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The Journal of Immunology, 2006, 176: 3767-3773.
Copyright © 2006 by The American Association of Immunologists

A Quantitative Trait Loci Analysis to Map Genes Involved in Lipopolysaccharide-Induced Inflammatory Response: Identification of Macrophage Scavenger Receptor 1 as a Candidate Gene1

William B. Fulton*, Roger H. Reeves{dagger}, Motohiro Takeya{ddagger} and Antonio De Maio2,*,{dagger}

* Division of Pediatric Surgery, Department of Surgery, and {dagger} Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205; and {ddagger} Second Department of Pathology, Kumamoto University School of Medicine, Kumamoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Septic shock, which is a major complication observed after trauma and other human diseases, is likely the product of a prolonged and poorly controlled systemic inflammatory response. Symptoms of sepsis can be partially reproduced by injection of bacterial LPS in mice. Differences in mortality between C57BL/6Jhigh and A/Jlow mice after LPS injection have been previously observed and correlated with differences in the inflammatory response between these two inbred strains. In the present study, we have mapped four loci responsible for differences in levels of LPS-induced IL-10, named modifier of IL-10, between the two strains. A locus within mouse chromosome 8 was confirmed using chromosome 8 consomic mice. This locus was further reduced in size by haplotype analysis and evaluated by the presence of potential candidate genes. The macrophage scavenger receptor 1 (Msr1) within this locus emerged as a candidate gene based on differences at the expression and structural levels between C57BL/6J and A/J mice. In comparison with wild-type (C57BL/6J) mice, Msr1 knockout mice displayed reduced levels of LPS-induced IL-10, but not of TNF-{alpha} or IL-6, confirming a specific role for this gene in the regulation of IL-10. These results suggest that Msr1 is involved in the regulation of the anti-inflammatory process, thus offering a new perspective on the molecular mechanisms involved in endotoxemia and sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Sepsis is a major health problem in the United States, with an occurrence of ~750,000 cases per year (1). This condition is triggered by an array of insults, including infection, trauma, burn injury, pancreatitis, and even elective surgery. The mortality rate for sepsis ranges from 35 to 50%, primarily due to the development of multiple organ dysfunction syndrome (1, 2). Despite tremendous advances in the care of critically ill patients, therapy for sepsis remains supportive and extremely costly. A major obstacle to the development of treatments for sepsis is the immense heterogeneity of the clinical profiles observed in septic patients. Moreover, there are no reliable predictors for this condition. Although the etiology of sepsis is poorly understood, this condition has been associated with a prolonged and inadequately controlled systemic inflammatory response. An important component of the inflammatory process is secretion of cytokines by macrophages, monocytes, and polymorphic nuclear leukocytes. Proinflammatory cytokines, such as TNF-{alpha} and IL-6, modulate gene expression in a variety of organs (e.g., acute phase genes in the liver). Anti-inflammatory cytokines, such as IL-10, are necessary to control the inflammatory process. Specifically, IL-10 is involved in the down-regulation of TNF-{alpha} (3, 4). The imbalance between pro- and anti-inflammatory reactants is likely to be responsible for the development of exaggerated inflammatory response conditions, such as those observed in sepsis.

