| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Curriculum in Genetics and Molecular Biology and
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599; and
Department of Respiratory, Allergy, Immunology, Inflammation and Infectious Diseases, Central Research Division, Pfizer, Groton, CT 06340
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The contribution of LT to a specific inflammatory response depends both on the ability of cells present in the inflammatory lesion to produce a particular LT and the response of the tissue to these bioactive lipids. 5-LO is expressed predominantly by cells of myeloid origin, particularly neutrophils, eosinophils, monocytes/macrophages, and mast cells (3, 4). The enzyme has also been detected at low levels in B cell lines (5, 6) and in extrahemopoietic cells types, including keratinocytes (7) and colonic epithelia (8). Similarly, the GST LTC4 synthase is expressed in a limited number of cell types, predominantly eosinophils, monocytes/macrophages, mast cells, and some leukemic cell lines (9).
In contrast to 5-LO and LTC4 synthase, LTA4 hydrolase expression is ubiquitous with high levels of enzyme seen in several tissues. In particular, abundant message is detected in small intestine epithelial cells, lung, and aorta (10). Moderate expression is observed also in leukocytes, particularly neutrophils (11).
Contribution of the AA metabolites produced by the enzymes of the 5-LO pathway to inflammatory pathophysiology is supported by the identification of high levels of LT in both acute and chronic inflammatory lesions and by the demonstration that introduction of LT into normal tissue can elicit a number of the primary signs of inflammation. Such experiments also support a differential function for LTB4 and cysteinyl LT during the inflammatory response. LTB4 stimulates adhesion of leukocytes to vascular endothelia, thus facilitating the migration of these cells into adjacent tissue. Furthermore, it has been shown to initiate PMN degranulation (12). The cysteinyl LT, predominantly secreted by eosinophils, mast cells, and macrophages, cause vasodilation, increase postcapillary venule permeability, and potently stimulate bronchoconstriction and mucous secretion (13).
Interestingly, LTC4 production by endothelial cells (14), vascular smooth muscle, and platelets has been demonstrated (9). Because these cells do not express 5-LO, this finding suggests that exogenous LTA4 can serve as substrate for LTC4 synthase via intercellular transfer.
As with LTC4, LTB4 production has been verified in cells that are unable to synthesize their own LTA4, and transfer of LTA4 into these cells has been described (15, 16). The differential expression of 5-LO, LTC4 synthase, and LTA4 hydrolase, and particularly the presence of LTA4 hydrolase in tissues unable to synthesize LTA4, suggests an important regulatory role for intercellular transfer of LTA4 in LTB4 synthesis. It is possible that this process is important for the amplification, regulation, and/or progression of the inflammatory response.
An alternate explanation for the high levels of LTA4 hydrolase present in tissues that do not express 5-LO became apparent with the isolation of the cDNAs encoding this enzyme (17, 18). Examination of the primary sequence of LTA4 hydrolase identified a region homologous to the active sites of aminopeptidases, enzymes critical for protein maturation, digestion, and regulatory turnover (19). It is possible, therefore, that LTA4 hydrolase is a bifunctional enzyme, because it possesses an in vitro aminopeptidase activity in addition to its epoxide hydrolysis of LTA4 (20, 21). No target for the aminopeptidase function of LTA4 hydrolase has been identified in vivo, although there is some evidence that such a target may be similar or identical to the intercellular target of the lethal factor of Bacillus anthracis (which is also unidentified) (22). A search for naturally occurring peptides that can be cleaved by the peptidase activity of LTA4 hydrolase led to the demonstration that the opioid dynorphin fragment 1–7 is a good substrate for the enzyme (23), suggesting a role for LTA4 hydrolase in neuropeptide homeostasis.
A direct method for determining the dependence of a particular inflammatory response on LT production was made possible by the generation of mouse lines deficient in 5-LO and FLAP (24, 25). Analysis of these mice has shown that LT production is critical for edema formation and neutrophil migration in a number of acute models of inflammation. For example, edema formation and neutrophil migration were largely abolished in topical AA-induced inflammation of the skin. Similarly, the time course of edema formation and neutrophil migration is altered in 5-LO- and FLAP-deficient animals in a model of zymosan A-induced peritonitis.
We describe in this work the generation of mice deficient in the enzyme LTA4 hydrolase and compare the inflammatory responses of these animals with those of wild-type controls and mice deficient in 5-LO. This comparison provides a means of directly assessing the contribution of LTB4 and the cysteinyl LT to inflammatory processes in vivo. Furthermore, any phenotypic differences between LTA4 hydrolase-deficient, 5-LO-deficient, and wild-type mice could provide insight into an in vivo aminopeptidase activity for LTA4 hydrolase.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An 882-bp murine LtA4
hydrolase cDNA fragment was generated by RT-PCR (primer 1,
5'-ACGAATTCAACCAGAGGGTTCC-3'; primer 2,
5'-AAGAATTCCTCTTTGGCAGTAACCC-3') and then used as a probe to screen a
genomic DNA library in the Lambda FIX II vector generated from the 129
mouse strain (Stratagene, La Jolla, CA). Six genomic clones were
isolated and purified to homogeneity, and one of these was subcloned
into the NotI site of pBluescript II SK+ (Stratagene) for
mapping analysis. The mapped LtA4
hydrolase clone, 
5111, was found to possess a coding
region flanked on either side by several kb of noncoding DNA and was
useful, therefore, in the creation of a targeting construct at this
locus.
Generation of LTA4 hydrolase-deficient mice
The murine genomic clone 5111 was used to construct a
targeting vector for the LtA4
hydrolase gene. Two genomic fragments, 4.25 and 3.99 kb,
respectively, were inserted 5' and 3' of the neo gene of
pJNS2 (26) such that coding material would be replaced by
neo after homologous recombination of the plasmid with the
endogenous locus. Sequence analysis of the fragment replaced through
this targeting strategy showed that successful targeting would
interrupt an exon at the BglII site at position 738 of the
LtA4 hydrolase cDNA and would
eliminate the remainder of the exon (bases 739–853). This construct
was electroporated into E14TG2a embryonic stem (ES) cells established
from the 129/Ola mouse line (27), and colonies resistant
to neomycin and ganciclovir were identified, as described previously
(26). Southern blot analysis was performed on
EcoRV-digested genomic DNA to identify correctly targeted
resistant ES cell lines. An 871-bp EcoRI/EcoRV
fragment downstream of the regions of homology was used to screen for
recombinants (Fig. 1
A). Verified recombinant ES cell lines
were microinjected into mouse blastocysts, and chimeric mice were
generated. Chimeras were bred to 129/SvEv mice, and offspring were
genotyped by Southern blot analysis of tail DNA digested with
EcoRV, as described above. Heterozygotes in the
F1 generation were then mated to give rise to
animals homozygous for the disrupted allele in the
F2 generation.
