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d in IgG Immune Complex-Induced Rat Lung Injury1 ,2
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and ICOS Corporation, Bothwell, WA 98021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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d is a newly cloned adhesion molecule that forms a heterodimer
with CD18. The requirement for d in IgG immune complex-induced lung
injury in rats has been evaluated by the use of blocking polyclonal and
monoclonal antibodies to rat d. Using whole lung extracts, Northern
and Western blot analyses have revealed up-regulation of mRNA and d
protein in inflamed lungs. Immunostaining has revealed the presence of
d in lung tissue and in alveolar macrophages as early as 1 h
after initiation of the inflammatory reaction. When polyclonal rabbit
Ab to rat d was coinstilled into lung together with Ab to BSA, lung
injury (as determined by leakage of [125I]albumin into
lung parenchyma) was significantly diminished. In parallel, there was
reduced accumulation of neutrophils recoverable in bronchoalveolar
lavage (BAL) fluids. These findings were associated with reduced levels
of TNF- as well as
NO2-/NO3- in BAL
fluids. A hamster mAb to rat d was also protective in this lung
injury model. Anti-d inhibited in vitro production of
NO2-/NO3- by rat
alveolar macrophages (stimulated with LPS and IFN-
) by approximately
60%. These data suggest that, in the lung inflammatory model employed,
d up-regulation occurs in lung macrophages and is necessary for
expression of TNF-, recruitment of neutrophils, and full development
of lung injury. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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d has been cloned
and, as part of the heterodimeric complex, d/CD18, it appears to be
a counter-receptor for ICAM-3 (22), although the in vivo role of
d/CD18 is not known.
In models of acute lung injury in rats, the recruitment of neutrophils
from the intravascular space to the alveolar compartment has been the
focus of recent investigations. Depending on the inflammatory model,
this process has been shown to be dependent on engagement of a series
of endothelial cell adhesion molecules (e.g., E-selectin, P-selectin,
ICAM-1, PECAM-1 (platelet endothelial cell adhesion molecule)) as well
as neutrophil adhesion molecules (L-selectin,
ß2-integrins) (23, 24, 25, 26). The recent cloning and expression
of rat d has allowed us to explore the role of this novel
ß2-integrin in the setting of the acute lung inflammatory
response triggered by deposition of IgG immune complexes. Up-regulation
of d at both the mRNA level (as determined by Northern blot
analysis) and protein level (as demonstrated by Western blot analysis
and immunohistochemical staining) was found. Blocking of d function
with Abs resulted in diminished inflammatory responses, as measured by
pulmonary vascular leak and content of neutrophils in bronchoalveolar
lavage (BAL) fluids. Furthermore, in vivo blocking of d appeared to
result in decreased activation of lung macrophages, as evidenced by
substantial decreases in both TNF- and
NO2-/NO3- content.
These data suggest that d plays a critical role in the activation of
macrophages in the context of IgG immune complex-initiated lung injury
in rats.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except where noted, all products were purchased from Sigma Chemical Co., (St. Louis, MO).
IgG immune complex-induced alveolitis
Male Long-Evans (specific, pathogen-free) rats (250 to 350
g, Charles River Breeding Laboratories, Portage, MI) were used for all
studies. Intraperitoneal injections of ketamine (2.5 to 5.0 mg/100 g
body weight) were given for sedation and anesthesia. IgG immune complex
lung injury was induced and quantified as previously described (24).