Sepsis is likely modified by multiple factors, including the type and extent of the initiating insult, the environment, and the genetic make-up, sex, and age of the patient (5). Although this assumption has been sustained by many clinical observations, it has not been experimentally tested in humans. Studies in experimental animal models have offered strong support for this hypothesis. For instance, a robust inflammatory response can be induced by injection of LPS. Indeed, mice maintained in identical environmental conditions and injected with Escherichia coli LPS displayed an inflammatory response that differed among the various inbred strains (6, 7). The frequency of mortality was also different among inbred mouse strains following injection of LPS (6). The genetic contribution to sepsis has been further supported by studies using a model of polymicrobial sepsis (8, 9). A direct correlation between extremely elevated anti-inflammatory (IL-10) cytokine levels and increased mortality has been observed after peritonitis (8, 9). Such studies, conducted on animals housed in identical environments and subjected to the same initiating insult, clearly illustrate that the genetic background is an important modifier of the inflammatory response and outcome. The question that emerges is whether these genetic differences can be exploited to identify genes that modulate the inflammatory response. The identification of such genes would shed light on the possible pathways activated in response to sepsis. In addition, this knowledge could result in the detection of genetic markers to be used as predictors for the development of sepsis. Mapping quantitative trait loci (QTL)3 is a direct approach to the identification of genes that modify the inflammatory response (5). Using an intercross strategy, we previously identified loci on mouse chromosomes (Chr) 13 and 5 showing an epistatic relationship for the infiltration of polymorphonuclear leukocytes in the liver after LPS challenge (7). In the present study, we mapped several loci for plasma levels of IL-10 as part of the inflammatory response induced by LPS. A candidate gene associated with LPS-induced IL-10 plasma levels was identified using a combination of genetic mapping and informatics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Experimental animal model of endotoxemia

Male mice (A/J, AKR/J, BALB/cJ, B6, DBA/2J, B6AF1/J (BXA), AXB/BXA recombinant inbred, B6-Chr 8A/NaJ, and B6-Chr 9A/NaJ) were obtained from The Jackson Laboratory. AB6F1/J (AXB) mice were bred in our animal facility, and macrophage scavenger receptor (Msr1) knockout mice were donated by Dr. M. Freeman (Massachusetts General Hospital, Boston, MA). Mice were maintained under identical environmental conditions in a pathogen-free animal facility. All mice used in this study were Helicobacter negative. All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University School of Medicine. At 8 wk of age, animals were fasted for 16 h before injection of E. coli LPS serotype 026:B6 (15 mg/kg; Difco Laboratories) under aseptic conditions. After injection, animals were given food and water ad libitum. At 1.5 h postinjection, mice were sacrificed under isoflurane anesthesia; blood was drawn by cardiac puncture using a 22-gauge needle, placed into K2EDTA Microtainer tubes (BD Biosciences), and centrifuged for 5 min. Plasma (supernatant) was removed, aliquoted, and frozen at –80°C until use. Plasma cytokine levels were measured by ELISA (BioSource International).

Linkage analysis

Linkage analysis was performed using MapManager QTXb20 (10). IL-10 plasma levels were averaged from five animals, log-transformed, and entered as QTL. Mean values of IL-10 were weighted by SE (log-transformed) and compared against the AXB/BXA set for QTL mapping (11) that was corrected based on updated genomic mapping. Suggestive, significant, and highly significant threshold levels were determined by the permutation test function of MapManager, which is based on the statistical methods developed by Churchill and Doerge (12). A likelihood ratio statistic (LRS) value of 4.6 is equivalent to one logarithm of the odds (LOD). A two-LOD support interval was defined for loci as the region at which the LRS value was within 9.2 U (2 LOD U) of the peak value.

Isolation of mouse peritoneal macrophages (PM{phi})

Mice were euthanized by cervical dislocation under isoflurane anesthesia, injected with 5 ml of cold RPMI 1640 medium supplemented with 1 U/ml heparin. The medium was gently flushed five times within the peritoneum and withdrawn. Cells were centrifuged and resuspended at a concentration of 1 x 106 cells/ml in RPMI 1640 medium (Mediatech Cellgro), supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 50 U/ml penicillin, 50 µg/ml streptomycin (Invitrogen Life Technologies), plated in 10-cm2 tissue culture dishes, and maintained at 37°C in 5% CO2, 95% incubator air for 1 h. Nonadherent cells were washed off, and PM{phi} were lysed with cold RIPA buffer (20 mM HEPES (pH 7.4), 1% Nonidet P-40, 50 mM NaCl, 1 mM EGTA, 5 mM beta-glycerophosphate, 30 mM sodium pyrophosphate, 100 mM sodium orthovanadate, 0.1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A). The lysate was centrifuged at 2000 x g for 2 min and the supernatant was used for SDS-PAGE and Western blotting. Proteins immobilized on a polyvinylidene difluoride (PVDF) membrane were quenched for 1 h with TBS-TBST (20 mM Tris, 500 mM NaCl, 0.1% Tween 20) containing 5% powdered milk, and incubated with anti-mouse MSR1 (1/5,000) for 16 h at 4°C. This Ab was raised in rabbit against a synthetic peptide (KEEQAHVEQEVKQEVR) corresponding to the {alpha} helical coiled-coil domain of mouse MSR1 common to various mouse strains (13). Membranes were washed three times with TBST and incubated with HRP-conjugated anti-rabbit-IgG (1/25,000; Amersham Pharmacia Biotech) in blocking buffer for 1 h. Membranes were washed again with TBST and the signal was visualized using Super Signal West Dura (Pierce Biotechnology).

