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*
Department of Medicine, University of North Carolina, Chapel Hill, NC 27599; and
Division of Nephrology, Department of Medicine, Duke University and Durham Veterans Affairs Medical Centers, Durham, NC 27705
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LT can elicit many of the physiological events associated with inflammation, including edema formation, vasopermeability, and cellular infiltration (2, 11, 12, 13). LTB4 is the most potent natural chemoattractant for neutrophils, eosinophils, and monocytes (14, 15, 16). In addition, it has been shown that LTB4 stimulates adhesion of leukocytes to vascular endothelia for extravasation into adjacent tissue (12, 17). LTB4 also initiates neutrophil aggregation and degranulation (12). The cDNA encoding the LTB4 receptor (B-LT receptor) has been cloned recently and, as predicted by pharmacological studies, this receptor is a seven-membrane-spanning G-protein-coupled receptor (18). Studies have shown that the B-LT receptor is highly expressed in peripheral blood leukocytes and, to a lesser degree, in spleen and thymus. However, its expression is limited since most other tissues examined showed no or insignificant expression of mRNA for the B-LT receptor (18). Activation of B-LT receptors results in mobilization of Ca2+, accumulation of inositol trisphosphate, and inhibition of adenylyl cyclase (18).
Although cysteinyl-LT do not directly stimulate migration of leukocytes to inflammatory lesions, they play an important role in edema formation during the inflammatory response. LTC4 and its metabolites, LTD4 and LTE4, are secreted in vivo by eosinophils, mast cells, and macrophages (12, 13). These mediators act directly on the microvasculature by inducing vasoconstriction and increasing postcapillary venule permeability, thereby allowing leakage of fluid and proteins and facilitating migration of leukocytes into the inflammatory site (12, 13, 17). The cysteinyl-LT mediate their activities through at least two distinct receptors termed Cys-LT1 (formerly the "LTD4 receptor") and Cys-LT2 (formerly the "LTC4 receptor") (19, 20). The recent cloning of Cys-LT1 indicates that this receptor, unlike the B-LT receptor, has a broad tissue distribution, including peripheral blood leukocytes, spleen, and lung, suggesting that LTC4 could influence the function of many cell types (21).
A direct means of determining the contribution of LT to an inflammatory response was made available by the generation of mouse lines deficient in either the 5LO enzyme or the 5-lipoxygenase-activating protein (22, 23, 24, 25). Using these mouse lines, we and others have defined a role for LT in several models of acute inflammation (22, 23, 24). However, these studies revealed variability in both the intensity of inflammatory responses in wild-type mice and the extent to which loss of LT production affected these responses. We suspected that these differences were linked to the fact that both 5LO-deficient mice and wild-type controls were F2 generation animals, containing a mixture of genes from the 129/Ola-derived embryonic stem (ES) cells and the B6D2F1 (F1 offspring of C57BL/6 x DBA/2 crosses) animals to which chimeras were bred. Therefore, we set out to determine whether modifier genes altered the magnitude of inflammatory responses and also to determine the contribution of LT to these processes.
The influence of genetic background on immunological processes in mice, although poorly understood in most instances, has been recognized since the development of inbred mouse strains. In recent years, it has become apparent that genetic differences between strains of mice can result in qualitative and quantitative differences in immune responses. For example, it is now well accepted that T cell responses are dominated by Th2-type cells in the BALB/c mouse, whereas the Th1 cell is prevalent in immune responses in the C57BL/6 (B6) mouse. Studies have also shown significant variability in the airway responsiveness phenotype of various inbred mouse strains (26). In addition, the multiple intestinal neoplasia (Min) mouse, a model for human familial adenomatous polyposis, demonstrates the modification of disease by genetic background. Min mice differ greatly in the number of intestinal tumors, depending on the mouse strain carrying this mutation (27, 28). Recently, studies have shown that the gene encoding the group II secretory nonpancreatic PLA2 (sPLA2) enzyme is naturally disrupted in a number of inbred mouse strains and has been identified as a candidate gene for the modifier of the Min 1 (Mom1) locus (27, 28). Loss of sPla2 gene function results in susceptibility to the Min phenotype and the formation of multiple intestinal polyps, whereas mice expressing an active sPla2 gene are resistant to polyp formation. The mouse strains B6 and 129/Sv were found to be homozygous for the defective sPla2 gene, whereas DBA/1 and DBA/2 mice had a normal sPla2 genotype at this locus (27, 28).
