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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2341-2346
Rat Neutrophils Express  4 and  1 Integrins and Bind to
Vascular Cell Adhesion Molecule-1 (VCAM-1) and Mucosal Addressin
Cell Adhesion Molecule-1 (MAdCAM-1)
By
Kelly L. Davenpeck,
Sherry A. Sterbinsky, and
Bruce S. Bochner
From the Department of Medicine, Division of Clinical Immunology, The
Johns Hopkins University School of Medicine, JHAAC, Baltimore, MD.
 |
ABSTRACT |
The  4 integrins, which are constitutively expressed on all human
leukocyte subtypes except neutrophils, interact with vascular cell
adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule (MAdCAM-1) on endothelium to mediate selective recruitment of
leukocyte subpopulations, other than neutrophils, to sites of
inflammation. However, here we report that a different paradigm of
leukocyte recruitment may exist in the rat. Flow cytometric analysis of
rat neutrophils using a panel of monoclonal antibodies which recognize
rat 4 and  1 integrins showed consistent, low levels of
expression. Although 4 was expressed at lower levels on neutrophils
than all other rat leukocytes, this level of expression was sufficient
to mediate significant levels of 4- and 1-dependent neutrophil
adhesion to rat and human VCAM-1, and 4-dependent, but
1-independent, adhesion to human MAdCAM-1. These data suggest that
rat neutrophils, unlike other species, may use 4 integrins to
traffic to sites of inflammation in vivo.
| |
INTRODUCTION |
THE INTEGRINS, WHICH mediate
leukocyte-endothelial and leukocyte-matrix interactions, are a complex
family of heterodimeric glycoproteins consisting of and subunit
pairs. To date, at least 16 and eight subunits have been
described and combined to generate over 20 integrin molecules on human
cells.1,2 The 4 integrin subunit, first described by
Hemler et al3 on T-lymphoblastoid cell lines, has been
shown to pair with both the 1 and 7 subunits. By virtue of their
ability to interact with endothelial expressed ligands, the 4
integrins 41 (very late antigen-4 [VLA-4, CD49d/CD29]) and
47 (lymphocyte-Peyer's patch adhesion molecule-1 [LPAM-1,
CD49d/CD103]), are believed to play a major role in the recruitment of
leukocytes during inflammation. In humans, both 41, which binds
to endothelial vascular cell adhesion molecule-1 (VCAM-1), and
47, which interacts with both VCAM-1 and mucosal addressin cell
adhesion molecule-1 (MAdCAM-1), are constitutively expressed on the
surface of eosinophils, basophils, and lymphocytes, but are not
detected on neutrophils, whereas monocytes only express
41.4,5 This limited pattern of 4 integrin
expression has been theorized to contribute to the selective recruitment of leukocyte subtypes other than neutrophils to sites of
inflammation. In addition, the interactions of 41 and 47 with their endothelial ligands are unique in that unlike the 2 integrins, 4 integrins can mediate both leukocyte rolling and firm
adherence to the endothelial surface.6-9
Based on the use of blocking antibodies, numerous in vivo studies
suggest that 4 integrins can play a role in selective leukocyte recruitment in inflammatory disease processes such as allergic inflammation,10-12 arthritis,13 and
delayed-type hypersensitivity.14 Although antibodies to
both 4 integrins and VCAM-1 have been used to study selective
leukocyte recruitment in various animal models, there has been no
thorough analysis performed to establish whether 4 integrin
expression on leukocytes in various species is the same as that
observed in humans. Though guinea pig and sheep neutrophils do not
express 4 integrins,12,15,16 some studies in rats and
mice have unexpectedly found that antibodies to 4 integrins can
affect neutrophil recruitment responses and neutrophil-dependent
inflammation in vivo.17,18 Previously, these findings have
been attributed to 4 integrin antibody effects on other leukocytes,
which in turn may affect neutrophil recruitment. However, Issekutz et
al19 have recently shown that, unlike human neutrophils,
rat neutrophils constitutively express low levels of 4 integrins,
and that administration of an anti-4 monoclonal antibody (MoAb), in
conjunction with an anti-2 integrin MoAb, inhibits neutrophil
migration into arthritic joints in the rat. Although these findings
strongly suggest a role for neutrophil-expressed 4 integrins, the
investigators did not confirm neutrophil interaction with the
endothelial ligands VCAM-1 or MAdCAM-1.19 In the present study, we confirm and extend the findings of Issekutz et
al19 by showing that rat neutrophils consistently express
41 integrins and use 41 integrins to bind VCAM-1, whereas
only 4 integrins are used to bind MAdCAM-1 in vitro.