Immunostaining

PM{phi} were fixed with 4% paraformaldehyde for 10 min, then washed twice with PBS. Nonspecific binding was blocked by incubation with 10% FCS in PBS for 30 min at 25°C. Fixed cells were incubated with anti-mouse MSR1 (1/200) in PBS-5% FCS for 1 h at 25°C. Cells were washed three times with PBS and incubated with anti-rabbit IgG Cy3 Conjugate F(ab')2 (1/1000; Sigma-Aldrich) in PBS-5% FCS for 40 min at 25°C. Cells were washed three times with PBS, then mounted and visualized with a fluorescent microscope.

FACS analysis

PM{phi} were isolated as described above, washed twice with PBS, and resuspended in PBS containing 5% FCS and 1 mm NaN3. Cells were incubated at 25°C with anti-mouse MSR1 (1 µg/1 x 106 cells) for 1 h and washed twice with PBS. Cells were resuspended in PBS containing 5% FCS and 1 mm NaN3 and incubated with anti-rabbit IgG FITC Conjugate F(ab')2 (1/1000; Sigma-Aldrich) for 40 min at 25°C. Cells were then washed twice with PBS, resuspended in PBS-EDTA, and analyzed by flow cytometry.

Data analysis

Statistical significance was determined by one-way ANOVA with Dunn’s correction or ANOVA on Ranks with Dunn’s correction or Student-Newman-Keuls method and Mann-Whitney U rank sum test or the Student t test; *, value of p < 0.05 was considered significant. Data are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Several QTLs for LPS-induced IL-10 plasma levels were identified using recombinant inbred mouse strains

We previously showed that the frequency of mortality of C57BL/6J (B6) mice was higher than that of A/J mice after injection with LPS. This difference in mortality was correlated with differences in the inflammatory response between the two strains (6). Because cytokines are excellent markers of the inflammatory process, we evaluated the plasma levels of IL-10 in A/J and B6 mice 1.5 h after LPS injection. This time point corresponds to the maximal detectable levels of this cytokine after administration of LPS. Significantly higher levels of LPS-induced IL-10 plasma levels were observed in B6 in comparison with A/J mice (Fig. 1). This analysis was performed several times over a period of a year to overcome any potential seasonal variability in LPS-induced cytokine levels. The difference in response between B6 and A/J mice was observed regardless of any seasonal variability. Analysis of the first generation (F1) between B6 and A/J mice (AXB/BXA) showed that LPS-induced IL-10 levels were similar to those of B6 mice, suggesting that this trait is neither sex-linked nor imprinted and is likely B6 dominant. Recombinant inbred (RI) mouse strains were used to map the loci contributing to these phenotypes. Male mice from the AXB/BXA set (26 RI strains) were injected with LPS and levels of IL-10 were measured (Fig. 2). The log-transformed data, weighted for variance, was analyzed by MapManager QTXb20. Several contributing loci, named modifier of IL-10 (Milt), were obtained for the IL-10 plasma level phenotype. Two loci, one on Chr 8 (Milt3) and the other on Chr 13 (Milt4), displayed significant linkages, whereas loci on Chr 2 (Milt1) and 4 (Milt2) exhibited suggestive linkages (Table I).