Polyclonal rabbit IgG containing 2.5 mg anti-BSA was instilled (in
a volume of 300 µl) into the lungs via a tracheal cannula. The Ag,
BSA (10 mg), was injected i.v. immediately thereafter in a volume of
0.5 ml. Rats were killed at the indicated times. Lung injury was
quantified at 4 h by measuring increases in lung vascular
permeability (extravascular accumulation of 125I-labeled
BSA). For blockade, either 300 µg preimmune rabbit IgG or
anti-d polyclonal rabbit IgG were instilled intratracheally with
the anti-BSA. When hamster monoclonal IgG was employed, 200 µg
hamster monoclonal anti-TNP IgG (Armenian hamster IgG clone
G235–2356) or 200 µg hamster mAb to rat d (clone 205c) was
instilled with the anti-BSA.
d-specific polyclonal rabbit sera
Rabbit anti-rat d polyclonal sera was generated to a
recombinant rat d "I" domain/human IgG (rd/huIgG) fusion
protein. The Ag was derived by subcloning cDNA from the "I" domain
of rat d (encompassing base pairs 469–1125) into an expression
vector containing cDNA from the Fc region of human IgG4. The
rd/huIgG fusion protein was expressed in COS 7 cells and isolated
from the cell supernatants by passage over a protein A column. The
material was eluted with 0.1 M glycine buffer, pH 3.0, dialyzed against
sterile PBS (pH 7.3), and found to be 
85% pure by SDS-PAGE
analysis. Rabbits were initially immunized s.c. with the rd/huIgG
fusion protein emulsified in CFA, while subsequent boosts were
administered using IFA. Ig from the anti-rat d polyclonal rabbit
sera was purified on a protein A column, then absorbed by passing it
over a human IgG4 CNBr-Sepharose column to remove anti-human IgG
reactivity. To test the specificity of the rabbit polyclonal sera,
immunoprecipitations were done with rat spleen lysates. The rabbit
polyclonal Ab was found to recognize only the rat d/CD18 heterodimer
(-chain of 145 kDa) and not the other ß2 integrins
(see below).
Biotinylated cell lysates
Biotinylated bone marrow cell lysates were prepared from a Lewis
rat. Briefly, femurs were excised and bone marrow cells were flushed
from the bone with a 20-gauge needle and a 10-ml syringe containing
PBS. The cells (2 x 108) were labeled for 15 min
at 25°C with 0.1 mg/ml normal human serum-sulfobiotin in 40 ml PBS
followed by three consecutive washes with 50 ml PBS. The cells were
pelleted and lysed in 2 ml lysis buffer (1% Nonidet P-40, 50 mM Tris
(pH 8.0), 0.5 M NaCl, and 10 mM EDTA) containing 0.1 mM PMSF. Lysates
were incubated 5 min at 25°C, vortexed for 30 s, then placed on
ice for 15 min. The lysates were centrifuged to remove insoluble
material.
Immunoprecipitation
Prior to the immunoprecipitation, 200 µl of protein
A-Sepharose bead slurry (1:2 beads to liquid) were added to 1 ml of
cell lysates and mixed on an end-over-end rotator overnight at 4°C.
The beads were pelleted and the precleared lysates aliquoted into
100-µl samples. For each immunoprecipitation, 10 µg of one of the
following purified mAbs were added: (515F = mouse anti-rat
CD11a (ICOS, Bothell, WA); OX42 = mouse anti-rat CD11b
(Serotech, Raleigh, NC); 100G = hamster anti-rat CD11c (ICOS);
205C = hamster anti-rat d (ICOS); 20C5B = mouse
anti-rat CD18 (ICOS); and purified mouse IgG (Cappel, Durham, NC).
The Ab was allowed to mix with the cell lysates on an end-over-end
rotator for 2 h at 4°C.
To facilitate immunoprecipitations done with murine mAbs, protein A-Sepharose was armed with rabbit anti-mouse IgG. Approximately 0.5 ml protein A-Sepharose beads was mixed with 2 mg rabbit anti-mouse IgG and the slurry was allowed to mix for 30 min at 25°C. The protein A-Sepharose was washed and resuspended in 1 ml PBS. A total of 100 µl armed protein A-Sepharose slurry were added to each immunoprecipitation tube containing a mouse Ab. A total of 100 µl unarmed protein A-Sepharose were added to lysates containing hamster mAbs. The tubes were incubated end-over-end at 25°C for 30 min, the protein A-Sepharose was then pelleted and the cell lysates were removed. The protein-A Sepharose beads were washed three times in cold wash buffer (10 mM HEPES, 50 mM Tris (pH 8.0), 0.5 M NaCl, and 1% Triton X-100), resuspended in 20 µl 2 x SDS (containing 10% 2-ME) buffer, and boiled 5 min. The beads were pelleted and the liquid was run on a prepoured 8% SDS gel (Novex, San Diego, CA).