| |
MATERIALS AND METHODS |
Rat leukocyte isolation.
Whole blood leukocytes and enriched neutrophil populations were
isolated from pentobarbital-anesthetized male Sprague-Dawley rats
(Charles River Labs Inc, Wilmington, MA and Harlan Sprague Dawley,
Indianapolis, IN) weighing 275 to 300 g. EDTA-anticoagulated arterial
blood was obtained via cannulation of the right carotid artery. For
whole blood leukocytes, a leukocyte-rich buffy coat was obtained by
centrifugation at 400g for 20 minutes at 22°C. Contaminating
red blood cells (RBC) were removed via hypotonic lysis performed at
4°C. Cell differentials were determined by Diff-Quick staining
(Baxter Scientific Products, McGaw, IL) and viability was confirmed by
erythrosin B dye exclusion.
Enriched neutrophils populations (polymorph-nuclear leukocyte [PMN])
were obtained via density gradient centrifugation methods, in a manner
similar to that described for human neutrophil isolation.20 In brief, EDTA-anticoagulated whole blood was layered over Percoll (specific gravity, 1.085 g/L) and centrifuged for 20 minutes at 22°C,
followed by hypotonic lysis of RBC at 4°C. In preparations in which
contaminating lymphocytes made up more than 5% of the cell population,
the cells underwent a second centrifugation step over Percoll (specific
gravity, 1.085 g/L) to remove these cells. Neutrophil populations were
93.1% ± 0.6% pure with 5.5% ± 0.5% contaminating eosinophils
and 1.5% ± 0.3% contaminating lymphocytes (n < 20). Cell
viability for all flow cytometry and adhesion experiments was greater
than 97%.
In addition to isolating neutrophils, mixed populations of rat
mononuclear cells (MNC), consisting of lymphocytes and monocytes, were
obtained by harvesting the upper layer from the Percoll gradient. These
cells were washed twice and subjected to hypotonic lysis to remove any
contaminating platelets or RBC. This mononuclear cell population was
used as a positive control for analysis of 4 and 1 expression and
in VCAM-1 and MAdCAM-1 adhesion assays (see below), and consisted of
7.8% ± 1.5% monocytes and 92.3% ± 1.5% lymphocytes (n = 6).
Flow cytometric analysis of leukocyte adhesion molecules.