Figure 1
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  		FIGURE 1. IL-10 plasma levels following LPS injection in A/J, B6, and F1 generation. Male A/J (n = 45), B6 (n = 47), AXB (n = 15), and BXA (n = 15) mice were fasted for 16 h and injected i.p. with LPS (15 mg/kg). Blood was drawn via cardiac puncture 1.5 h after the injection, and IL-10 levels were measured in plasma by ELISA. IL-10 plasma levels in B6, AXB, and BXA mice were significantly higher than in A/J mice (*, p < 0.05 by ANOVA on Ranks, Dunn’s correction). Each group represents three or more experiments to account for seasonal variation.

 

Figure 2
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  		FIGURE 2. QTL analysis for differences in IL-10 plasma levels in RI mouse strains. Eight-week-old, male mice from 26 AXB/BXA RI strains (n = 5 per strain), and A/J (n = 8) and B6 (n = 9) mice, were fasted for 16 h and injected i.p. with LPS (15 mg/kg). Blood was drawn via cardiac puncture 1.5 h after the injection, and IL-10 levels were measured in plasma by ELISA. On average, five animals per strain were used for phenotyping and the experiments were performed over a 6-mo period.

 

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  		Table I. QTLs that modify the LPS-induced IL-10 response

 
We focused on the two loci that displayed significant QTL (Milt3 and Milt4). Consomic mice were used to confirm these loci for Chr 8 (B6-Chr8A/NaJ) and Chr 13 (B6-Chr13A/NaJ), respectively. These consomic mice contained the chromosome of interest from A/J and all remaining chromosomes from B6 mice (14). Observed LPS-induced IL-10 plasma levels in B6-Chr8A/NaJ mice were very similar to those of A/J mice (Fig. 3), whereas B6-Chr13A/NaJ behaved as B6 mice (data not shown). An unrelated consomic mouse strain, B6-Chr9A/NaJ, also displayed a response similar to B6 mice. These observations confirm the influence of a gene within Milt3 on levels of LPS-induced IL-10. In contrast, Milt4 may have an epistatic relationship with other genes.


Figure 3
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  		FIGURE 3. IL-10 plasma levels in consomic mice. Eight-week-old, B6-Chr8A/NaJ (n = 18), and B6-Chr9A/NaJ (n = 4), A/J (n = 35), and B6 (n = 37) male mice were fasted for 16 h and injected i.p. with LPS (15 mg/kg). Blood was drawn via cardiac puncture 1.5 h after the injection, and IL-10 levels were measured in plasma by ELISA. IL-10 plasma levels in B6 and B6-Chr9A/NaJ mice were significantly higher than A/J and B6-Chr8A/NaJ (*, p < 0.05 by ANOVA on Ranks, Dunn’s correction).

 
Refinement of Milt3 position by haplotype/single nucleotide polymorphism analysis

Based on the consomic mouse results, Milt3 was further refined because this locus can be analyzed independently of other alleles. The number of plausible candidate genes contained within the 95% confidence interval around this locus (40 cM) is very large, minimizing the chance for successful systematic analysis. Consequently, we attempted to reduce the number of candidate genes by using a haplotype comparison among inbred mouse strains (<http://snp.gnf.org>) (15). We analyzed the IL-10 response in inbred strains (AKR/J, BALBc/J, DBA/2J, 129S1/SvImJ(129/SJ)) that shared haplotype information on Chr 8 with A/J but differed from B6 mice (15). LPS-induced IL-10 plasma levels were similar among AKR/J, BALBc/J, DBA/2J, 129/SJ with respect to A/J mice and significantly lower than B6 mice (Fig. 4A). Based on the haplotype comparison among "A/J-like" strains, we were able to reduce the size of Milt3 from 40 cM to ~24 cM, decreasing the number of potential candidate genes from ~160 to 68 (Fig. 4B).