The ability to generate mice that carry a null allele for proteins involved in the synthesis of inflammatory mediators, and the subsequent generation of congenic mouse lines carrying the null allele, makes it possible to identify a role for modifier genes in specific inflammatory responses. To investigate the effect of genetic factors on the contribution of LT to acute inflammation, we generated three congenic lines of 5LO-deficient mice. The disrupted 5lo allele was maintained on a relatively inbred 129 genetic background and was also backcrossed onto several other inbred strains, including B6 and DBA/1Lac (DBA). Although no differences were seen in the general health or development of these three mouse lines, significant variations in the contribution of LT to AA-induced ear inflammation and peritonitis were observed. Moreover, the relative contribution of modifier genes to AA-induced inflammatory responses were shown to be tissue specific. We also demonstrated that the variation in the intensity of inflammation between strains results primarily from differences in the ability of a particular mouse strain and a specific tissue to respond to LTC4.
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Materials and Methods |
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Chimeric animals were created by microinjecting 5lo-targeted embryonic stem cells derived from the 129/Ola mouse strain into B6 host blastocysts (23). The 5lo mutation was maintained on a relatively inbred 129 genetic background by breeding chimeras with 129/SvEv mice. Inbred B6 mice homozygous for the mutant 5lo allele were generated by backcrossing the 5lo mutation onto the B6 mouse strain for 12 generations and then intercrossing the resulting N12 B6-5lo+/- mice. 5LO-deficient DBA mice were created by repeatedly backcrossing animals heterozygous for the targeted 5lo gene mutation to DBA mice for a minimum of eight generations. Intercrosses of DBA mice heterozygous for the disrupted 5lo allele produced DBA-5lo-/- and DBA-5lo+/+ animals. 129/SvEv, B6, and DBA mice used for breeding were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were screened for the 5lo mutation by Southern blot analysis of EcoRI-digested tail genomic DNA as described previously (23). All mice studied were at least 8 wk old and were bred and maintained in specific pathogen-free animal barrier facilities at the University of North Carolina.
Induction of inflammatory responses in mouse ear tissue
Animals were injected i.v. with either 0.5% Evans blue dye (Sigma, St. Louis, MO) dissolved in PBS (pH 7.5; 10 ml of dye solution/kg body wt) or with indomethacin (INDO; 1 mg/ml in 0.1 M Na2CO3 and 0.15 M Na2HPO4, pH 7.4; Sigma) combined with 0.5% Evans blue dye (10 mg INDO/kg body weight). The inside of the left ear of each mouse was painted with 20 µl of AA (100 µg/µl in acetone; Sigma), while the right ear was treated with an equal amount of acetone alone. In related experiments, the left ear of each mouse was injected intradermally with either 5 µl of LTC4 (20 ng/µl in 1:4 ethanol:PBS; Cayman Chemicals, Ann Arbor, MI) or 20 µl of PGE1 (Cayman Chemicals) combined with bradykinin (BK; Sigma; 25 ng/µl in 1:19 ethanol:PBS). As a control, the right ear was injected with equal amounts of vehicle alone. At 1 h after AA or LTC4 treatment, or 30 min after PGE1 plus BK intradermal injections, mice were sacrificed and an 8-mm-diameter disc of tissue was punched from the center of each ear.
Edema and vascular permeability measurements in mouse ear tissue
Edema was measured by determining the wet weight of the ear punches. To extract extravasated Evans blue dye, ear biopsies were incubated in 1 ml of formamide at 55°C for 48 h. Evans blue was quantified by measuring the absorbance of the formamide extracts at 610 nm with a spectrophotometer (29).
Induction of peritoneal inflammation
For the zymosan A peritonitis experiments, inflammation was induced using a modification of the procedure of Rao et al. (30). Zymosan A (Sigma) was dissolved in warm (55°C) PBS to a final concentration of 1 mg/ml and mixed until it passed freely through a 30-gauge syringe needle. Mice were injected i.p. with 1 ml of zymosan A solution. In related experiments, mice received i.p. injections of 0.5 ml of either LTC4 (100 ng in 1:499 ethanol:PBS), AA (2 mg in 1:24 acetone:PBS), PGE1 plus BK (1 µg of each in 1:249 ethanol:PBS), or equal volumes of vehicle alone. All mice received i.v. injections of 0.5% Evans blue dye solution immediately before administration of inflammatory stimuli.