The following 4 integrin MoAbs were purchased and used at the
indicated saturating concentrations: TA-2 (immunoglobulin
[Ig]G1, mouse anti-rat, 1 µg/mL; Seikagaku America,
Inc, Rockville, MD), MR4 (IgG2b, mouse anti-rat, 5 µg/mL; Pharmingen, San Diego, CA), and L25 (IgG1, mouse
anti-human, found to cross-react with rat, 5.8 µg/mL;
Becton-Dickinson, Mountain View, CA). 1 integrin staining on rat
leukocytes was examined using the hamster anti-mouse MoAb Ha2/5 (IgM, 3 µg/mL; Pharmingen) and the hamster anti-mouse MoAb HM1-1 (IgG, 3 µg/mL; Pharmingen) found to cross-react with rat. Staining was also
attempted with the murine anti-human 7 integrin MoAb ACT-1 (IgG, 1 µg/mL) generously provided by David J. Erle (University of
California, San Francisco). Murine anti-rat MoAbs recognizing CD11a
(WT.1, IgG2a, 5 µg/mL), CD11b/c (OX-42,
IgG2a, 1 µg/mL), CD18 (WT.3, IgG1, 5 µg/mL), and CD3 (G4.18, IgG3, 3.1 µg/mL) were also
purchased from Pharmingen, and control, nonbinding, isotype-matched
mouse IgG1 and hamster IgM were obtained from Coulter
(Hialeah, FL) and Pharmingen, respectively. A mouse anti-human L-selectin MoAb LAM1-116 (IgG2a, 3 µg/mL),
cross-reactive with rat, was generously provided by Drs Thomas Tedder
and Douglas Steeber (Duke University, Durham, NC).
Labeling of cells for indirect immunofluorescence was performed as
described4 using saturating concentrations of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody (BioSource International, Camarillo, CA) for all preparations except
those in which Ha2/5 or HM1-1 was the primary MoAb, in which case an
FITC-conjugated goat anti-hamster IgG (H + L) antibody (Jackson
Immunoresearch Laboratories, Inc, West Grove, PA) was used. Cells were
immediately analyzed unfixed using an EPICS Profile flow cytometer
(Coulter Corporation, Hialeah, FL). Monocyte and lymphocyte populations were distinguished via their scatter and CD3:CD11b/c staining characteristics. Neutrophil and eosinophil populations were easily distinguished from each other via their light
scatter and 4 integrin staining characteristics (ie, eosinophils have higher forward scatter and higher 4 integrin expression than
neutrophils). To examine whether 4 integrin expression could be
upregulated by neutrophil activation, enriched neutrophil populations were incubated with either phorbol myristate acetate (PMA; 10 ng/mL),
fMLP (10 6 mol/L), or C5a (100 ng/mL) for 20 minutes at
37°C before incubation with primary antibodies.
Irrelevant isotype-matched control staining with murine
IgG1, IgG2a, or IgG3, or hamster
IgM, typically yielded mean fluorescence values of 2 to 4. Data are
presented as fold mean fluorescence above the respective control to
facilitate comparisons among various cell types.
Neutrophil labeling with 51Cr and static adhesion
assays.
For adhesion assays, rat neutrophils, mononuclear cells, and human
Jurkat cells were labeled with 51Cr as described for human
leukocytes.20 The Jurkat human T-lymphocytic cell line, a
generous gift of Dr Vincenzo Casolaro (Johns Hopkins Asthma and Allergy
Center, Baltimore, MD), was used as a control for adhesion assays
because these cells are known to constitutively express high levels of
41, but they do not express 47 (21 and data not
shown). The Jurkat T cells were passaged every 3 to 5 days in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal
bovine serum (FBS; Hyclone Laboratories, Inc, Logan, UT), 100 U/mL
penicillin G, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B
(GIBCO-BRL). Chinese hamster ovary (CHO) cells and CHO cells stably
transfected with rat or human VCAM-1 (known to bind to both human and
rat 422; generously supplied by Dr Roy Lobb, Biogen Inc,
Cambridge, MA) were grown to confluence as previously
described23 using MEM alpha medium (GIBCO-BRL)
supplemented with 10% FBS and methotrexate (500 nmol/L), in 24-well
plates for use in static adhesion assays. CHO cells stably transfected
with human MAdCAM-1, generously provided by Dr Michael Briskin
(LeukoSite, Inc, Cambridge, MA), were grown in a manner identical to
VCAM-1-transfected CHO cells except that methotrexate was omitted.