Figure 4
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  		FIGURE 4. Refining the Milt3 locus by haplotype analysis. A, Eight-week-old male mice from the inbred strains AKR/J (n = 8), BALB/cJ (n = 8), DBA/2J (n = 7), and 129S1/SvImJ (n = 6), which share haplotypes in a portion of the Milt3 region with A/J, A/J (n = 8), and B6 (n = 8) were fasted for 16 h and injected i.p. with LPS (15 mg/kg). Blood was drawn via cardiac puncture 1.5 h after the injection, and cytokine levels were measured in plasma by ELISA. IL-10 plasma levels in B6 mice were significantly higher than the other strains (*, p < 0.05 by ANOVA on Ranks, Dunn’s correction). B, Markers were chosen at sites where B6 differed from all A/J-like haplotypes. If a region contained at least one A/J haplotype similar to B6, that area was discarded. The black bar denotes areas that differed between B6 and all the A/J haplotypes. This combined region spans 24 cM.

 
Msr1 as a candidate gene

Evaluation of possible candidate genes within the reduced 24 cM region on Milt3 indicated only five genes with known polymorphisms between A/J and B6: Msr1, Rasl5–2, Nat1, Lpl, and Ucp1. Msr1 (SR-A) was chosen as the primary candidate gene for several reasons. Msr1 encodes for a cell surface glycoprotein that is specifically expressed in macrophages (16). These cells are central to the activation and regulation of the inflammatory response. MSR1 is apparently involved in the clearance of numerous substances, including LPS, pathogens, and apoptotic cells (17). Polymorphisms within the coding region of Msr1 have already been identified between B6 and A/J mice, although no functional differences were previously attributed to these allelic variants (18). To investigate the possible role of MSR1 in the differential response to LPS, Msr1–/– mice bred onto a B6 background for at least six generations (provided by Dr. M. Freeman; Ref.19) were injected with LPS, and IL-10 plasma levels were measured 1.5 h postinjection. LPS-induced IL-10 plasma levels were reduced by ~50% in the knockout mice compared with B6 mice (wild type). Moreover, LPS-induced IL-10 levels in the null mice were similar to those observed in A/J mice (Fig. 5A). In contrast, LPS-induced TNF-{alpha} and IL-6 plasma levels were similar between Msr1–/– and B6 mice (Fig. 5, B and C). These results specifically segregate the effect of MSR1 on LPS-induced IL-10 from that on other cytokines.


Figure 5
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  		FIGURE 5. Cytokine plasma levels following LPS injection in A/J, B6, and Msr1–/– mice. Male mice (8-wk old) were fasted for 16 h and injected i.p. with LPS (15 mg/kg). Blood was drawn via cardiac puncture 1.5 h (TNF-{alpha} and IL-10) or 6 h (IL-6) after the injection, and cytokine levels were measured in plasma by ELISA. A, IL-10 plasma levels in B6 mice were significantly higher as compared with A/J and Msr1–/– mice (*, p < 0.05 by ANOVA on Ranks, Dunn’s correction, n = 10 per strain). For TNF-{alpha} (B) and IL-6 (C) plasma levels, A/J mice were significantly different compared with B6 and Msr1–/– mice (#, p < 0.05 by one-way ANOVA by Student-Newman-Keuls Method for TNF-{alpha}, n = 5 per strain; and {ddagger}, p < 0.001 for IL-6, n = 8 per strain).