Quantification of edema and vascular permeability in peritonitis inflammatory models
Thirty minutes after zymosan A or LTC4 i.p. injections, or 1 h after AA or PGE1 plus BK administration, mice were euthanized and injected i.p. with 4 ml of PBS. The abdomen was massaged for a few seconds to mix its contents, and lavage fluids were collected into polypropylene tubes on ice through a small incision. Samples that were visibly red and therefore contaminated with blood were not included in analyses. Lavage fluid (1 ml) was centrifuged at 40,000 x g for 10 min and supernatants were collected. The extravasation of Evans blue dye in the lavage fluid was quantified by spectrophotometric analysis (610 nm wavelength).
Statistical analysis
Data are presented as the mean ± SEM. Statistical significance for comparisons between groups was determined using an unpaired two-sample t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate 5LO-deficient mouse lines, chimeras created by injection of 5lo-targeted ES cells into B6 host blastocysts (23) were bred to either 129/SvEv, B6, or DBA mice. The parental ES cell line, E14TG2a (31), from which the 5lo-targeted line was generated, was derived from the 129/Ola mouse strain and therefore the 5lo mutation could be maintained on a relatively inbred 129 genetic background by breeding chimeras with 129/SvEv mice. B6 and DBA mice congenic for the 5lo mutation were generated by repeatedly crossing 5lo heterozygotes to B6 and DBA mice for 12 generations. Intercrosses between B6 and DBA mice that were heterozygous for the 5lo mutation produced wild-type (5lo+/+) B6 and 5LO-deficient (5lo-/-) B6 mice and DBA-5lo+/+ and DBA-5lo-/- animals. No differences were seen in the growth and development, survival, and overall health of mice deficient in 5LO on any of the three genetic backgrounds.
AA-induced inflammation in ears of 129, B6, and DBA wild-type mice
Previous studies using F2 generation mice of a mixed background
consisting of genes from 129, B6, and DBA mouse strains showed large
animal to animal variation in the intensity of the inflammatory
responses to topically applied AA (23). To determine
whether this variation reflected differences in the ability of the
three strains to respond to AA, we examined the effect of this stimulus
on the ear tissue of 129, B6, and DBA wild-type inbred mice.
Inflammation was measured by determining both the change in weight of
an ear biopsy and the extravasation of plasma protein. Protein
extravasation was assayed by injecting the mice with Evans blue, a
dye which binds to serum proteins, before the administration of AA and
quantifying the amount of dye in the ear tissue. As shown in Fig. 1
, topical application of AA resulted in
a significant increase in edema and vascular permeability, as measured
by changes in ear weight and plasma protein extravasation,
respectively, in treated tissue of both 129 and DBA inbred mice. The
intensity of the AA-induced inflammatory responses was similar in
wild-type mice of 129 and DBA strains (Fig. 1, p =
0.225 and p = 0.331, respectively). In contrast, only a
modest increase in ear weight in response to AA was observed in inbred
B6-5lo+/+ mice. AA-stimulated edema in this
group was significantly less than that seen in both 129- and
DBA-5lo+/+ animals (Fig. 1A,
p = 2.65 x 10-7 and
8.05 x 10-7, respectively). A
corresponding difference in the amount of protein extravasation in
B6-5lo+/+ mice compared with both wild-type
129 and DBA animals was also noted (Fig. 1B,
p = 3.09 x 10-7 and
0.00015, respectively). These data demonstrate that the vascular
response of inbred wild-type B6 mice to AA is significantly less
vigorous than the response seen in either wild-type 129 or DBA inbred
mouse strains.
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Using 5LO-deficient mice of a mixed genetic background, we and others have shown that LT play an important role in AA-induced inflammatory responses (22, 23). These studies also indicated that, while loss of 5LO decreased the edema and vascular permeability elicited by AA, a residual LT-insensitive response remained in the 5LO-deficient animals. To determine whether the variation in this inflammatory model among the 129, B6, and DBA strains reflects differences in this LT-independent response or in the role of LT in these three mouse strains, we compared the effect of topically applied AA on ear tissue of wild-type 129, B6, and DBA mice to that of congenic animals deficient in 5LO.