Rat leukocytes and Jurkat T cells were incubated for 30 minutes at
4°C in PAG-Mn buffer (PIPES buffer; 25 mmol/L Piperazine-N, N -bis-[2-ethanesulfonic acid], 110 mmol/L NaCl, 5 mmol/L KCl [containing 0.003% human serum albumin], 0.1% D-glucose, and 1 mmol/L MnCl2 [Sigma Chemical Co, St Louis, MO]) to
enhance 4 avidity.24 Leukocyte aliquots (100 µL,
2.5 × 105 cells/well) were added in
duplicate to each well and allowed to adhere for 10 minutes. All
adhesion assays were performed at 4°C to diminish 2 integrin
interactions. Nonadherent cells were removed by washing with PAG-Mn.
Adherent cells were then lysed with 1 mol/L NH4OH for 30 minutes, the supernatant removed, and radioactivity counted on a gamma
counter. Total counts (ie, total radioactivity) added per well were
determined by counting separate aliquots of 2.5 × 105
labeled cells. Percent adhesion was obtained by dividing counts for
bound cells by the total counts. In some experiments, cells were
preincubated for 30 minutes with blocking antibody to rat 4 (TA-2, 1 µg/mL), 1 (Ha2/5 or HM1-1, 3 µg/mL),4 or CD18 (WT.3, 3 µg/mL) to show the specificity of the adhesion interaction. Preincubation with the human VCAM-1 MoAb 2G7 (F(ab)2, 10 µg/mL)4 was also used to show adhesion specificity in
assays using human VCAM-1-transfected CHO cells. All experiments were
performed in duplicate and data are presented as mean adhesion for four
to seven individual experiments.
Because enriched neutrophil populations contained approximately 7%
contaminating cells, we performed additional experiments to determine
if adherent cells were neutrophils or contaminating eosinophils or
lymphocytes. In some experiments, non-51Cr-labeled
neutrophil preparations were allowed to adhere to rat or human
VCAM-1-transfected CHO cells as described. After removal of
nonadherent cells, PAG-EDTA (5 mmol/L) was added to the wells for 2 minutes to remove adherent cells. These cells were collected and cell
differentials were determined by Diff-Quick staining.
Statistical analysis.
All leukocyte adhesion data are presented as mean ± SEM. Data were
compared by analysis of variance (ANOVA) using post hoc analysis with
Fischer's corrected t-test. Probabilities of .05 or less were
considered statistically significant.
| |
RESULTS |
Rat neutrophils express 4 and 1
integrins.
Immunofluorescent staining and flow cytometric analysis were performed
on rat whole blood leukocytes and enriched neutrophil populations using
a panel of murine anti-4 MoAbs. Expression of 4 was examined on
neutrophils, lymphocytes, monocytes, and eosinophils. Differences in
neutrophil and eosinophil scatter in the rat were confirmed in
experiments using enriched neutrophil populations in which neutrophils
made up approximately 94% of cells, with the remainder being
eosinophils. In these experiments two distinct populations, with
percentage values corresponding to neutrophils and eosinophils,
respectively, could be visualized based on light scatter, and these
populations were found to have distinct staining characteristics for
4. These differing scatter characteristics made it possible to
independently gate on neutrophils or eosinophils without additional
antibody labeling.
Contrary to findings with human neutrophils, rat neutrophils
constitutively expressed 4 integrins as confirmed by staining with
MoAb TA-2, MR4, and L25 (Fig 1A and C).Expression of 4 integrins on rat neutrophils was relatively low
compared with levels on other cell types, but expression was detectable
in all animals examined (Fig 1B). The brightest staining for rat 4
on all cell types was observed with the MoAb TA-2 (Fig 1C). The
anti-rat 4 MoAb MR4 and the anti-human MoAb L25 provided similar
levels of staining. Incubation of enriched neutrophil populations with PMA, fMLP, or C5a, at concentrations sufficient to upregulate 2
integrin expression, did not increase expression of 4 as determined by staining with MoAb TA-2 (data not shown).
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| Fig 1.