|
Animals of the indicated genotypes were euthanized, and the femurs of each mouse were exposed in a sterile field. Cuts were made through the knee and hip joints to isolate the bone and to expose the marrow. A total of 3 ml serum-free media was flushed through each femur into a petri dish to isolate the marrow. Cells from one leg were centrifuged and used for RNA isolation, as indicated below. Marrow cells collected from the other leg of each animal were processed to isolate bone marrow-derived mast cells (BMMC), according to Rottem et al. (28). Total RNA was extracted from bone marrow cells and BMMC by resuspension of cell pellets in RNAzol B (Tel-Test, Friendswood, TX), according to the protocol supplied by the manufacturer, and was separated by gel electrophoresis (15–20 µg/lane). Northern blot analysis was conducted according to standard techniques using the 882-bp murine LtA4 hydrolase cDNA fragment described above as a probe to detect expression of LtA4 hydrolase mRNA. A 429-bp probe upstream of the disrupted exon was also hybridized with these Northern blots to insure that no spliced or partial message was being produced in the LtA4 hydrolase-/- tissue. Finally, a probe for murine ß-actin was used as a positive control (Stratagene).
Inflammatory responses induced by AA
129 strain LTA4 hydrolase-deficient mice were injected i.v. with 0.5% Evans blue dye (10 ml of dye solution/kg of body weight) dissolved in PBS (2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4, pH 7.5) and filtered through a 0.2-µm nitrocellulose membrane. The pinna of the left ear was coated with 20 µl of AA (100 mg/ml) in acetone (Sigma, St. Louis, MO) to induce an inflammatory response, while the right ear received acetone alone on its inner surface. After 1 h for edema assays or 7 h for myeloperoxidase (MPO) analysis, mice were euthanized, and an 8-mm-diameter disc of tissue was obtained from the center of each ear for analysis. For comparison, experiments were conducted in parallel with age- and gender-matched 129 wild-type, 5-lo-/-, and Flap-/- mice.
Inflammatory responses induced by PMA
Mice of the indicated genotypes were injected i.v. with 0.5% Evans blue dye in identical fashion to the AA experiments above. The left ear was then coated with 1 µg of the diacylglycerol analogue PMA in acetone, while the right ear received acetone alone as a control. After 6 h, 8-mm-diameter discs of ear tissue were biopsied, and edema and plasma protein extravasation were assessed.
Quantification of edema and vascular permeability
The wet weight of each ear biopsy was determined to assess edema formation. Each ear disc was then incubated in 1 ml formamide at 55°C for 24 h. Extravasation of Evans blue dye was quantified by spectrophotometric analysis of the formamide extracts at 610 nm (29). Data are expressed as mean differences between experimental and control ears for each animal.
MPO assay
Ear punches (8 mm diameter) were homogenized and analyzed, according to a previously described protocol (24) modified from Bradley et al. (30). MPO present in the lysed cell supernatants was quantified by comparison with a standard curve derived from serial dilution of commercial MPO (Calbiochem, San Diego, CA).
Zymosan A-induced peritoneal inflammation
Peritoneal inflammation was induced in LtA4 hydrolase-/-, Flap-/-, 5-lo-/-, and wild-type animals using a protocol modified from Rao et al. (31). Each mouse received 1 ml zymosan A suspension (1 mg/ml in PBS, pH 7.5) by i.p. injection. After 3 h, mice were killed, and a peritoneal lavage was performed with 4 ml of ice-cold PBS. After lavage fluid was collected, 1 ml was transferred to microcentrifuge tubes and centrifuged at 40,000 x g for 6 min. Supernatants were collected for direct enzyme immunoassay (EIA) analysis of LTC4 and PGE2 levels (Cayman Chemical, Ann Arbor, MI). Aliquots of the collected supernatants were also purified by HPLC (see below), and LTB4 levels were measured in the eluted fractions by EIA (Cayman Chemical). Cell pellets, meanwhile, were resuspended in 1 ml PBS/0.5% hexadecyltrimethylammonium bromide (Sigma), and the cells were lysed by sequential freeze-thaw cycles. Cellular debris was removed by centrifugation, and the presence of neutrophils in the cell pellets was determined by detection of MPO in the lysis buffer using an enzymatic assay, as described above. MPO analysis of cell pellets was also performed in this way at time points of 0, 1, 6, and 12 h following zymosan A treatment.
Total cell counts were made for lavage fluid collected at the 3-h time point using a hemocytometer. Approximately 2.5 x 105 cells were then mounted on glass slides by cytospin centrifugation and were stained with Diff-Quik (Dade Diagnostics, Aguada, PR). The percentage of PMN present in the lavage fluid was determined by microscopy.
In related experiments, plasma protein extravasation into the peritoneal cavity was examined. Mice received 0.5% Evans blue dye (10 ml of dye solution/kg of body weight) in PBS i.v. immediately before injection of zymosan A suspension. Mice were euthanized at time points of 10, 20, 30, 60, 180, and 360 min and underwent peritoneal lavage with 4 ml of cold PBS, as described above. Cells were centrifuged out of the lavage fluid, and Evans blue dye extravasation was assessed by light spectrophotometry of supernatants at 610 nm.
HPLC purification of zymosan A-stimulated lavage fluid
Peritoneal lavage fluid was collected from zymosan A-treated animals after 3 h, as detailed above. Lavage fluid (0.5 ml) was spiked with 100 µg indomethacin in 10 µl methanol as an internal standard with or without LTB4 standard (25 ng; Biomol, Plymouth Meeting, PA), and protein was precipitated by addition of 1 ml acetonitrile, followed by centrifugation at 8000 x g for 10 min. The supernatant was acidified and diluted in water/acetic acid (100/0.1, v/v) containing 1 mM disodium EDTA and passed through a 100 mg BondElut C18 solid phase extraction cartridge (Varian, Harbor City, CA). The column was washed twice with methanol/water (80:20, v/v) and then eluted with 0.5 ml methanol/triethylamine (100:0.1, v/v). The entire eluate was diluted with 4.1 ml water/acetic acid (100:0.1, v/v) containing 1 mM disodium EDTA, and 5 ml was injected into the HPLC column (a Perkin-Elmer (Norwalk, CT) CR-C18 cartridge column, 3 x 0.46 cm, 3 µm). The column was equilibrated with methanol/water/acetic acid (20:80:0.1, v/v/v). After sample injection, a mobile phase gradient was established from 100% methanol/water/triethylamine (20:80:0.1, v/v/v) to 50% methanol/water/triethylamine (20:80:0.1, v/v/v) and 50% methanol over 20 min at a flow rate of 1.5 ml/min. The column eluant was monitored by two Perkin-Elmer LC-95 UV detectors in series at 280 and 235 nm. Eluate fractions were collected every 0.2 min, dried under a vacuum, and redissolved in 250 µl EIA buffer for EIA detection of LTB4 (Cayman Chemical).