The protein was transferred to nitrocellulose and standard Western blot techniques were applied using a 1:10,000 dilution of Streptavidin-horseradish peroxidase (HRP) (Boehringer Mannheim, Indianapolis, IN) and the enhanced chemiluminescence detection kit (Pierce, Rockford, IL).
Rat d transfectants
The rat d cDNA was cloned from a rat spleen library,
purchased from Clonetech Laboratories, (Palo Alto, CA). The library was
screened under low stringency conditions using a 5' probe generated
from the human d cDNA clone. A rat clone, designated 684.3, was
identified in the screen, sequenced, and found to have 65% homology
with the human d cDNA.
DG44 CHO cells were cotransfected with a pDC1 plasmid containing full
length rat d and a pRC plasmid containing full length human CD18.
The cells were plated onto a 150-cm tissue culture dish containing 20
ml culture medium (DMEM-F12, 10% FBS, 1 mM sodium phosphate, 2 mM
L-glutamine, 100 U each of penicillin and streptomycin/ml,
0.1 mM hypoxanthine, and 0.016 mM thymidine). After 2 days the cells
were transferred to selective media (DMEM-F12, 10% dialyzed FBS, 1 mM
sodium phosphate, 2 mM L-glutamine, 100 U each of
penicillin and streptomycin/ml, and 400 µg/ml G418/ml) and allowed to
grow. When colonies were established, cells were split 1:2 every 3 to 5
days. Expression of a rat d/huCD18 heterodimer was confirmed by FACS
staining using the TS1/18.1 mAb (anti-human CD18) and the
anti-rat d polyclonal rabbit antisera. To increase the
expression of the rat d/CD18 complex, transfected CHO cells were
subjected to several rounds of sorting of FACS using the rabbit
anti-rat d polyclonal antisera.
Generation of mAb to rat d
Monoclonal antibody to rat d was made in Armenian hamsters
using the rd/huIg fusion protein as the immunogen. The fusion wells
were initially screened by FACS analysis using the d/human
CD18-transfected CHO cells. Positive fusion wells were subsequently
screened for the ability to immunoprecipitate d from biotin-labeled
rat spleen lysates. The mAb, designated 205C, was identified in an
early fusion and further characterized. To verify the specificity of
the 205C mAb, rat spleen lysates were first precleared of all other
-chains of the ß2 integrin family, including CD11a,
CD11b, and CD11c, by immunoprecipitation. Following this step, 205C
continued to immunoprecipitate a 145 kDa/95 kDa heterodimer consistent
with the known size of the d/CD18 complex. To complete the
characterization, the mAb was used to affinity purify d from rat
splenocyte lysates. The N-terminal sequence analysis from the
affinity-purified protein was found to be consistent with amino acid
sequence predicted by the d cDNA clone (data not shown).
In vitro production of NO2-/NO3-
Rat alveolar macrophages were recovered as previously described
(27). Cells (1 x 105/well) were cultured in 96-well
tissue culture plates in DMEM and nonadherent cells removed following
1-h incubation at 37°C in 7.5% CO2 in air. Cells were
stimulated with murine IFN- (25 U/ml) and 10 µg bacterial LPS at
37°C for 18 h and supernatant fluids collected and analyzed for
NO2-/NO3-.