Indirect immunofluorescence and flow cytometric analysis
of the surface expression of 4 and 1 integrins on rat leukocytes. (A) Representative histograms of rat neutrophil staining with TA-2 and
Ha2/5 as compared with IgG1 or IgM control, respectively. (B) Actual fluorescence intensity (FI) values for 4 and 1
staining on neutrophils with MoAb TA-2 and Ha2/5 for six rats. *Mean FI values are significantly (P < .05) increased over
mean FI values for IgG1 or IgM controls. (C) 4 and 1
expression was examined on neutrophils (PMN,  ), lymphocytes (LYMPH,
 ), monocytes (MONO,  ) and eosinophils (EOS,  ) using the murine
anti-rat 4 MoAb TA-2, MR4, the murine anti-human 4 MoAb L25,
and the hamster anti-murine 1 MoAb Ha2/5. Data are presented as fold
increase in mean fluorescence intensity (MFI) over IgG or IgM control
(n = 6).
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To determine if neutrophils also expressed 1 integrins, rat
neutrophils, and other leukocyte types were first labeled with the
anti-1 MoAb Ha2/5 (Fig 1A). Rat neutrophils consistently showed low,
but significant levels of 1 staining (n = 6), as did other
leukocyte types (Fig 1A and B). Levels and patterns of staining for
1 on all cell types were similar when the hamster anti-mouse MoAb
HM1-1 was used (data not shown). Staining for rat 7 was attempted
using the murine anti-human 7 MoAb ACT-1, but staining on all rat
cell types was negative, implying a lack of MoAb cross-reactivity with
rat. As seen in Fig 2, the relative amounts
of 4 expressed on all rat leukocyte subtypes, as compared with the
2 integrins and L-selectin, was low, even for eosinophils, which
showed the strongest 4 integrin staining.
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| Fig 2.
Rat 4 expression as compared with the 2 integrins,
CD3, and L-selectin. Indirect immunofluorescence and flow cytometric analysis of the surface expression of 4 (TA-2), CD11a, CD11b/c, CD18, CD3, and L-selectin on rat neutrophils (PMN, ), lymphocytes (LYMPH, ), monocytes (MONO, ), and eosinophils (EOS, ). Data are presented as in Fig 1 (n = 5).
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Rat neutrophils adhere to VCAM-1 and MAdCAM-1.
To determine if the levels of 4 integrins on neutrophils were
sufficient to mediate neutrophil adhesion to the known ligands for
41 and 47, we first examined rat neutrophil adhesion to rat
and human VCAM-1-transfected CHO cells. As shown in Fig
3a, both neutrophils and MNC exhibited
significant adherence to rat VCAM-1 CHO cells, as compared with
nontransfected CHO cells (eg, for rat neutrophils, 15.8% ± 3.2%
v 3.4% ± 0.7% adhesion respectively, P < .01,
n = 7). Affinity of binding was relatively low, because neutrophil
adhesion to VCAM-1 was not consistently seen in the absence of
Mn2+ (data not shown). Adhesion of both cell populations to
rat VCAM-1 was completely inhibited by incubation of the cells with the
mouse anti-rat 4 blocking antibody TA-2 (1 µg/mL) (PMN,
2.6% ± 0.6%; MNC, 2.9% ± 0.8%). Adhesion was
only partially inhibited by incubation of the leukocytes with the
anti-1 MoAb Ha2/5 (PMN, 9.2% ± 2.1%; MNC, 10.4% ± 1.7%).
Similar results were obtained with the 1 MoAb HM1-1 (n = 2,
data not shown). The anti-CD18 MoAb WT.3 did not significantly block
neutrophil or MNC adhesion to rat VCAM-1 (n = 2, data not shown).
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| Fig 3.