Contact hypersensitivity to topical FITC
A method adapted from that of Furue and Tamaki (32) was used to assess the ability of LTA4 hydrolase-deficient mice to mount a contact hypersensitivity response. Briefly, LtA4 hydrolase-/- and wild-type mice of the 129 strain were sensitized by application of a 100 µl topical dose of either 0.5% or 2.5% FITC (Sigma) in 1:1 acetone:dibutyl phthalate (w/v) to the shaved abdomen. After 6 days, a hypersensitivity reaction was elicited by topical application of 20 µl of a corresponding concentration of FITC to the left ear. Nonsensitized animals were also treated with 0.5% and 2.5% FITC as a control. The right ear of each mouse received vehicle alone. Twenty-four hours later, mice were euthanized, and ears were collected and weighed, as described above. The differences in wet weight between experimental (left) and control (right) ears were compared for mice of each genotype.
Passive systemic IgE-induced anaphylaxis
Passive systemic anaphylaxis was induced as previously described (26). LTA4 hydrolase-deficient animals and wild-type controls received 20 µg of a monoclonal mouse anti-mouse DNP IgE in 200 µl of PBS i.v. After 24 h, baseline body temperatures were determined using a rectal probe, and mice received an i.v. injection of 1 mg DNP-human serum albumin (HSA) and 1% Evans blue (w/v) in 200 µl PBS to induce anaphylaxis. Thirty minutes following this injection, body temperature was measured again. Mice were then euthanized, and Evans blue dye extravasation was quantitated in 8-mm-diameter ear punches, as described above.
PAF-induced systemic shock
LTA4 hydrolase-deficient and wild-type control mice received i.v. injections of PAF (50 µg/kg of body weight) in PBS containing BSA (at 2.5 mg/ml) and propranolol (at 0.05 mg/ml). Survival was assessed for 24 h following the injection of PAF.
Endotoxic shock
Survival after endotoxin treatment was determined using a protocol adapted from Ogata et al. (33). LTA4 hydrolase-deficient, 5-LO-deficient, FLAP-deficient, and wild-type mice received an i.p. dose of 5 mg iota (type V) carrageenan (Sigma) in 500 µl PBS. After 24 h, mice were injected i.v. with 30 µg LPS (from Escherichia coli serotype 0111:B4; Sigma) in 300 µl PBS and were monitored for survival for the subsequent 72 h.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fragments of DNA corresponding to the mouse
LtA4 hydrolase gene were isolated
from a genomic library. These fragments were used to prepare a plasmid
that was expected to disrupt the coding sequence of this gene upon
homologous recombination with the endogenous locus (Fig. 1
A). The region of the gene
replaced by the neomycin-resistance cassette (neo) upon
homologous recombination (see Fig. 1A) was subcloned and
sequenced. This analysis indicated that successful targeting would
result in the integration of neo into the BglII
site present at bp 738 of the mouse LtA4
hydrolase cDNA. In addition, the recombination event would
remove 114 bp of coding sequence immediately 3' of this restriction
site (bases 739–853 of the mRNA).
The LTA4 hydrolase-targeting plasmid was
electroporated into E14TG2a ES cells (27), and transfected
clones were selected by growth in the presence of G418 and ganciclovir.
Resistant colonies were picked, expanded, and screened for the presence
of homologous recombination events by Southern blot analysis of
EcoRV-digested genomic DNA using a probe corresponding to
DNA present 3' of the DNA used in the preparation of the targeting
construct (Fig. 1A). Because the neo cassette
introduces a novel EcoRV site into the recombinant locus,
this probe identifies an 8.7-kb DNA fragment from the endogenous
LtA4 hydrolase locus and a 5.1-kb DNA
fragment from the targeted locus (Fig. 1B). Two targeted ES
cell lines were identified and microinjected into mouse blastocysts to
produce chimeric mice capable of transmitting the targeted allele to
their offspring. Intercrossing of these heterozygous animals produced
LtA4 hydrolase-/-
mice in expected numbers based on Mendelian inheritance of the targeted
locus. Thus, loss of LTA4 hydrolase expression
had no effect on the fetal development or perinatal survival of the
animals. The LTA4 hydrolase-deficient animals could not be
distinguished from LtA4
hydrolase+/- or wild-type littermates by
simple observation or on histological analysis of major organ
systems.
Lymphocytic development in the LTA4-deficient mice was examined using two-color FACS analyses of cells isolated from spleen, thymus, and lymph nodes with Abs to murine CD4 and CD8. Similar numbers of CD4+, CD8+, double-positive (CD4+8+), and double-negative (CD4-8-) thymocytes and thymocyte precursors were counted in cells collected from LtA4 hydrolase-/- and control animals. FACS analysis of these cells using B220 and Thy-1.2 Abs also revealed no difference in the ratios of cells bearing these B and T cell markers in the two populations of mice (data not shown). Differential counts of hemopoietic cells in blood smears also failed to identify any differences between the wild-type and LTA4 hydrolase-deficient mice.
The mRNA encoding LTA4 hydrolase is abundant in
leukocytes (11). Therefore, to determine whether the
homologous recombination event had resulted in a null
LtA4 hydrolase allele, total RNA was
isolated from bone marrow cells and BMMC obtained from
LtA4 hydrolase-/-
and wild-type mice, and expression of LtA4
hydrolase was examined. Northern blot analysis using the
882-bp LtA4 hydrolase cDNA fragment
used to isolate the initial LtA4
hydrolase genomic clones (Fig. 1C) indicated high
levels of LtA4 hydrolase mRNA in RNA
prepared from wild-type animals. In contrast, expression of this gene
could not be detected in RNA samples prepared from bone marrow cells
and BMMC of LtA4
hydrolase-/- mice. This finding was
confirmed using a cDNA probe corresponding to coding regions located
entirely outside of the region of the gene altered by the homologous
recombination event. Again, no LtA4 hydrolase
message was detected in the LTA4
hydrolase-deficient animals (data not shown).
LTB4 production in LTA4 hydrolase-deficient animals
An experimental peritonitis was elicited in
LtA4 hydrolase-/-
and wild-type mice by injection of zymosan A into the peritoneal
cavities of these animals. HPLC-purified fractions of peritoneal lavage
fluids that coeluted with known LTB4 standard
were analyzed for the presence of LTB4 by EIA.
