Accepted for publication January 3, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in the pathogenesis of the T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis. J. Immunol. 155:5003.[Abstract]
and MIP-1ß. J. Immunol. 161:5663.: a novel chemotactic cytokine for macrophages in rheumatoid arthritis. J. Clin. Invest. 93:921.
expression in interstitial lung disease. J. Immunol. 151:2852.[Abstract]
, and MCP-1 into asthmatic airways following endobronchial allergen challenge. Am. J. Respir. Crit. Care Med. 156:1377. (MIP1) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am. J. Respir. Cell Mol. Biol. 13:738.[Abstract]
. J. Biol. Chem. 271:17161. and MIP-1ß. Science 260:355. (MIP-1) and MIP-1ß chemokines attract distinct populations of lymphocytes. J. Exp. Med. 177:1821. induce the migration and activation of normal human eosinophil granulocytes. J. Exp. Med. 176:1489., MCP-1, and RANTES, but
not MIP-1ß induced the chemotaxis of NK and IL-2-activated NK (IANK)
cells, as determined in microchemotaxis assay. J. Immunol.
153:4969.
on neutrophils mediated by CCR1. J. Immunol. 162:4938. and -ß. J. Immunol. 150:4550.[Abstract]
activates basophils and mast cells. J. Exp. Med. 176:781., or human interleukin 8. J. Exp. Med. 178:1913. are most efficient in recruiting T cells into the skin in vivo. J. Invest. Dermatol. 111:1040.[Medline]
in human neutrophils. J. Immunol. 162:474. within human platelets and their release during storage. Int. Arch. Allergy Immunol. 107:541.[Medline]
occurs in respiratory distress syndrome and is associated with poor outcome. Pediatr. Res. 40:490.[Medline]
in vivo. Blood 79:221.
and monocyte chemoattractant peptide-1 elicit immediate and late cutaneous reactions and activate murine mast cells in vivo. J. Immunol. 152:1298.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  C. A. Reichel, M. Rehberg, M. Lerchenberger, N. Berberich, P. Bihari, A. G. Khandoga, S. Zahler, and F. Krombach Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1787 - 1793. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Dal-Secco, T. M. Cunha, A. Freitas, J. C. Alves-Filho, F. O. Souto, S. Y. Fukada, R. Grespan, N. M. N. Alencar, A. F. Neto, M. A. Rossi, et al. Hydrogen Sulfide Augments Neutrophil Migration through Enhancement of Adhesion Molecule Expression and Prevention of CXCR2 Internalization: Role of ATP-Sensitive Potassium Channels J. Immunol., September 15, 2008; 181(6): 4287 - 4298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Deruaz, A. Frauenschuh, A. L. Alessandri, J. M. Dias, F. M. Coelho, R. C. Russo, B. R. Ferreira, G. J. Graham, J. P. Shaw, T. N.C. Wells, et al. Ticks produce highly selective chemokine binding proteins with antiinflammatory activity J. Exp. Med., September 1, 2008; 205(9): 2019 - 2031. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Moreno, C. Nicaise, T. Gustot, E. Quertinmont, N. Nagy, M. Parmentier, H. Louis, and J. Deviere Chemokine receptor CCR5 deficiency exacerbates cerulein-induced acute pancreatitis in mice Am J Physiol Gastrointest Liver Physiol, December 1, 2006; 291(6): G1089 - G1099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. P. Gladue, S. H. Cole, M. L. Roach, L. A. Tylaska, R. T. Nelson, R. M. Shepard, J. D. McNeish, K. T. Ogborne, and K. S. Neote The Human Specific CCR1 Antagonist CP-481,715 Inhibits Cell Infiltration and Inflammatory Responses in Human CCR1 Transgenic Mice. J. Immunol., March 1, 2006; 176(5): 3141 - 3148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bhatia, R. D. Ramnath, L. Chevali, and A. Guglielmotti Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis Am J Physiol Gastrointest Liver Physiol, June 1, 2005; 288(6): G1259 - G1265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R. Veillard, B. Kwak, G. Pelli, F. Mulhaupt, R. W. James, A. E.I. Proudfoot, and F. Mach Antagonism of RANTES Receptors