Adhesion of rat neutrophils (PMN) and mononuclear cells
(MNC, lymphocytes and monocytes) to untransfected and rat (panel a, n = 7) or human (panel b, n = 6) VCAM-1-transfected CHO cells. Adhesion was tested to nontransfected CHO cells, or CHO cells transfected with VCAM-1 in the presence or absence of 4 MoAb TA-2.
Adhesion of rat neutrophils to rat VCAM-1 was also tested in the
presence of the 1 antibody Ha2/5. 1 blocking studies were not
performed for neutrophil adhesion to human VCAM-1. Data are presented
as mean percent adherence ± SEM. *(P < .05) and **(P < .01) indicate values significantly different from
percent adhesion to VCAM-1-transfected CHO-cells. (), CHO; (),
VCAM-1 CHO; (), VCAM-1 CHO + anti-4 MoAb; ( ), VCAM-1 CHO + anti-1 MoAb.
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Rat neutrophils and MNC also adhered to human VCAM-1-transfected CHO
cells (Fig 3b). Although adhesion was less than that observed with rat
VCAM-1 CHO cells (eg, for rat neutrophils, 5.5% ± 1.1% adhesion,
n = 6), binding was shown to be 4 specific as MoAb TA-2 completely
inhibited adhesion. Adhesion of rat neutrophils and MNC was also
completely inhibited by pretreatment of VCAM-1 CHO cells with the mouse
anti-human VCAM-1 MoAb 2G7 (n = 6, data not shown). Although rat
neutrophils showed consistent adherence to rat and human VCAM-1,
neutrophil adherence in both cases was less than that observed for
mononuclear cells (Fig 3a and b), consistent with the higher levels of
4 expression on rat MNC. Jurkat cells, which were used as a control
cell population, adhered avidly to human VCAM-1 (Fig 3b), consistent
with their high levels of 41 expression. Because enriched
neutrophil populations contained approximately 7% contaminating cells,
we performed additional experiments to determine whether the cells
adhering to VCAM-1 were neutrophils or contaminating eosinophils or
lymphocytes. For both rat and human VCAM-1, neutrophils were found to
make up greater than 86% of the adherent cells, with eosinophils
making up 10.0% ± 2.5% and lymphocytes 3.0% ± 0.8% (n = 5).
Because 1 integrin blockade only partially inhibited adhesion to
VCAM-1, and because we were unable to directly identify 7 integrin
expression by flow cytometry, we determined if rat leukocytes could
adhere to MAdCAM-1, an 47 ligand. As shown in Fig
4, both rat neutrophils and MNC exhibited
significant adherence to human MAdCAM-1-transfected CHO cells (eg,
neutrophil adhesion 15.9% ± 4.3%), as compared with untransfected
CHO cells (4.3% ± 1.2% adhesion; Fig 4). Again, the mouse
anti-rat 4 MoAb TA-2 was used to show the 4 specificity of rat
neutrophil and MNC adhesion to MAdCAM-1. Adhesion of rat neutrophils
and MNC to MAdCAM-1 CHO cells was completely blocked by the addition of
MoAb TA-2 to cell preparations. The 1 MoAb Ha2/5 did not have any
significant effect on neutrophil adhesion to MAdCAM-1 CHO cells,
although it did significantly inhibit MNC adhesion to MAdCAM-1. Similar
results were observed for both cell types with MoAb HM1-1
(n = 2, data not shown).
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| Fig 4.
Adhesion of rat neutrophils (PMN, n = 5), mononuclear
cells (MNC, n = 5), or Jurkat cells (n = 4) to untransfected
and human MAdCAM-1-transfected CHO cells. Adhesion was tested to
nontransfected CHO cells, or CHO cells transfected with MAdCAM-1 in the
presence or absence of 4 MoAb TA-2 or 1 MoAb Ha2/5. Data are
presented as mean percent adherence ± SEM. **(P < .01)
indicate values significantly different from percent adhesion to
MAdCAM-1-transfected CHO cells. (), CHO; (), MAdCAM-1 CHO;
(), MAdCAM-1 CHO + anti-4 MoAb; (), MAdCAM-1 CHO + anti-1 MoAb.