While LTB4 was easily detected in the lavage
fluid of wild-type animals, LTB4 could not be
detected in samples obtained from the LtA4
hydrolase-/- animals (Fig. 2A). In contrast, levels of
LTC4 in the peritoneal fluid from the
LtA4 hydrolase-/-
animals were equal, if not higher, than those measured in samples from
wild-type animals (Fig. 2B). LTA4
hydrolase-deficient mice produced 
3.75 times more
LTC4 in this assay (3015 ± 916.3 pg/ml)
than wild-type mice (801.2 ± 290.8 pg/ml); however, this increase
did not reach statistical significance (p =
0.074). The loss of LTA4 hydrolase expression
had no apparent effect on the activity of the cyclooxygenase pathway in
this peritoneal inflammatory response, as wild-type and
LTA4 hydrolase-deficient mice produced similar
levels of PGE2 in this assay (Fig. 2C).
|
Topical application of AA onto the inner surface of the mouse ear causes an intense acute inflammatory reaction with both vascular and cellular components. This inflammatory response results from the conversion of the applied AA into eicosanoids, primarily metabolites of the 5-LO and cyclooxygenase pathways, with proinflammatory properties. The contribution of LT to both the cellular and vascular components of this response has been demonstrated using animals deficient in expression of 5-LO and FLAP (24, 25, 34). Because 5-LO-deficient animals are unable to synthesize both LTB4 and the cysteinyl LT, comparison of the responses in these mice with those observed in the LTA4 hydrolase-deficient mice should allow determination of quantitative and qualitative differences in the contribution of these two groups of LT to the inflammatory response. Therefore, we treated LtA4 hydrolase-/-, 5-LO-deficient, and control animals with 2 mg of AA, biopsied ear tissue, and examined the impact of loss of LTB4 and cysteinyl LT on both the vascular and cellular components of the inflammatory response. Edema formation was assessed by comparison of the weight of ear biopsies from the ear treated with AA with the vehicle-treated control ear of each animal. To measure alterations in plasma exudation, mice received an i.v. injection of Evans blue dye before treatment with AA. Because this dye binds to serum proteins, quantification of the dye present in ear biopsies provides a reliable means of measuring changes in vascular permeability (29). The cellular component of this inflammatory response was quantitated by measuring the levels of MPO, an enzyme specific to cells of the myeloid lineage, including neutrophils, present in the ear biopsies (35).
The increase in ear weight following topical AA treatment was
attenuated in LTA4 hydrolase-deficient mice
compared with age- and gender-matched wild-type controls (Fig. 3A). Although the response of
the LTA4 hydrolase-deficient mice was
significantly lower than that observed in wild-type animals
(p = 1.5 x 10-5),
the response remained more vigorous than that seen in age- and
gender-matched 5-LO-deficient mice (p =
0.00045). Changes in vascular permeability and plasma protein
extravasation in response to AA stimulus paralleled these findings,
with LTA4 hydrolase-deficient animals displaying
responses intermediate between those of the wild-type and
5-LO-deficient animals. The decrease in protein extravasation into the
AA-treated tissue observed in the LTA4
hydrolase-deficient mice compared with wild-type animals was highly
significant (p = 0.009). Protein leakage was
significantly greater, however, than that measured in the
5-LO-deficient animals (p = 0.0046) (Fig. 3B). These results indicate that edema formation and plasma
protein extravasation in response to AA are dependent in part, either
directly or indirectly, on the synthesis of LTB4
by LTA4 hydrolase.
|
C). We
have reported previously that this response is significantly reduced in
the 5-LO-deficient animals. In contrast to the intermediate vascular
response seen on treatment of the LTB4-deficient
mice with AA, neutrophil infiltrate in LtA4
hydrolase-/- ear tissue was decreased to
the same low level observed in the
5-lo-/- mice. No significant difference
was seen between these two groups of animals.
The loss of 5-LO or LTA4 hydrolase did not alter
either the vascular or cellular components of the inflammatory response
to topical application of PMA (Fig. 3, A–C). Exposure to
PMA results in general stimulation of cellular activity mediated by
protein kinase C (36) including induction of cytokine
production and cyclooxygenase expression in endothelial cells
(37). PMA also activates T cells (38),
neutrophils, platelets, basophils, and mast cells (39). In
the experiments described in this work, the inflammatory properties of
PMA are not diminished by the absence of LT.
LtA4 hydrolase-/- mice show blunted PMN recruitment, but normal plasma protein influx in an experimental model of peritonitis
To further examine the roles of LTB4 and the
cysteinyl LT in the inflammatory process, we measured plasma protein
extravasation and PMN infiltration in response to i.p. injection of
zymosan A. To assess extravasation of plasma proteins into the
peritoneal cavity, mice were injected with Evans blue dye before
treatment with zymosan A. Peritoneal lavage was performed 0, 10, 20,
30, 60, 120, 180, and 360 min after treatment. As shown in Fig. 4A, plasma protein leakage in
response to this irritant was identical in LTA4
hydrolase-deficient and wild-type animals. In contrast, and consistent
with previous reports, the rate at which serum proteins accumulated in
the peritoneal cavity after initiation of the inflammatory response was
altered in 5-LO-deficient animals. These data indicate that cysteinyl
LT synthesis alone is sufficient to allow LtA4
hydrolase-/- mice to exhibit a normal
rate of plasma protein influx into the peritoneal cavity in response to
zymosan A.
|
6 h after treatment. Although the level of MPO increased in lavage
fluids collected from the 5-LO-deficient animals, it remained
significantly lower than levels seen in wild-type mice at all time
points examined after injection of zymosan A (Fig. 4B). The
influx of PMN in the LtA4
hydrolase-/- animals was significantly
decreased in comparison with wild-type animals. The level of MPO in the
lysates prepared from lavage fluid from these animals, however,
indicated that PMN influx into the peritoneal cavity of the
LtA4 hydrolase-/-
was greater than that observed in the 5-LO-deficient animals. This
difference between the LtA4
hydrolase-/- and
5-lo-/- mice was verified by direct
enumeration of the numbers of neutrophils present in the lavage fluid
obtained from animals of each genotype 3 h after treatment with
zymosan A. Consistent with the results obtained on examination of MPO
activity, the number of neutrophils present in the
LtA4 hydrolase-/-
mice was significantly less than that seen in the wild-type mice, but
remained higher than that observed in the lavage fluid collected from
the 5-lo-/- animals (data not shown). Dermal contact hypersensitivity to FITC is unaltered in the absence of LTA4 hydrolase
Because high levels of LTA4 hydrolase are
found in mature T cells, we examined the possibility that the
aminopeptidase activity of this enzyme might contribute to T
cell-dependent responses in vivo. Contact hypersensitivity to FITC, a
form of delayed-type hypersensitivity reaction, was induced in
wild-type and LTA4 hydrolase-deficient animals by
topical application of FITC. The T cell-dependent response of the two
groups of animals was quantitated by topical application of FITC to the
ears of sensitized animals 1 wk later and then measuring the change in
wet weight of the ear tissue 24 h after challenge. As shown in
Fig. 5, LTA4
hydrolase-deficient mice are able to mount a normal contact
hypersensitivity response to Ag.