A significant reduction in AA-induced edema and vascular permeability,
as measured by increases in ear weight and extravasation of serum
proteins, was seen in the inbred
129-5lo-/- animals compared with 129
wild-type mice (Fig. 1
, p = 1.61 x
10-13 and 1.68 x
10-12, respectively). Loss of 5LO activity also
significantly reduced these responses in inbred DBA mice (Fig. 1, p = 5.97 x 10-6 and
1.87 x 10-5, respectively). The
5lo mutation on each of these two genetic backgrounds
resulted in a significant attenuation of AA-stimulated acute
inflammation compared with wild-type counterparts. However, the
AA-induced increase in ear weight was slightly lower in
129-5lo-/- mice than in
DBA-5lo-/- animals (Fig. 1A,
p = 0.005). Serum protein extravasation, as determined
by the amount of dye in the ear tissue, was reduced to a similar level
in both 129-5lo-/- and
DBA-5lo-/- animals (Fig. 1B,
p = 0.119). In both strains, however, the loss of 5LO
did not completely eliminate the inflammatory response to AA.
In contrast to the decreased inflammatory responses to AA in the
5LO-deficient 129 and DBA mice, the loss of 5LO had no impact on the
inflammatory response elicited by AA in the B6 mouse strain. 5LO
deficiency strongly inhibited AA-induced ear inflammation in the 129
(76–77%) and DBA (59–69%) strains, but had no effect on these
responses in B6 (-3 to -5%) animals (Table I). Alterations in ear weight and dye
extravasation in response to AA were similar in wild-type and
5LO-deficient B6 mice (Fig. 1, p = 0.423 and 0.375,
respectively). AA-induced edema, as measured by ear weight increases,
was higher in B6-5lo-/- mice than in 129-
and DBA-5lo-/- animals (Fig. 1A, p = 3.45 x
10-5 and 0.183, respectively). Also, protein
extravasation in B6-5lo-/- mice was
significantly increased compared with 5LO-deficient mice of both the
129 and DBA strains (Fig. 1B, p = 0.00019
and 0.0295, respectively). Taken together, these data clearly show that
the loss of 5LO activity significantly reduced AA-induced inflammation
in the ears of 129 and DBA mice, but had no effect on this response in
the B6 strain.
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We showed previously that the AA-induced inflammatory response
remaining in 5LO-deficient mice of a mixed genetic background is
sensitive to the COX inhibitor INDO (23). Therefore, we
determined whether the LT-independent inflammatory response seen in the
B6 mice also results from the synthesis of prostanoids, the products of
the COX pathways, by treating wild-type and 5LO-deficient 129, B6, and
DBA mice with INDO before the application of AA. As can be seen in Fig. 2A, AA-induced edema was
slightly lower in INDO-treated 129 wild-type mice than in untreated 129
mice, but this difference was not significant
(p = 0.294 and Table II). The amount of dye extravasation was
also comparable in INDO-treated and untreated wild-type mice of the 129
strain (Fig. 2B, p = 0.056, and Table II).
Similarly, inhibition of the COX pathway by treatment with INDO had no
effect on AA-induced inflammation, measured by either ear weight or dye
extravasation, in wild-type DBA mice (Fig. 2, p = 0.295
and 0.196, respectively, and Table II).
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A, p = 0.0013) and vascular
permeability (49%) (Fig. 2B, p = 0.029) in
129–5LO-deficient mice (Table II). However, these responses were only
reduced by 20–25% by INDO in DBA-5lo-/-
mice (Fig. 2, p = 0.104 and 0.207, respectively, and
Table II). These data suggest that prostanoids contribute to the
residual AA-induced inflammation in 5LO-deficient 129 and DBA mouse
lines, although the relative role of these lipid mediators in this
model is dependent on the genetic background of the mice being
studied.
The inflammatory response seen in B6 mice is significantly reduced by
treatment of the mice with INDO before application of AA, regardless of
5LO activity. As shown in Table II, edema and vascular permeability
were inhibited by 69 and 38%, respectively, in B6 wild-type mice and,
moreover, these responses were greatly reduced (73–77%) by INDO in
B6-5lo-/- animals. These data indicate
that the vascular component of the AA inflammatory model in the B6
mouse strain is dependent primarily on prostanoid synthesis.