Immunostaining techniques
For immunostaining of frozen sections, lungs from injured rats
were frozen in OCT compound (Miles Co., Elkhart, IN) and stained with
hamster mAb 205C diluted 1:1000 in PBS containing 0.1% BSA for 1
h in a humidified chamber. Slides were then washed two times in PBS and
incubated for 1 h with HRP-conjugated rabbit anti-hamster
IgG-specific Ab (Rockland, Gilbertsville, IL) diluted 1:10,000 in PBS.
Slides were washed two times in PBS, dried, and incubated with
HRP-specific substrate True Blue (Kirkegard & Perry, Gaithersburg, MD)
for 5 min. Slides were then dipped in 100% ethanol. When BAL
macrophages were stained for rat d, cytospin preparations were used
employing similar immunostaining methods.
Western blot analysis
Lung homogenates were prepared from rats undergoing IgG immune
complex-induced lung injury at times 0, 1, 2, and 4 h and
separated by electrophoresis on SDS-polyacrylamide gels (15%).
Homogenate protein levels were determined by Bio-Rad Protein Assay
(Bio-Rad, Hercules, CA). A total of 100 µg obtained from each time
point were loaded per lane under nonreducing conditions. Separated
proteins were transblotted to polyvinylidene difluoride membrane
(Westran, Schleicher and Schuell, Keene, NH) for 1 h at 10 V. The
membrane was blocked overnight at 4°C with 5% nonfat dry milk (NFDM)
in PBS and then washed three times with 0.05% Tween-20 in PBS (PBS-T).
The membrane was incubated for 1 h at room temperature with
primary Ab (rabbit anti-mouse d) at a dilution of 1/100 in 1%
NFDM in PBS. After washing three times in PBS-T, secondary Ab (goat
anti-rabbit IgG HRP-conjugated Ab; Bio-Rad) was added at a final
dilution of 1/10,000 in 1% NFDM-PBS and incubated for 1 h at room
temperature. After washing, the membrane was developed by enhanced
chemiluminescence technique according to the manufacturer’s protocol
(Amersham Co., Little Chalfont, U.K.).
Northern blot analysis
Following IgG immune complex deposition, rats were sacrificed at
2-h intervals from 0 to 4 h. Whole lungs were dissected and frozen
in liquid nitrogen for Northern blot analysis of IL-6 mRNA. RNA was
extracted using a guanidinium-isothiocyanate method as described
previously (27, 28). Twelve micrograms of cytoplasmic RNA were
fractionated electrophoretically in a 1% formaldehyde gel and
transferred to a nylon blot (Zetabind, CUNO Laboratories, Meriden, Ct).
Equal loading of samples was confirmed by methylene blue staining of
18S and 28S rRNA bands. Rat d cDNA clone was used as a template to
generate a PCR probe encompassing base pairs 2014–2871. The region of
the gene was chosen for lack of homology to the other integrin
-chains. The cDNA for rat d was
[32P]dCTP-radiolabeled (NEN-DuPont, Boston, MA) by PCR to
generate the cDNA probe that was applied to the Northern blot.
Hybridization was performed at 65°C for 18 h and the
autoradiogram was developed on Kodak X-Omat film.
For 18S band labeling, the Northern blot was prehybridized for 4 h
on 6x SSC with 1% SDS. An oligomer for the 18S band
(5'-GACAAGCATATGCTACTGGC-3') was labeled with
[-32P]ATP (10 pmol) using T4 polynucleotide kinase (10
U, Life Technologies) incubated for 30 min at 37°C. A second aliquot
of kinase (10 U) was added and the reaction was incubated an additional
15 min. The reaction was terminated with 2 ml 0.5 M EDTA.
Unincorporated [-32P]ATP was removed by spin-column
chromatography (BioSpin 6 column, BioRad). Labeled probe was combined
with 8 mg of unlabeled oligonucleotide and heated for 1 min at 95°C
prior to hybridization with the Northern blot. The blot was incubated
for 18 h at 42°C. Following hybridization, the blot was washed
twice with 3x SSC with 1% SDS for 15 min each at 50°C and developed
using a phosphor imaging screen.