|
PAF is a potent mediator of systemic shock characterized by
microvascular leakage, vasodilation, contraction of smooth muscle,
endothelial adhesion, and activation of neutrophils, macrophages, and
eosinophils. The ability of PAF to stimulate LT production and the
effectiveness of LT inhibitors in diminishing the physiological changes
seen on treatment of animals with PAF suggested that many of the
effects attributed to PAF are dependent on the secondary production of
leukotrienes. Direct evidence supporting this mechanism has come from
the demonstration that mice deficient in FLAP and 5-LO have an
increased resistance to PAF (24, 25, 34). To determine
whether this change in sensitivity to PAF results from loss of
production of LTB4 or the cysteinyl LT,
leukotrienes, 13 LtA4
hydrolase-/-, and 14 control mice
received i.v. injections of PAF and were monitored for survival over
24 h. Survival of LtA4
hydrolase-/- animals was significantly
higher (92.3%) than that observed in the control group, in which 7 of
the 14 mice died. Analysis of these results using the
2 test indicates a difference in
susceptibility to PAF-induced shock between the two groups with a
significance level of 0.016. This suggests that production of
LTB4 contributes to physiological changes leading
to death on exposure to high levels of i.v. PAF.
In contrast to results obtained on treatment of 5-LO- and FLAP-deficient animals with PAF, no difference was reported in the sensitivity of LT-deficient mice to LPS-induced shock (34). To verify this and to determine whether loss of the aminopeptidase activity of LTA4 hydrolase might alter the sensitivity of the animals to LPS, wild-type, LtA4 hydrolase-/-, 5-LO-deficient, and FLAP-deficient mice were treated with carrageenan followed by LPS challenge. After 72 h, it was found that mortality following LPS treatment was identical in mice of all four genotypes (data not shown).
Systemic IgE anaphylaxis in the mouse is characterized by
bronchoconstriction, plasma extravasation, hypotension with resultant
tachycardia, and drop in body temperature. These physiological changes
result from the release of proinflammatory mediators stored in mast
cells after activation of the high affinity IgE receptor as well as the
de novo synthesis of lipid mediators from AA (26). Passive
systemic anaphylaxis was induced in LTA4
hydrolase-deficient and wild-type mice by injection of monoclonal mouse
anti-mouse DNP IgE. Twenty-four hours later, DNP-HSA and Evans blue
were administered. No difference in this response was observed between
normal and 5-LO- and FLAP-deficient mice in previous studies. Again, to
determine whether the aminopeptidase activity of
LTA4 hydrolase contributes to IgE-mediated
inflammation, we compared this response in LTA4
hydrolase-deficient and wild-type mice. Changes in body temperature and
vascular permeability were similar in these groups of animals after
treatment with Ag (Fig. 6).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
One well-characterized inflammatory model in which LT have been
implicated involves topical application of AA to the murine ear. This
treatment initiates an inflammatory response characterized by both a
cellular and a vascular component. The cellular component of this
response consists of leukocyte accumulation at the site of
inflammation. The production of both LTB4
(40) and LTC4 (41, 42)
has been reported in murine ear tissue following topical AA treatment,
each peaking 15 min after stimulation. Histological examination and
enzymatic analysis for MPO (40, 42) have shown that few
neutrophils are present in murine ears 15 min after AA treatment. PMN
begin to accumulate 30–60 min post-AA exposure, with the highest
numbers seen 4–7 h after initiation of the response
(40, 41, 42). It is likely, therefore, that cells resident in
the skin produce the LT measured immediately following induction of
this inflammatory response. Neutrophils attracted to the inflamed ear
by these eicosanoids may then contribute to further accumulation of
cells by production of additional LT as well as other chemotactic
mediators. Direct evidence for a role for LT in the recruitment of PMN
to inflamed tissue in this model has come from the observation that the
level of MPO in the ear tissue of 5-LO-deficient mice is significantly
lower than that seen in wild-type mice. We show in this study that the
reduction in MPO activity in the tissue from LTA4
hydrolase-deficient mice is similar to that seen in the 5-LO-deficient
animals. Thus, while both LTB4 and
LTC4 typically are produced in ear tissue
immediately following AA exposure, only LTB4
appears to contribute to the cellular component of the resulting
inflammatory response.
Measurements of edema and plasma extravasation, which represent the
vascular component of the AA-induced response, indicate the involvement
of both LTB4 and the cysteinyl LT. Formation of
edema in the LTA4 hydrolase-deficient mice,
although reduced in comparison with wild-type animals, did not reach
the low levels measured in 5-lo-/- mice
(Fig. 3, A and B). Similar findings have been
reported in this model after treatment of mice with a high affinity
LTB4 receptor antagonist (43). This
in vivo study supports previous reports that have identified distinct
mechanisms by which the cysteinyl LT and LTB4
contribute to edema formation. Increases in postcapillary venule
permeability in response to the cysteinyl LT have been shown to result
from plasma protein leakage through gaps between contracted endothelial
cells (44). In contrast, these studies suggested that
LTB4 contributes to edema formation by initiating
leukocyte adhesion to endothelium and diapedesis into the interstitial
space (45, 46). Plasma leakage accompanies this process
and is dependent upon leukocyte migration (47). Finally,
marginating leukocytes may themselves produce factors to enhance or
augment edema formation.
A second inflammatory response that is characterized by production of
both LTB4 (31) and
LTC4 (48) is elicited in response to
peritoneal administration of zymosan A. LTC4
production peaks rapidly between 30 and 60 min after zymosan A exposure
and slowly subsides over several hours. LTB4
levels increase in a biphasic manner with an early plateau followed by
an additional increase, which reaches a maximum 2–3-h postzymosan A
treatment (31). Both peritoneal macrophages and
infiltrating neutrophils have been shown to synthesize
LTB4 during phagocytosis of zymosan A particles
(31). The biphasic pattern of LTB4
production is, therefore, believed to result from an initial burst of
synthesis by phagocytic resident macrophages, followed by a further
increase in LTB4 synthesis by PMN recruited to
the site of inflammation 2 h after zymosan injection
(31).
Mice deficient in either FLAP or 5-LO show a decreased rate of edema formation and PMN migration into the peritoneal cavity after treatment with zymosan A. Unlike the observation made in AA-induced ear inflammation, a more pronounced decrease in influx of neutrophils was seen in 5-LO-deficient mice than in animals lacking only in LTB4 synthesis. This suggests that in this inflammatory model, LTC4 synthesis can contribute to the influx of neutrophils. To the contrary, however, i.p. injection of LTC4 alone does not result in PMN recruitment into the peritoneal cavity (49). A possible explanation for this finding in LTA4 hydrolase-deficient mice is that cysteinyl LT exert their effects on neutrophil influx by causing endothelial cell contraction, thereby enhancing neutrophil recruitment by any chemotactic factors in the microenvironment, including those released by resident macrophages and mast cells upon stimulation by zymosan A. Because LTB4 is thought to be the predominant chemotactic factor produced in topical AA-induced inflammation, loss of LTA4 hydrolase reduces influx of neutrophils with the same impact as that seen in the 5-LO-deficient animals. LTC4 would be unable to enhance the migration of PMN in the topical AA model because there are no accessory chemotactic factors beyond LTB4 produced in the microenvironment.