Reduces Atherosclerotic Plaque Formation in Mice Circ. Res., February 6, 2004; 94(2): 253 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P. Gladue, L. A. Tylaska, W. H. Brissette, P. D. Lira, J. C. Kath, C. S. Poss, M. F. Brown, T. J. Paradis, M. J. Conklyn, K. T. Ogborne, et al. CP-481,715, a Potent and Selective CCR1 Antagonist with Potential Therapeutic Implications for Inflammatory Diseases J. Biol. Chem., October 17, 2003; 278(42): 40473 - 40480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Frenette, N. Chbinou, C. Godbout, D. Marsolais, and P. S. Frenette Macrophages, not neutrophils, infiltrate skeletal muscle in mice deficient in P/E selectins after mechanical reloading Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2003; 285(4): R727 - R732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Phillips, V. E. L. Stubbs, M. R. Henson, T. J. Williams, J. E. Pease, and I. Sabroe Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter J. Immunol., June 15, 2003; 170(12): 6190 - 6201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Arase, H. Arase, S. Hirano, T. Yokosuka, D. Sakurai, and T. Saito IgE-Mediated Activation of NK Cells Through Fc{gamma}RIII J. Immunol., March 15, 2003; 170(6): 3054 - 3058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Menzies-Gow, S. Ying, I. Sabroe, V. L. Stubbs, D. Soler, T. J. Williams, and A. B. Kay Eotaxin (CCL11) and Eotaxin-2 (CCL24) Induce Recruitment of Eosinophils, Basophils, Neutrophils, and Macrophages As Well As Features of Early- and Late-Phase Allergic Reactions Following Cutaneous Injection in Human Atopic and Nonatopic Volunteers J. Immunol., September 1, 2002; 169(5): 2712 - 2718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Forssmann, H.-J. Magert, K. Adermann, S. E. Escher, and W.-G. Forssmann Hemofiltrate CC chemokines with unique biochemical properties: HCC-1/CCL14a and HCC-2/CCL15 J. Leukoc. Biol., September 1, 2001; 70(3): 357 - 366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. E. H. Low, I. A. Drugea, L. A. Duffner, D. G. Quinn, D. N. Cook, B. J. Rollins, E. J. Kovacs, and L. A. DiPietro Wound Healing in MIP-1{alpha}-/- and MCP-1-/- Mice Am. J. Pathol., August 1, 2001; 159(2): 457 - 463. [Abstract] [Full Text]
|
|
|
|
|
|
L. R. Hall, E. Diaconu, R. Patel, and E. Pearlman CXC Chemokine Receptor 2 But Not C-C Chemokine Receptor 1 Expression Is Essential for Neutrophil Recruitment to the Cornea in Helminth-Mediated Keratitis (River Blindness) J. Immunol., March 15, 2001; 166(6): 4035 - 4041. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. L. Makepeace, P. J. Watt, J. E. Heckels, and M. Christodoulides Interactions of Neisseria gonorrhoeae with Mature Human Macrophage Opacity Proteins Influence Production of Proinflammatory Cytokines Infect. Immun., March 1, 2001; 69(3): 1909 - 1913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Fahy, H. Porte, S. Senechal, H. Vorng, A. R. McEuen, M. G. Buckley, A. F. Walls, B. Wallaert, A.-B. Tonnel, and A. Tsicopoulos Chemokine-Induced Cutaneous Inflammatory Cell Infiltration in a Model of Hu-PBMC-SCID Mice Grafted with Human Skin Am. J. Pathol., March 1, 2001; 158(3): 1053 - 1063. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
1%
eosinophil contamination, eosinophils were added back to HUVECs to
obtain a final concentration of 1% (lane 2). The
amplified products were the predicted size of 251 bp, as determined by
comparison with a DNA ladder of known m.w. PCR for ß-actin was
performed to confirm the integrity of the RNA for each sample (data not
shown). Surface expression of CCR1 (B) and CD16
(C) on granulocytes from healthy human donors
(representative of six donors) is shown. Histograms of flow cytometric
analysis with an anti-CCR1 (2D4) or anti-CD16 (3G8) mAb are
represented by open histograms and the IgG1 isotype controls are
depicted by the filled histograms.