BAL fluid neutrophil counts and TNF- content
BAL fluids were collected from rats sacrificed at 4 h
following commencement of injury by instilling and withdrawing 9 ml
sterile Dulbecco’s PBS (without Ca/Mg) three times from the lungs via
an intratracheal cannula. Total white cell counts were determined using
a Coulter counter (Coulter Electronics, Hialeah, FL). Specimens for
cell differentials were prepared using cytospin centrifugation
(700 x g for 7 min) on BAL fluids. Specimens were fixed and
stained with Diff-Quik products (Baxter Co., Miami, FL) for
determination of percentage of neutrophils and macrophages/monocytes.
The total numbers of neutrophils for each BAL sample were then
determined according to the volume of BAL recovered. Remaining BAL
samples were centrifuged at 1500 x g for 10 min and
the supernatant fluids frozen and subsequently evaluated for TNF-
activity using a standard WEHI cell cytotoxicity assay as previously
reported (29). Time course studies of TNF- expression have shown
4 h to be a time point of peak expression.
Measurement of NO2-/NO3- in BAL fluids and culture supernatant fluids
Nitrite was measured with the Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, and 25% hydrochloric acid) which forms a chromophore absorbing at 543 nm (27). Any nitrate present was reduced to nitrite with nitrate reductase (EC 1.6.2.2) from Aspergillus sp., to which 2.5 nM NADPH was added. Absorbance was then measured as NO2- (the combination of nitrite and reduced nitrate being designated as NO2-/NO3-). These measurements were presented as nanomoles per milliliter of BAL fluid or nanomoles in culture supernatant fluids.
Statistical analysis
All values were expressed as mean ± SEM. All statistical comparisons were made between treatment groups and positive controls after values obtained from negative controls had been subtracted from each data point. Two-way analysis of variance was determined, together with use of the Scheffe t test and the Protected Least Significant Difference test. Statistical significance was defined as p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rat bone marrow cells were biotinylated and subjected to
immunoprecipitation techniques described above. The results, appearing
as Western blots and using mAb (205c), are shown in Figure 1
. In each case, bands consistent with
the m.w. of CD18 appeared in the region between the marker positions of
85 and 115 kDa. Bands representing the higher m.w. -chain proteins
were present, in the region between 130 and 180 kDa. In the case of
d, a band in the position of CD18 was found, together with a higher
m.w. protein of 145 kDa. The reference murine IgG demonstrated the
expected heavy and light chains.
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d mRNA and d protein in inflamed rat lung
Lungs from animals undergoing intrapulmonary deposition of IgG
immune complexes were extracted for RNA and evaluated by Northern blot
analysis for d mRNA and by Western blot for d protein. The
results of these studies are shown in Figures 2 and 3.
Faint, constitutive expression of mRNA could be detected in lung at
time 0, but increases in mRNA were found between 0.5 and 2.0 h.
Thereafter, a progressive reduction in d mRNA was found. In the
upper part of Figure 2, equal loading of RNA was confirmed
by the use on oligonucleotide that detects 18S ribosomal RNA. As shown
in Figure 3, using Western blot analyses, homogenates from immune
complex-injured lungs at 0, 1, 2, 3, and 4 h revealed slight
constitutive d (at 0 h) and clear and increasing amounts of
d, peaking at 3 h, followed by a reduction at 4 h. These
findings indicate that IgG immune complex-induced lung inflammation in
rats causes up-regulation of lung mRNA and protein for d in a
time-dependent manner, but that the up-regulation is not sustained.
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d
Frozen sections of rat lungs were obtained at 0, 1, 2, and 4
h after intrapulmonary deposition of IgG immune complexes and stained
for d. As shown in Figure 4
(A–D), little reaction product (appearing as a
brown stain) was found in lungs at 0 h (A). By 1 h
(B), more reaction product was found. It was markedly
accentuated at 2 h (C) and appeared in interstitial
areas and along alveolar surfaces. By 4 h (D), there
was a dramatic reduction. Alveolar macrophages were retrieved by BAL
procedures at the same time points and stained for d. Very little
reaction product was found at 0 time (E). At 1 and 2 h,
positive staining of alveolar macrophages could be seen (F
and G, respectively). By 4 h (H) very little
reaction product was found, consistent with the immunostaining of lung
sections (A–D) and the Western blot (Fig. 3
).