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| |
DISCUSSION |
Previous studies have shown that normal human, guinea pig, and sheep
neutrophils do not constitutively express 4
integrins.4,12,15,16 Although 4 integrins are not
constitutively present on human neutrophils, Kubes et al25
have shown that under certain experimental conditions such as treatment
with dihydrocytochalasin B or after in vitro transendothelial
migration, human neutrophils can be induced to express 4 integrins
and can adhere to stimulated endothelial cells under static and flow
conditions.9,25 However, previous data19 and
data presented here indicate that rat neutrophils, unlike other
species, constitutively express 4 integrins. Flow cytometric
analysis of rat neutrophils using the mouse anti-rat 4 MoAbs TA-2
and MR4, as well as the mouse anti-human 4 MoAb L25, showed a
consistent, low level of 4 expression (eg, 2.3 ± 0.3 mean fold
fluorescence above background IgG with TA-2). These data confirm and
expand the findings of Issekutz et al19 in which low levels
of 4 expression were shown on the surface of rat neutrophils using a
single MoAb TA-2. We show a similar level of 4 expression on
neutrophils using MoAb TA-2, as well as with the other MoAbs which bind
rat and human 4 integrins. Direct comparison of 4 expressed on
neutrophils to that on other rat leukocytes via flow cytometry shows
that although they are consistently present, neutrophils express the
lowest levels of 4 integrins, with rat eosinophils expressing the
highest levels.
In addition, we show low levels of 1 integrin expression on rat
neutrophils. Flow cytometric analysis of rat 1 expression using MoAb
Ha2/5 revealed consistent, low-level 1 expression on neutrophils,
with greater expression on lymphocytes and eosinophils, and the
greatest expression on monocytes. However, the partial to minimal
inhibitory activity seen with 1 integrin blockade in the VCAM-1 and
MAdCAM-1 adhesion assays may suggest the presence of an additional 4
integrin subunit, such as 7. It is possible that
neutrophil-expressed 47 could account in part for neutrophil adherence to both VCAM-1 and MAdCAM-1, because 47 is a ligand for
both molecules. Unfortunately, the lack of antibodies which cross-react
with rat 7, and the inability to obtain eosinophil-free preparations
of neutrophils for immunoprecipitation experiments makes it impossible
to determine the exact heterodimeric composition of rat neutrophil 4
integrins at this time. Interestingly, the ability of the anti-1
MoAb to significantly block MNC adhesion to MAdCAM-1 may suggest that
MNC-expressed 41 integrins can interact with MAdCAM-1 in the rat.
In mouse and human cell systems, 4 integrin interactions with
MAdCAM-1 have been seen only with 47, not 41.