Although loss of 5-LO significantly altered the rate of peritoneal edema after treatment with zymosan A, no reduction in edema was seen in the LTA4 hydrolase-deficient animals. This clearly implicates the cysteinyl LT in the vascular permeability mediated by LT in this response. This differs from the observations made in the topical AA model, in which a decrease in edema formation (albeit smaller than that seen in the 5-LO-deficient mice) was observed in the absence of LTA4 hydrolase.
The differential contribution of LTB4 to edema formation in the two models may reflect the complexity of cells and inflammatory mediators each possesses. The numbers and types of inflammatory cells present in the peritoneal cavity certainly vary from those stimulated by epidermal exposure to AA. Peritoneal macrophages and circulating PMN, as well as other leukocytes, are available to act on zymosan granules and produce a variety of bioactive mediators in our model of peritonitis. Again it is important that the milieu of inflammatory mediators produced following zymosan exposure is not likely to be limited to AA metabolites, while eicosanoids probably predominate in the response to topical AA. As mentioned above, measurable levels of neutrophil influx are seen in the 5-LO- and LTA4 hydrolase-deficient mice after i.p. injection of zymosan A, presumably because of production of multiple chemotactic agents in this inflammatory model. This influx of neutrophils may be sufficient to potentiate the effects of LTC4 on vascular permeability in the absence of LTB4-induced chemotaxis. In the topical AA model, the role of LTB4 in potentiation of edema formation is more apparent, because neutrophil influx in this model is dependent on the synthesis of this bioactive lipid.
It is also important to note that the impact of transcellular LTA4 metabolism on the cellular and vascular components of inflammation could differ according to the site of the response. Available LTA4 carries the potential to be shunted to cysteinyl LT or to lipoxin formation in the absence of the hydrolase. The effects of abundant LTA4 on cysteinyl LT or lipoxin concentration and function in an inflammatory focus will again depend on the cells resident in a particular tissue and their ability to use LTA4. Variable LT or lipoxin synthesis could contribute to the differences we have seen in the vascular and cellular components of inflammation in the two models described above.
The third type of inflammatory response in which LT have been implicated involves PAF-induced systemic shock. Although the mechanism by which exposure to high levels of PAF leads to shock and death is poorly understood, direct evidence for a role for LT in this response comes from the observation that both 5-LO- and FLAP-deficient animals display a decreased sensitivity to PAF (24, 25). The high percentage of the LTA4 hydrolase-deficient animals surviving PAF-induced shock in this study suggests that loss of synthesis of LTB4 is responsible for the increased survival of the 5-LO-deficient animals. Thus, stimulation of LTB4 synthesis by neutrophils and pulmonary sequestration of these activated neutrophils may be important factors in determining survival after exposure to PAF. It is also possible that the absence of LTA4 hydrolase shifts the availability of free LTA4 such that production of lipoxins or other potentially protective substances is up-regulated. Neither the LTB4- nor 5-LO-deficient animals are completely protected from the effects of PAF, suggesting that, while alteration of the LT profile contributes to the physiological changes seen on treatment of animals with PAF, other pathways also influence mortality. In contrast with the clear role LTA4 hydrolase plays on PAF-induced shock, no protection was seen in IgE-mediated passive anaphylaxis or LPS-induced systemic shock in the LTA4 hydrolase-deficient animals compared with wild-type. Similar results have been obtained on examination of 5-LO- and FLAP-deficient animals in these assays.
LTA4 hydrolase is a bifunctional enzyme, possessing an in vitro aminopeptidase activity in addition to its role in AA metabolism (20, 21). The significance of this activity in vivo is not known, although some naturally occurring opioid peptides, particularly the dynorphin fragment 1–7, have been shown to be cleaved by the enzyme (23). Such cleavage can inactivate the peptides, making it impossible for them to bind specific receptors (50). A potential role for the neuropeptides, which can cause mast cell degranulation, vascular permeability, LTC4 and thromboxane B2 release, and neutrophil accumulation, has been suggested in inflammatory diseases of the skin (51). Thus, it has been hypothesized that the loss of LTA4 hydrolase could affect the inflammatory process through alteration of opioid peptide catabolism.
We have seen reductions in neutrophil recruitment, edema formation, and plasma protein extravasation in LTA4 hydrolase-deficient animals stimulated by either topical AA or i.p. zymosan A, which appear to correlate with the failure of these mice to synthesize LTB4. In inflammatory responses in which no role for LT could be established using 5-LO- and FLAP-deficient animals, there were also no differences noted in responses of LTA4 hydrolase-deficient mice. For example, despite the high LTA4 hydrolase expression in T cells, no alteration was seen in the contact hypersensitivity response to the Ag FITC. It is possible that future studies using the hydrolase-deficient mice in models of psoriasis and skin disease will reveal a role for opioid peptide cleavage by LTA4 hydrolase in the initiation and maintenance of an inflammatory response. In addition, the LTA4 hydrolase-deficient mice will continue to be useful in distinguishing between the specific functions of LTB4 and the cysteinyl LT in complex inflammatory reactions and as a model to study intercellular transfer and metabolism of LTA4.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Beverly H. Koller, Department of Medicine, 7007 Thurston-Bowles Building, University of North Carolina, CB# 7248, Chapel Hill, NC 27599-7248. E-mail address:
3 Abbreviations used in this paper: LT, leukotriene; AA, arachidonic acid; BMMC, bone marrow-derived mast cell; EIA, enzyme immunoassay; ES, embryonic stem; FLAP, 5-lipoxygenase-activating protein; HSA, human serum albumin; LO, lipoxygenase; MPO, myeloperoxidase; PAF, platelet-activating factor; PMN, polymorphonuclear leukocyte.