 
The expression of Msr1 is different between B6 and A/J mice

We compared the presence of MSR1 in isolated naive PM{phi} from A/J and B6 mice. Western blot analyses of protein lysates from PM{phi} isolated from these mouse strains were performed using Abs specific for mouse MSR1 that did not differentiate between genetic variants (13). Higher levels of MSR1 were consistently observed in B6 with respect to A/J-isolated PM{phi} (1.6-fold, p < 0.005; Fig. 6, A and B). In addition to differences in expression level, we observed a difference in the electrophoretic mobility of MSR1 between B6 and A/J mice in which the glycoprotein derived from A/J mice migrated faster (Fig. 6A). This observation raises the possibility of differential glycosylation or some other posttranslational modification between the two strains. Polymorphisms within the coding region of Msr1 (18) result in a different number of hypothetical N-linked glycosylation sites between B6 and A/J MSR1, in particular, a substitution of 198H for N. Protein extracts derived from B6 and A/J PM{phi} were treated with endoglycosydase F, which cleaves N-linked carbohydrate moieties of glycoproteins (Fig. 6C). This treatment resulted in a decrease in the apparent m.w. of MSR1, as well as sharper appearance of the band, consistent with the presence of oligosaccharides in this molecule. In addition, the difference in electrophoretic mobility between MSR1 derived from B6 and A/J mice disappeared, while the difference in the amount of MSR1 was maintained between the two strains. Finally, we investigated the presence of MSR1 by immunostaining. The cell surface localization was the same in PM{phi} isolated from A/J and B6 mice, but B6 displayed apparently higher levels of MSR1 than A/J-derived PM{phi} (Fig. 7A). This observation was further corroborated by FACS analysis of isolated A/J or B6 derived PM{phi} using the Ab specific for MSR1. We found 78% of B6 cells positive for MSR1 in contrast to 31% for A/J-derived PM{phi} (Fig. 7B). These observations provide another potential explanation for a difference in functional ability of the allelic variants.


Figure 6
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  		FIGURE 6. Msr1 protein levels in PM{phi} from A/J and B6 mice. PM{phi} from A/J and B6 mice were obtained by peritoneal lavage. Macrophages were allowed to attach for 1 h, washed with PBS, and then lysed in RIPA buffer containing PMSF and complete antiprotease mixture. A, Protein was separated on a 10% polyacrylamide gel, transferred onto PVDF membranes, and incubated with an Ab against mouse MSR1 that recognized B6 and A/J variants. B6 MSR1 migrated slower than A/J MSR1. The same blot was stripped and reprobed using Abs against beta-actin. The blot is representative of at least three experiments. B, MSR1 signal was quantitated and normalized to the corresponding signal for beta-actin. B6 protein levels were significantly higher than A/J levels (*, p = 0.003 by Mann-Whitney rank sum test, n = 10 per strain). C, Proteins (40 µg) were treated with PNGaseF (1000 U) in the presence of 10% Nonidet P-40 and G7 buffer for 75 min at 37°C and separated on a 7.5% polyacrylamide gel, transferred onto PVDF membranes, and incubated with an Ab against mouse MSR1. Notice that the difference in electrophoretic migration between A/J and B6 MSR1 disappeared whereas the difference in intensity was preserved.

 

Figure 7
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  		FIGURE 7. Comparison of MSR1 surface expression on PM{phi} isolated from A/J and B6 mice. A, PM{phi} isolated from A/J and B6 mice were harvested by peritoneal lavage and allowed to attach to glass coverslips. Cells were immunostained with an anti-mouse MSR1 Ab and visualized by Cy3 IgG F(ab')2. Figure depicts a representative field under x100 objective. B, PM{phi} isolated from A/J and B6 mice were harvested by peritoneal lavage and analyzed by FACS. Top row, Cells stained with anti-mouse MSR1 and as a secondary anti-rabbit IgG FITC conjugate F(ab')2; bottom row, with secondary alone. Numbers in the top right quadrant represent number of cells gated that were positive for MSR1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We used QTL mapping in RI mouse strains as a direct approach to the identification of modifier genes of the inflammatory process, in particular, LPS-induced IL-10 plasma levels. IL-10 is an important component of the regulation of the inflammatory response. Four loci were identified by this approach, with only two, Milt3 (Chr 8) and Milt4 (Chr 13), displaying statistically significant levels. Milt3 was confirmed to be involved in the regulation of LPS-induced IL-10 levels by using recently developed consomic mice (14). In contrast, Milt4 could not be confirmed using the respective consomic mice, probably because it is involved in an epistatic relationship with other genes. Previous studies have mapped QTL to the same region on mouse Chr 8 for susceptibility to arthritis (20), colitis (21, 22), and experimental allergic encephalomyelitis (23). It is likely that these diseases share an inflammatory component with endotoxemia. The boundaries of Milt3 were delimited based on SNP haplotypes (<http://snp.gnf.org>; Ref.15) and only five genes within this region were found to display polymorphisms (<www.informatics.jax.org>) between A/J and B6 mice. Of these, Msr1 was selected as a candidate gene because it is specifically expressed in cells involved in the inflammatory process (i.e., macrophages). In addition, previous studies have suggested that Msr1 plays a role in the inflammatory response (24, 25, 26).