Beyond showing the expression of 4 and 1 on the neutrophil
surface, we also showed that neutrophil-expressed 4 integrins can
mediate neutrophil, as well as mononuclear cell, adhesion to VCAM-1 and
MAdCAM-1 expressed on transfected CHO-cells. Isolated rat neutrophils
incubated in Mn2+-containing buffer specifically adhered to
both rat and human VCAM-1-transfected CHO cells and
MAdCAM-1-transfected CHO cells at 4°C. Neutrophils did not
consistently adhere to VCAM-1 or MAdCAM-1 in the absence of
Mn2+, suggesting that these cells expressed low levels of
activated 4 integrins. This may in part explain differences between
our findings and those of Andrew et al26 in which they were
unable to show 47-mediated adhesion to VCAM-1 at 4°C. Adhesion
to both VCAM-1 and MAdCAM-1 was completely blocked by anti-4 MoAb
TA-2. Our findings that rat neutrophils bind VCAM-1 and MAdCAM-1 in an
4-dependent manner support in vivo data from Issekutz et
al19 which indicate that the MoAb TA-2 may effect
neutrophil recruitment in the rat. Although these
findings19 strongly suggest a role for neutrophil-expressed
4, the investigators did not confirm that neutrophil 4 integrins
were expressed at sufficient levels to mediate interaction with the
endothelial ligands VCAM-1 or MAdCAM-1. However, adhesion data from our
studies clearly indicate that the in vivo effect of diminished
neutrophil recruitment observed with the administration of MoAb TA-2 is
likely the result of antibody blockade of neutrophil interaction with
VCAM-1. Furthermore, the ability of 4 integrin MoAb to block rat
neutrophil adhesion to MAdCAM-1 suggests that 4 integrin MoAb may
also be capable of blocking neutrophil trafficking to the gut. Blocking
rat 4 integrins may also inhibit neutrophil interaction with the
matrix protein fibronectin, because emigrated rat neutrophil binding to
cardiac myocytes has been shown to be 4 integrin and fibronectin
dependent.27
Studies by Issekutz et al19 provide the most direct
evidence that neutrophil-expressed 4 integrins may be important for neutrophil recruitment in the rat, but earlier data from Mulligan et
al,17 published before 4 integrin identification on rat neutrophils, also support this. In these experiments, the rat 4
antibody TA-2 was found to significantly reduce neutrophil infiltration, changes in lung permeability, and hemorrhage in a model
of intrapulmonary IgG deposition. These investigators have previously
shown this model of lung injury to be almost exclusively neutrophil
mediated, with some role for alveolar
macrophages.28,29 In their discussion of the
data the investigators speculate that the effects observed in this
model, with the antibody TA-2, may be attributed to a role for 4 in
macrophage cytokine release.17 Although the potential
effects of MoAb TA-2 on macrophage function can not be discounted, our
findings would suggest that the inhibition of neutrophil infiltration
is more likely a direct effect of the antibody on neutrophil
interaction with 4 integrin ligands. Examples of 4 MoAb reduction
of neutrophil recruitment also exist in the mouse. Chisholm et
al18 found reduced neutrophil-dependent edema with an 4
MoAb treatment in a mouse model of T-cell-dependent contact
hypersensitivity. Here the investigators again speculate that the
decreased neutrophil recruitment is the result of decreased T-cell
infiltration and thus decreased mediator release. It is likely that
these observations are in part correct, but the presence of 4 on
mouse neutrophils has not been examined, and therefore a direct effect
of the 4 MoAb on neutrophil recruitment can not be ruled out.
In conclusion, we have shown that rat neutrophils, unlike
neutrophils from most other species, constitutively express low levels
of functional 4 and 1 integrins. The low level expression of 4
integrins can mediate neutrophil binding to both rat and human VCAM-1
as well as human MAdCAM-1. These data show a novel role for 4
integrins in rat neutrophil recruitment and suggest that MoAbs reacting
with 4, 1, VCAM-1, MAdCAM-1, or perhaps 7 administered in vivo
in rat models of cell recruitment may directly affect neutrophil
recruitment.
| |
FOOTNOTES |
Submitted July 30, 1997;
accepted November 17, 1997.
Supported by National Institutes of Health (NIH) Grants No. HL-49545
and AI-07056, the Burroughs Wellcome Fund, and with funds from the
Office of Naval Research through the Asthma and Allergy Foundation of
America.
Address reprint requests to Bruce S. Bochner, MD, Johns Hopkins Asthma
and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We gratefully acknowledge Drs Thomas Tedder, Douglas Steeber,
David J. Erle, and Walter Newman for providing MoAbs; Dr Roy Lobb for
the VCAM-1-transfected CHO cells; Dr Michael J. Briskin for the
MAdCAM-1-transfected CHO cells; and Dr Vincenzo Casolaro for the
Jurkat T-cell line.
| |
REFERENCES |
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Leukocyte-endothelial adhesion molecules.
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Abstract
Introduction
Abstract