LTC4-induced cutaneous inflammation in wild-type inbred mouse strains
We examined the possibility that the differential role of LT in
the inflammatory models described above reflects variation in the
ability of mouse strains to respond to LT. To test this hypothesis, we
studied the effect of exogenously administered
LTC4 on edema formation and vascular permeability
in ear tissue of high- and low-responding mouse strains. After labeling
serum proteins with i.v. Evans blue, as described above, we injected
LTC4 intradermally in the left ear of B6 and 129
wild-type mice. As seen in Fig. 3,
LTC4 was effective in causing both edema and
increased vascular permeability, as measured by increases in ear weight
and dye extravasation, respectively, in 129 mice. In contrast to the
responses of 129 mice, only small increases in edema and
vasopermeability were observed in ear biopsies from B6 wild-type mice
after exposure to LTC4 (Fig. 3, p
= 0.0008 and 0.0115, respectively).
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, intradermal injections of
PGE1 plus BK resulted in significant increases in
both edema and vascular permeability in ear biopsies of 129 and B6
mice. However, the intensity of inflammation induced by this stimulus
was similar in the 129 and B6 mouse strains (Fig. 3, p
= 0.151 and 0.140, respectively), suggesting that gross physiological
differences between the two strains do not account for the variation
observed in the cutaneous response to LTC4. AA-induced inflammatory responses in the peritoneal cavities of 5lo+/+ and 5lo-/- mice
To determine whether the variation in these inflammatory responses
between mouse strains is specific to cutaneous tissue, we also examined
the effect of i.p. administration of AA in 129, B6, and DBA mice.
Animals were injected i.v. with Evans blue, and the levels of this dye
in lavage fluid after AA challenge were quantitated as a measurement of
vascular permeability in the peritoneal cavity. Baseline levels of
Evans blue dye extravasation in lavage fluids from wild-type and
5LO-deficient mice injected i.p. with PBS were not significantly
different among the three strains (data not shown). Therefore, only
data for wild-type mice treated with PBS are shown in Fig. 4. AA treatment resulted in increased dye
extravasation in all three mouse strains. However, the vascular
response of DBA-5lo+/+ mice was
significantly lower than that seen in the 129 (Fig. 4, p = 0.0002) and B6 (Fig. 4, p = 0.053)
wild-type mouse strains. Although a greater increase in vascular
permeability was seen in the B6 wild-type mice compared with the DBA
strain, this level of inflammation was significantly lower than that
observed in 129 mice (Fig. 4, p = 0.0163). These
results are contrary to those of the experiments described above, in
which the response of B6 mice to topically applied AA was significantly
lower than that of both 129 and DBA mouse strains.
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, we observed a significant decrease in
Evans blue dye extravasation in the inbred
129-5lo-/- mice compared with
129-5lo+/+ animals
(p = 0.0008). This response was also
significantly reduced in 5LO-deficient B6 mice (Fig. 4, p = 0.0195). In contrast, the 5lo mutation
had no effect on AA-induced inflammation in the DBA mouse strain (Fig. 4, p = 0.450). The loss of 5LO activity significantly
inhibited AA-induced peritonitis in the 129 (22%) and B6 (14%) mouse
strains, but had no impact on this inflammatory response in the DBA
mice (-1.3%, Table III). The increased
levels of plasma protein extravasation stimulated by AA were not
significantly different (p > 0.05) among the
three 5LO-deficient mouse lines, suggesting that the 5LO-independent
component of this response is similar in the 129, B6, and DBA mouse
strains.
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To determine whether the strain differences observed in the
responses to AA extended to other inflammatory responses, we examined
zymosan A-induced peritonitis in wild-type and
5lo-/- 129, B6, and DBA animals. We have
shown previously, using the inbred 129–5LO-deficient mice, that plasma
protein extravasation induced by zymosan A is almost completely
dependent on the production of LT by 5LO during the early phases of the
response (24). As can be seen in Fig. 5, we observed a significant decrease
in zymosan A-stimulated inflammation, as measured by Evans blue
dye extravasation, in the inbred
129-5lo-/- mice
(p = 3.23 x
10-12). The increase in dye extravasation
elicited by zymosan A was of similar magnitude in the
B6-5lo+/+ mice compared with 129 wild-type
animals (Fig. 5, p = 0.135) and a corresponding large
decrease in this response was seen in the
B6-5lo-/- animals compared with the
wild-type B6 mice (Fig. 5, p = 0.00011). Zymosan A
administration also resulted in an increase in dye extravasation in
DBA-5lo+/+ mice, although the intensity of
the response was significantly lower than that seen in the 129 and B6
wild-type mouse strains (Fig. 5, p = 1.27 x
10-6 and 0.00043, respectively). Although
zymosan A-induced inflammation was significantly inhibited by 34% in
the DBA-5lo-/- animals (Fig. 5, p = 0.0375), this percent reduction was less than that
observed in the 129 (72%) and B6 (67%) mice (Table IV).