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d on lung injury and lung content of
neutrophils and TNF-
Rats undergoing IgG immune complex-induced lung inflammation were
evaluated for the ability to block rabbit polyclonal Ab to rat d to
affect the outcome of lung inflammatory reactions. In data shown in
Figure 5A, the positive
control groups received, together with the intratracheally administered
anti-BSA, either 300 µg preimmune rabbit IgG or 300 µg rabbit
IgG anti-d. The effect on pulmonary vascular permeability (as
measured by albumin leak into lung) was then determined 4 h after
initiation of the reactions. The differences in the permeability
indices in the negative and positive (treated with preimmune IgG)
controls were approximately fivefold. In the presence of polyclonal
anti-d, there was a 55% reduction (p <
0.001) in the permeability index when compared with the positive
control group treated with 300 µg preimmune IgG. In a companion set
of animals, neutrophils retrieved by BAL were quantitated as a function
of treatment (Fig. 5B). When compared with negative controls
(receiving anti-BSA only with omission of the i.v. administration
of Ag), positive controls that received 300 µg preimmune IgG
intratracheally together with anti-BSA showed a sixfold increase in
numbers of BAL neutrophils, rising from 1.28 ± 0.14 x
106 in the negative controls to 7.04 ± 1.09 x
106 in the positive controls (p <
0.001). In rats receiving an intratracheal instillation of 300 µg IgG
anti-d together with the anti-BSA, the yield of neutrophils
fell by 44% (p = 0.03), to 3.92 ±
0.672 x 106.
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, a cytokine
that is required for up-regulation of lung vascular ICAM-1 and
E-selectin adhesion molecules, which are essential for recruitment of
neutrophils (24, 29, 30). Under the same experimental conditions
described above, negative controls had BAL TNF- levels of 316
± 51 pg/ml, while positive controls receiving 300 µg preimmune
rabbit IgG together with the anti-BSA had 973 ± 33 pg
TNF-/ml (Fig. 5C). In contrast, the presence of
anti-d in the anti-BSA preparation dramatically reduced
(86%, p < 0.001) the rise in TNF- levels (to
409 ± 56 pg TNF-/ml). These data indicate that in the
experimental lung inflammatory model employed d plays an important
role in neutrophil recruitment and development of lung damage, in part
by facilitating lung expression of TNF-.
A companion series of studies was undertaken to assess the effects of
hamster mAb to rat d. The experimental protocol was similar to that
described in Figure 5A. Negative controls received
anti-BSA intratracheally while positive controls received
anti-BSA together with 200 µg hamster irrelevant IgG mAb to
trinitrophenol. The other group of positive control animals received
200 µg hamster mAb (205C) to rat d, which was instilled
intratracheally together with the anti-BSA. Limitations in amounts
of available mAb precluded experiments with higher doses. The results
are shown in Figure 6. In this
experiment, the permeability rise in the positive control group, as
compared with the negative control group, was approximately fourfold
(rising from a value of 0.17 ± 0.01 to a value of 0.64 ±
0.007). In the presence of mAb to rat d, the permeability value fell
by 28% (p < 0.05), to a value of 0.51 ±
0.08. Thus, blockade of rat d by either polyclonal or monoclonal
antibody is protective in this model of lung injury, as reflected by
albumin leak.
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d on BAL fluid levels of
NO2-/NO3-
In rats undergoing IgG immune complex deposition, BAL fluids were
obtained at 4 h and evaluated for
NO2-/NO3- content.