Received for publication August 17, 1999. Accepted for publication September 23, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain gene. Cell 75:969.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. S. Lorrain, G. Bain, L. D. Correa, C. Chapman, A. R. Broadhead, A. M. Santini, P. Prodanovich, J. V. Darlington, J. H. Hutchinson, C. King, et al. Pharmacological Characterization of 3-[3-tert-Butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5-(pyridin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethyl-propionic Acid (AM103), a Novel Selective 5-Lipoxygenase-Activating Protein Inhibitor That Reduces Acute and Chronic Inflammation J. Pharmacol. Exp. Ther., December 1, 2009; 331(3): 1042 - 1050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Poeckel, K. A. Zemski Berry, R. C. Murphy, and C. D. Funk Dual 12/15- and 5-Lipoxygenase Deficiency in Macrophages Alters Arachidonic Acid Metabolism and Attenuates Peritonitis and Atherosclerosis in ApoE Knock-out Mice J. Biol. Chem., July 31, 2009; 284(31): 21077 - 21089. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miyahara, H. Ohnishi, S. Miyahara, K. Takeda, S. Matsubara, H. Matsuda, M. Okamoto, J. E. Loader, A. Joetham, M. Tanimoto, et al. Leukotriene B4 Release from Mast Cells in IgE-Mediated Airway Hyperresponsiveness and Inflammation Am. J. Respir. Cell Mol. Biol., June 1, 2009; 40(6): 672 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zarini, M. A. Gijon, A. E. Ransome, R. C. Murphy, and A. Sala Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation PNAS, May 19, 2009; 106(20): 8296 - 8301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Miyahara, H. Ohnishi, H. Matsuda, S. Miyahara, K. Takeda, T. Koya, S. Matsubara, M. Okamoto, A. Dakhama, B. Haribabu, et al. Leukotriene B4 Receptor 1 Expression on Dendritic Cells Is Required for the Development of Th2 Responses and Allergen-Induced Airway Hyperresponsiveness J. Immunol., July 15, 2008; 181(2): 1170 - 1178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-i. Yuhki, F. Ushikubi, H. Naraba, A. Ueno, H. Kato, F. Kojima, S. Narumiya, Y. Sugimoto, M. Matsushita, and S. Oh-ishi Prostaglandin I2 Plays a Key Role in Zymosan-Induced Mouse Pleurisy J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 601 - 609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Jordan, E. E. Moore, E. L. Sarin, S. S. Damle, S. B. Kashuk, C. C. Silliman, and A. Banerjee Arachidonic acid in postshock mesenteric lymph induces pulmonary synthesis of leukotriene B4 J Appl Physiol, April 1, 2008; 104(4): 1161 - 1166. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Liu, H.-E. Claesson, Y. Mahshid, G. Klein, and E. Klein Leukotriene B4 activates T cells that inhibit B-cell proliferation in EBV-infected cord blood-derived mononuclear cell cultures Blood, March 1, 2008; 111(5): 2693 - 2703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. F. P. Leite, J. Echevarria-Lima, S. C. Ferreira, J. B. Calixto, and V. M. Rumjanek ABCC transporter inhibition reduces zymosan-induced peritonitis J. Leukoc. Biol., September 1, 2007; 82(3): 630 - 637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Wan, A. Sabirsh, A. Wetterholm, B. Agerberth, and J. Z. Haeggstrom Leukotriene B4 triggers release of the cathelicidin LL-37 from human neutrophils: novel lipid-peptide interactions in innate immune responses FASEB J, September 1, 2007; 21(11): 2897 - 2905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Vroon, C. J. Heijnen, and A. Kavelaars GRKs and arrestins: regulators of migration and inflammation J. Leukoc. Biol., December 1, 2006; 80(6): 1214 - 1221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Chen, B. K. Lam, Y. Kanaoka, P. A. Nigrovic, L. P. Audoly, K. F. Austen, and D. M. Lee Neutrophil-derived leukotriene B4 is required for inflammatory arthritis J. Exp. Med., April 17, 2006; 203(4): 837 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Christmas, K. Tolentino, V. Primo, K. Z. Berry, R. C. Murphy, M. Chen, D. M. Lee, and R. J. Soberman Cytochrome P-450 4F18 Is the Leukotriene B4 {omega}-1/{omega}-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes: IDENTIFICATION AS THE FUNCTIONAL ORTHOLOGUE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE CYP4F3A IN THE DOWN-REGULATION OF RESPONSES TO LTB4 J. Biol. Chem., March 17, 2006; 281(11): 7189 - 7196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Suram, G. D. Brown, M. Ghosh, S. Gordon, R. Loper, P. R. Taylor, S. Akira, S. Uematsu, D. L. Williams, and C. C. Leslie Regulation of Cytosolic Phospholipase A2 Activation and Cyclooxygenase 2 Expression in Macrophages by the beta-Glucan Receptor J. Biol. Chem., March 3, 2006; 281(9): 5506 - 5514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. G. Werner, V. Godfrey, R. R. Arnold, G. L. Featherstone, D. Bender, J. Schlossmann, M. Schiemann, F. Hofmann, and K. B. Pryzwansky Neutrophil Dysfunction in Guanosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase I-Deficient Mice J. Immunol., August 1, 2005; 175(3): 1919 - 1929. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. G. Brock, Y.-J. Lee, E. Maydanski, T. L. Marburger, M. Luo, R. Paine III, and M. Peters-Golden Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L224 - L232. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sabirsh, A. Wetterholm, J. Bristulf, H. Leffler, J. Z. Haeggstrom, and C. Owman Fluorescent leukotriene B4: potential applications J. Lipid Res., June 1, 2005; 46(6): 1339 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gosselin, P. Borgeat, and L. Flamand Leukotriene B4 Protects Latently Infected Mice against Murine Cytomegalovirus Reactivation following Allogeneic Transplantation J. Immunol., February 1, 2005; 174(3): 1587 - 1593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Benjamim, C. Canetti, F. Q. Cunha, S. L. Kunkel, and M. Peters-Golden Opposing and Hierarchical Roles of Leukotrienes in Local Innate Immune versus Vascular Responses in a Model of Sepsis J. Immunol., February 1, 2005; 174(3): 1616 - 1620. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Z. Haeggstrom Leukotriene A4 Hydrolase/Aminopeptidase, the Gatekeeper of Chemotactic Leukotriene B4 Biosynthesis J. Biol. Chem., December 3, 2004; 279(49): 50639 - 50642. [Full Text] [PDF]
|
|
|
|
|
|