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We next determined whether the responses of the 129 and DBA mouse
strains to i.p. administration of LTC4 correlated
with the effects of other inflammatory stimuli, such as AA and zymosan
A, in this tissue. As seen in Fig. 6,
treatment with LTC4 resulted in a significant
increase in vascular permeability, as measured by Evans blue dye
extravasation, in the peritoneal cavities of 129 wild-type mice. These
data are consistent with those described above (Figs. 4 and 5), in
which a vigorous response is seen in 129 mice after i.p. administration
of either AA or zymosan A. In contrast, the vascular response of DBA
mice after exposure to LTC4 was significantly
less vigorous than the response to LTC4 observed
in the 129 strain (Fig. 6, p = 0.0165). As a control,
129 and DBA mice were injected i.p. with PGE1
plus BK, and the resulting alterations in vascular permeability were
similar in the DBA and 129 wild-type animals (Fig. 6, p
= 0.406).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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presents an overall summary of
differences among the three mouse strains in the various inflammatory
responses described above.
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Although LT appear to be the major mediators of AA-induced acute inflammation, it has been demonstrated that prostanoids produced via the COX pathways also play a role in this model (23, 32, 33, 34, 35, 36). Treatment of wild-type mice with the COX inhibitor INDO allowed us to examine the contribution of prostanoids to AA-induced cutaneous inflammation. Treatment of 5LO-deficient mice with INDO in this model allowed us to explore the potential shunting of AA between the 5LO and COX pathways in different mouse strains. Analysis of the AA-induced response in 129, B6, and DBA mice indicated differences among all three strains in the various pathways that contribute to this response. The level of inflammation induced by AA in 129 and DBA wild-type mice is similar and in both strains the response is largely mediated by LT, with only a small LT-independent component. The response to AA in wild-type mice of these two strains is largely unaffected by treatment with INDO, suggesting that prostanoids normally make only a minor contribution to the vascular component of this inflammatory model. However, consistent with our previous studies, the experiments described here show that prostanoids have a more significant effect on the inflammatory response to AA in the 5LO-deficient 129 and DBA mice than in their wild-type counterparts, suggesting that tissues in these strains may compensate for the loss of 5LO in part by altering the synthesis and/or activity of prostanoids to modulate the inflammatory response. Moreover, INDO pretreatment had a greater influence on the residual AA-induced inflammation in the 5LO-deficient 129 strain than in DBA-5lo-/- mice. These data suggest that edema and increased vasopermeability result largely from the actions of prostanoids in 129 mice that are deficient in 5LO and, therefore, unable to synthesize LT. In contrast, the AA-induced cutaneous response seen in the DBA-5LO-deficient mice is largely INDO insensitive. Presumably, residual inflammation in ear tissue of INDO-treated DBA-5lo-/- mice results from unidentified mediators that are generated independent of the expression of either 5LO or prostaglandin synthase enzymes. Therefore, prostanoids may not be able to initiate inflammation independent of other mediators in the DBA strain. Alternatively, it is possible that shunting of AA to the COX pathways is less efficient in the DBA strain.
In comparison to the responses of 129 and DBA mice, edema and changes in vascular permeability evoked by AA were attenuated in the B6 wild-type mice. Moreover, this response was largely 5LO independent and thus remains virtually identical to that observed in the B6–5LO-deficient animals. The response induced by topical application of AA in both wild-type and 5LO-deficient mice of the B6 strain is almost entirely prostanoid dependent.
Taken together, these results indicate that mouse strains can differ substantially in the relative contribution of LT to inflammatory responses and in the ability of prostanoids to induce inflammation in the absence of LT. These data suggest that the relative roles of LT and prostanoids and additional 5LO- and/or prostanoid-independent inflammatory mediators in this acute inflammatory response are strongly influenced by genetic background.