The groups included negative controls, which received anti-BSA
intratracheally in the absence of BSA, and positive controls, which
received 300 µg preimmune rabbit IgG or 300 µg of rabbit
anti-rat d IgG intratracheally together with the anti-BSA.
The results are shown in Figure 7. BAL
fluids from negative controls contained low levels of
NO2-/NO3- (1.63
± 1.49 nmol/ml), rising nearly threefold, to 3.29 ± 0.75 nmol/ml
in positive controls treated with preimmune rabbit IgG. In the presence
of anti-rat d, the increased production of
NO2-/NO3- was
significantly suppressed (p < 0.05) in BAL
fluids (falling to 1.71 ± 0.74 nmol/ml), indicating that d is
required for full lung production of · NO, an
intermediate known to be involved in development of lung injury in this
model (31).
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Experiments were done to determine if the Ab to d would affect
NO2-/NO3- production
in rat alveolar macrophages that had been stimulated with LPS (10
µg/ml) and murine IFN- (25 U/ml) at 37°C for 18 h. These
experiments were designed after preliminary observations suggesting
that d on rat macrophages was expressed after incubation with LPS
and IFN- (R. L. Warner and P. A. Ward, unpublished
observations). Supernatant fluids were collected at the end of the
incubation period and analyzed for
NO2-/NO3-. In
parallel sets of wells, increasing amounts (from 0 to 50 µg/ml) of
rabbit polyclonal IgG Ab to rat d were added at time 0. Other
controls included normal rabbit IgG at the concentration of 50 µg/ml.
The results are shown in Figure 8 where
anti-rat d caused a progressive reduction in
NO2-/NO3- formation
as a function of the Ig concentration. A plateau appeared to be reached
when the concentration of Ab was 10 µg/ml. The maximal amount of
inhibition of
NO2-/NO3- production
induced by anti-d was 63%. Irrelevant rabbit IgG had no
statistically significant effect on generation of
NO2-/NO3-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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d by normal cells and normal tissues appears to
be highly restricted. In normal tissues, d is expressed primarily by
macrophages in the splenic red pulp, cells in the bone marrow and in
medullary regions of lymph nodes, while in the blood d expression is
limited to a small subset of CD8+ cells (32). One of the
counter-receptors identified for human d is human ICAM-3, a member
of the Ig superfamily. Although intensive efforts have been made,
neither mouse nor rat ICAM-3 has been cloned (W. M. Gallatin,
personal communication). Although it seems likely that rodents contain
an ICAM-3 homologue (in view of the importance of ICAM-3 in activation
of human T cells) (7), the situation could be analogous to the absence
of IL-8 in rats or mice. Alternatively, it is possible that there
exists in rats (and mice) another counter-receptor for d. There are
suggestions that human d is reactive with VCAM-1 (W. M.
Gallatin and M. Van der Vieren, personal communication). Whether rat
VCAM-1 may function as a counter-receptor for rat d remains to be
determined. It seems likely that d/CD18, like other ß2
integrins, interacts with ICAM molecules, serves as an activation
marker, and participates in signal transduction events in stimulated
lymphocytes, monocytes, and macrophages. It has previously been shown
that full expression of lung injury in the IgG immune complex model in
rats requires both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18)
(33).
We have recently suggested that alveolar macrophages may be in adhesive
contact with ICAM-1 in surfaces of alveolar type II cells, the result
of which permits macrophages to optimally produce TNF- stimulated
with IgG immune complexes and complement activation products (34). This
conclusion is also based on the finding that intratracheally
administered blocking Ab to rat ICAM-1 suppresses lung inflammatory
injury in the same inflammatory model as described in this paper. A key
role for C5a has recently been shown in the IgG immune complex model,
since blockade of C5a by polyclonal Ab to rat C5a suppresses
inflammation and, surprisingly, substantially reduces up-regulation of
lung vascular ICAM-1. This outcome has been found to correlate with
greatly reduced levels of BAL fluid levels of TNF- (35).