J. L. Goulet, A. J. Pace, M. L. Key, R. S. Byrum, M. Nguyen, S. L. Tilley, S. G. Morham, R. Langenbach, J. L. Stock, J. D. McNeish, et al. E-Prostanoid-3 Receptors Mediate the Proinflammatory Actions of Prostaglandin E2 in Acute Cutaneous Inflammation J. Immunol., July 15, 2004; 173(2): 1321 - 1326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Rudberg, F. Tholander, M. Andberg, M. M. G. M. Thunnissen, and J. Z. Haeggstrom Leukotriene A4 Hydrolase: IDENTIFICATION OF A COMMON CARBOXYLATE RECOGNITION SITE FOR THE EPOXIDE HYDROLASE AND AMINOPEPTIDASE SUBSTRATES J. Biol. Chem., June 25, 2004; 279(26): 27376 - 27382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Takeshita, K. Sakai, K. B. Bacon, and F. Gantner Critical Role of Histamine H4 Receptor in Leukotriene B4 Production and Mast Cell-Dependent Neutrophil Recruitment Induced by Zymosan in Vivo J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 1072 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kavelaars, A. Vroon, R. P. Raatgever, A. M. Fong, R. T. Premont, D. D. Patel, R. J. Lefkowitz, and C. J. Heijnen Increased Acute Inflammation, Leukotriene B4-Induced Chemotaxis, and Signaling in Mice Deficient for G Protein-Coupled Receptor Kinase 6 J. Immunol., December 1, 2003; 171(11): 6128 - 6134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Chen, N. Li, S. Wang, N. Wu, J. Hong, X. Jiao, M. J. Krasna, D. G. Beer, and C. S. Yang Leukotriene A4 Hydrolase in Rat and Human Esophageal Adenocarcinomas and Inhibitory Effects of Bestatin J Natl Cancer Inst, July 16, 2003; 95(14): 1053 - 1061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cuzzocrea, A. Rossi, I. Serraino, E. Mazzon, R. Di Paola, L. Dugo, T. Genovese, B. Calabro, A. P. Caputi, and L. Sautebin 5-Lipoxygenase knockout mice exhibit a resistance to pleurisy and lung injury caused by carrageenan J. Leukoc. Biol., June 1, 2003; 73(6): 739 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Grenier, N. Flamand, J. Pelletier, P. H. Naccache, P. Borgeat, and S. G. Bourgoin Arachidonic acid activates phospholipase D in human neutrophils; essential role of endogenous leukotriene B4 and inhibition by adenosine A2A receptor engagement J. Leukoc. Biol., April 1, 2003; 73(4): 530 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Maekawa, K. F. Austen, and Y. Kanaoka Targeted Gene Disruption Reveals the Role of Cysteinyl Leukotriene 1 Receptor in the Enhanced Vascular Permeability of Mice Undergoing Acute Inflammatory Responses J. Biol. Chem., May 31, 2002; 277(23): 20820 - 20824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. F. Kachur, L. J. Askonas, D. Villani-Price, N. Ghoreishi-Haack, S. Won-Kim, C.-D. D. Liang, M. A. Russell, and W. G. Smith Pharmacological Characterization of SC-57461A (3-[Methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic Acid HCl), a Potent and Selective Inhibitor of Leukotriene A4 Hydrolase II: In Vivo Studies J. Pharmacol. Exp. Ther., February 1, 2002; 300(2): 583 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Eberhard, S. Jepsen, L. Pohl, H. K. Albers, and Y. Acil Bacterial Challenge Stimulates Formation of Arachidonic Acid Metabolites by Human Keratinocytes and Neutrophils In Vitro Clin. Vaccine Immunol., January 1, 2002; 9(1): 132 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-Z. Shi, B. Han, G. M. Habib, M. M. Matzuk, and M. W. Lieberman Disruption of {gamma}-Glutamyl Leukotrienase Results in Disruption of Leukotriene D4 Synthesis In Vivo and Attenuation of the Acute Inflammatory Response Mol. Cell. Biol., August 15, 2001; 21(16): 5389 - 5395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Wang, C. Feliciani, I. Freed, Q. Cai, and D. N. Sauder Insights into molecular mechanisms of contact hypersensitivity gained from gene knockout studies J. Leukoc. Biol., August 1, 2001; 70(2): 185 - 191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Haribabu, M. W. Verghese, D. A. Steeber, D. D. Sellars, C. B. Bock, and R. Snyderman Targeted Disruption of the Leukotriene B4Receptor in Mice Reveals Its Role in Inflammation and Platelet-Activating Factor-Induced Anaphylaxis J. Exp. Med., August 7, 2000; 192(3): 433 - 438. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Kanaoka, A. Maekawa, J. F. Penrose, K. F. Austen, and B. K. Lam Attenuated Zymosan-induced Peritoneal Vascular Permeability and IgE-dependent Passive Cutaneous Anaphylaxis in Mice Lacking Leukotriene C4 Synthase J. Biol. Chem., June 15, 2001; 276(25): 22608 - 22613. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. G. Brock, E. Maydanski, R. W. McNish, and M. Peters-Golden Co-localization of Leukotriene A4 Hydrolase with 5-Lipoxygenase in Nuclei of Alveolar Macrophages and Rat Basophilic Leukemia Cells but Not Neutrophils J. Biol. Chem., September 7, 2001; 276(37): 35071 - 35077. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Rudberg, F. Tholander, M. M. G. M. Thunnissen, and J. Z. Haeggstrom Leukotriene A4 Hydrolase/Aminopeptidase. GLUTAMATE 271 IS A CATALYTIC RESIDUE WITH SPECIFIC ROLES IN TWO DISTINCT ENZYME MECHANISMS J. Biol. Chem., January 4, 2002; 277(2): 1398 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Maekawa, Y. Kanaoka, B. K. Lam, and K. F. Austen Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing PNAS, February 27, 2001; 98(5): 2256 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ichinose, W. M. Zapol, A. Sapirstein, R. Ullrich, A. M. Tager, K. Coggins, R. Jones, and K. D. Bloch Attenuation of Hypoxic Pulmonary Vasoconstriction by Endotoxemia Requires 5-Lipoxygenase in Mice Circ. Res., April 27, 2001; 88(8): 832 - 838. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
),
5-LO-deficient (
), and wild-type (•) mice received an i.p.
injection of 1 ml zymosan A in PBS (1 mg/ml) to elicit peritoneal
inflammation. A, To measure plasma protein influx, mice
received 0.5% Evans blue dye i.v. before zymosan A treatment. Mice
were euthanized at time points of 10, 20, 30, 60, 180, and 360 min, and
peritoneal lavage was performed with 4 ml PBS. Cells were removed by
centrifugation, and wash fluid was analyzed for Evans blue dye
extravasation by light spectrophotometry at 610 nm. Totals:
n = 2–8 mice at 0 min, 4–11 mice at 10 min, 4–10
mice at 20 min, 4–11 mice at 30 min, 6–11 mice at 60 min, 7–10 mice
at 180 min, and 6–7 mice at 360 min. B, In related
experiments, mice of each genotype did not receive Evans blue dye
before zymosan A treatment. Peritoneal lavage was performed on these
animals at 0, 1, 6, and 12 h with 4 ml PBS. Cells were centrifuged
from the lavage fluids and were analyzed in an enzymatic assay for MPO
as an indicator of neutrophil content. Totals; n =
2–3 mice at 0 h, 5–7 mice at 1 h, 4–7 mice at 3 h,
3–9 mice at 6 h, and 3–5 mice at 12 h. Error bars indicate
SEM. *, p < 0.05; **, p
< 0.01; ***, p < 0.0005; ****,
p < 0.00005.