It is also evident that the effects of genetic factors on inflammatory processes depend upon the specific tissue being examined. The AA peritonitis model allowed us to determine whether genetic modifiers affect LT-sensitive responses elicited by the same inflammatory stimuli in different tissues. Significant interstrain variation is apparent in the response to AA injected into the peritoneal cavity of wild-type inbred mice. However, the 5lo mutation had no effect on AA-induced peritoneal inflammation in DBA mice, whereas LT play a major role in AA-induced cutaneous inflammation in this mouse strain. In contrast, as discussed above, the 5LO pathway does not contribute measurably to the response to topical AA in the B6 mouse strain. However, a significant difference in plasma protein extravasation was observed between wild-type and 5LO-deficient B6 mice in response to administration of AA into the peritoneal cavity. Our data demonstrated that a mouse strain which shows little or no response to AA when it is applied topically may react very strongly when a different tissue is exposed to the same stimulus. To determine whether the genetic background of the mouse would play a role in other inflammatory responses, we examined zymosan A-induced peritonitis in the same inbred mouse lines. A vigorous LT-dependent inflammatory response to zymosan A was seen in the B6 mouse and it was similar in magnitude to that of 129 wild-type animals. In contrast, inflammation in the peritoneal cavities of DBA mice was significantly blunted in response to both AA and zymosan A and had only a minor LT-sensitive component.
Interstrain differences in the contribution of LT to an inflammatory response could reflect alterations in the activity of the 5LO pathway and subsequent synthesis and metabolism of LT and/or in the response of a specific tissue to LT. We addressed these possibilities by treating wild-type inbred mice with exogenous LTC4. We found that the variation observed in other experimental models of inflammation correlates directly with the ability of a particular mouse strain and a specific tissue to respond to cysteinyl-LT, specifically LTC4. Examination of edema formation and changes in vascular permeability in response to LTC4 suggests that the ability of mice to respond to LTC4 is at least one of the factors underlying strain differences in these models of inflammation. However, these results do not exclude the possibility that differences in cysteinyl-LT biosynthesis also exist in mouse strains that respond poorly to LTC4. It is also possible that differences in the production of and/or response to LTB4 may contribute to the strain variation seen in the 5LO-dependent component of inflammatory responses. In humans, it has been suggested that increased production of cysteinyl-LT correlates with susceptibility to aspirin-intolerant asthma (37, 38). Recent studies have shown that variations in the synthesis of cysteinyl-LT result from a polymorphism in the LTC4 synthase gene promoter, which causes constitutive overexpression of this enzyme in aspirin-sensitive patients (37, 38, 39, 40). Differing expression levels of LTC4 synthase may account for variability observed in the response to LT inhibitors used in the treatment of asthma (37).
The molecular basis for this differential response to LTC4 is not clear. The responses of these mouse lines to another acute inflammatory stimulus did not differ, suggesting that the interstrain variability is not the result of anatomical or physiological differences. The simplest explanation is that a decrease in the expression of the receptor on specific populations of cells within the responding tissue alters the inflammatory response. In addition, genetic polymorphisms in the structure of the LTC4 receptor may alter the binding of LTC4 to this receptor and/or the activation of signal transduction pathways associated with this receptor. Alternatively, LTC4 may be more rapidly metabolized in these tissues, and thereby reduce the effective dose. This latter explanation seems less likely given the high concentrations injected into the tissue and the acute nature of the response. With the cloning of the LTC4 receptor gene, it should be possible to address some of these questions specifically in the future.
In summary, we have examined the effect of genetic background on acute inflammatory responses in several genetically distinct strains of mice: 129, B6, and DBA. We demonstrated that the intensity of responses to specific inflammatory stimuli varies among these three strains, in the presence and absence of 5LO activity. The relative contributions of LT and prostanoids to each of these inflammatory models appear to be dependent upon other undefined genetic factors, which vary among these strains. It is also apparent that careful consideration is required in choosing strains to use for immunological studies because of the natural variation among inbred strains in responsiveness to stimuli and disease susceptibility. Future studies, similar to those described here, designed to study the effect of specific gene mutations on the natural phenotypes of different mouse strains will help us to better understand the interactions and roles of genetic factors in directing the course of immune responses in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Beverly H. Koller, Department of Medicine, University of North Carolina, 7007 Thurston-Bowles Building, Chapel Hill, NC 27599-7248.
3 Abbreviations used in this paper: LT, leukotriene; 5LO, 5-lipoxygenase; AA, arachidonic acid; B6, C57BL/6; BK, bradykinin; COX, cyclooxygenase; DBA, DBA/1Lac; ES, embryonic stem; INDO, indomethacin; PL, phospholipase; s, secretory.
Received for publication September 9, 1999. Accepted for publication February 17, 2000.
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