The current studies extend knowledge regarding the roles of adhesion
molecules involved in neutrophil recruitment in the IgG immune
complex-induced injury model. As indicated above, both LFA-1 and Mac-1
are involved in this model of injury, with LFA-1 being tied to
neutrophil recruitment events in the vascular compartment and Mac-1
being involved in events (TNF- production) taking place in the
distal airway compartment of the lung (33, 34). LFA-1 appears to be
directly involved in neutrophil adhesion to the vascular endothelium,
probably through neutrophil LFA-1 interaction with up-regulated
vascular ICAM-1. A driving force for vascular ICAM-1 up-regulation
appears to be TNF- derived from lung macrophages. The role of Mac-1
may be related to alveolar macrophage Mac-1 interacting with alveolar
epithelial cell ICAM-1, resulting in maximal protection by adherent
macrophages of TNF-. Airway blockade of either Mac-1 (but not LFA-1)
or ICAM-1 reduces TNF- production, neutrophil recruitment, and lung
damage (34). We have also been shown that mAb to rat VLA-4 is
protective in the same model of lung injury, interfering with full
recruitment of neutrophils (33). Since rat neutrophils contain little,
if any, detectable VLA-4 (R. S. Warner and P. A. Ward,
unpublished observations), it is possible that lung macrophage VLA-4 is
reactive with macrophage VCAM-1 and that this adhesion-promoting
process facilitates optimal production of cytokines by macrophages. The
presence of VCAM-1 in macrophages has been reported (36, 37). As
described above, it is also possible (but not demonstrated) that rat
d is reactive with rat VCAM-1. The role of rat d in the IgG
immune complex model of lung injury suggests that, like ICAM-1, it is
required for full production of TNF- by lung macrophages, leading to
up-regulation of lung vascular ICAM-1 and neutrophil recruitment. The
fact that airway instillation of anti-rat d reduced BAL TNF-
levels by 86% (Fig. 5) is consistent with this possibility. What is
unclear is the counter-receptor in rat lung for rat d.
The data in this report indicate that the full recruitment of
neutrophils and expression of lung injury after deposition of IgG
immune complexes requires d which, like CD11b, plays an important
role in the full expression of macrophage-generated TNF-, a critical
molecule involved in vivo in the up-regulation of endothelial ICAM-1.
Additional in vivo roles of d remain to be determined. Thus, in the
IgG immune complex-induced model of lung injury, lung macrophage d,
like CD11b/CD18, facilitates the full production of TNF-, which
induces vascular ICAM-1 and E-selectin up-regulation and subsequent
neutrophil recruitment. It also appears that macrophage d subserves
another proinflammatory function, namely, facilitating production of
· NO, as indicated by measurements of
NO2-/NO3-. As
suggested above, both TNF- and · NO play important
lung-damaging functions, the former by facilitating neutrophil
recruitment and the latter (or its derivatives) by causing direct
damage to lung cells and extracellular matrix.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter A. Ward, Department of Pathology, The University of Michigan Medical School, M5240 Medical Science I, Box 0602, 1301 Catherine Road, Ann Arbor, Michigan 48109-0602, E-Mail:
3 Abbreviations used in this paper: ICAM, intercellular adhesion molecule; BAL, bronchoalveolar lavage; CHO, Chinese hamster ovary; NO2-/NO3-, nitrite/nitrate; NFDM, nonfat dry milk; rd/huIgG, recombinant rat d "I" domain/human IgG; PBS-T, phosphate-buffered saline with 0.05% Tween-20; VCAM, vascular cell adhesion molecule.
Received for publication April 29, 1997. Accepted for publication October 7, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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dß2, binds preferentially to ICAM-3. Immunity 3:683.[Medline]
(MIP-1) in acute lung injury in rats. J. Immunol. 154:4793.[Abstract]
dß2, is expressed by specific macrophage subpopulations in tissue and a minor CD8+ lymphocyte subpopulation in peripheral blood. J. Immunol. 155:35.[Abstract]
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