The involvement of Msr1 in LPS-induced IL-10 plasma levels was sustained by using homozygote knockout mice, which display LPS-induced IL-10 levels similar to A/J and different from B6 (wild-type) mice. These knockout mice were generated using 129-derived ES cells (A3-1), and were backcrossed to B6 for at least six generations, providing a 99.2% B6 genomic background. Although we cannot disregard the possibility that other genes within the congenic area are involved in the regulation of LPS-induced IL-10 levels, the potential contribution of genes within this 129 congenic region is less likely because 129 mice showed a different LPS-induced cytokine profile (TNF-{alpha} and IL6) than B6, A/J, and Msr1–/– mice.

MSR1 is an integral plasma membrane glycoprotein composed of three identical chains forming a collagenase-like structure. This glycoprotein is involved in the clearance of polyanionic macromolecules, including acetylated or oxidized low density lipoprotein, lipoteichoic acid, lipopolysaccharides, polynucleotides, and sulfated polysaccharides (17). In addition, MSR1 has been implicated in cell adhesion (27, 28) and the clearance of bacteria (24) and apoptotic cells (29). MSR1 is almost exclusively expressed on macrophages (16), which are the primary mediators of the inflammatory process. Polymorphisms within the Msr1 coding region have been reported between B6 and many other mouse strains, including A/J (18). We found that one of these polymorphisms resulted in a different number of N-glycosylation sites between the B6 and A/J variants, which may modify the function of this receptor. For example, an extra glycosylation site could affect the affinity of MSR1 for one or more of its several ligands or change the stability of the protein. The number of glycosylation sites observed in the B6 variant is conserved within other species, including humans (18). In addition, the content of MSR1 is higher in B6-derived PM{phi} than in A/J cells. Thus, the IL-10 hyperresponse observed in B6 may be related to elevated abundance of MSR1. This explanation is consistent with decreased LPS-induced IL-10 levels in the knockout mice.

We have correlated a hyperinflammatory response, in particular IL-10 levels, with an increase in mortality of B6 mice after endotoxic (6) and septic shock (8, 9), respectively. Other investigations have shown a correlation between overexpression of IL-10 and a detrimental outcome after infection (30). However, exogenous administration of IL-10 has been shown to be protective against LPS by down-regulating proinflammatory cytokines (3, 4). Kobayashi et al. (25) showed that the deletion of Msr1 resulted in protection from endotoxic shock, which was correlated with direct binding to LPS. This observation is consistent with the decrease in IL-10 levels observed in Msr1-deficient mice with respect to B6 wild-type mice. In contrast, Haworth et al. (31) showed that Msr1 knockout mice were more resistant to LPS after they were primed with bacillus Calmette-Guérin. The discrepancy between the studies could stem from the fact that the insults used were not identical, and that the genetic backgrounds of the null mice used in the two studies differed. In addition, these knockout mice have mixed genetic backgrounds of 129 and ICR due to a limited backcrossing strategy. In contrast, we used knockout mice that had been backcrossed to B6 mice for six generations, resulting in a more homogenous genetic background (19). Moreover, we did not find a correlation between TNF-{alpha} or IL-6 and Msr1, as proposed by others (31, 32). We are, however, in agreement with the results of studies that used the same knockout mice (33), indicating that MSR1 does not play a direct role in the TNF-{alpha}-signaling pathway.

Internalization of Brucella spp. was reported to be mediated by MSR1 and blocked by the addition of LPS from the same species or from Salmonella, but not from E. coli LPS (24). Msr1 knockout mice displayed decreased protection from infection with Listeria monocytogenes and herpes virus (26), which correlates with A/J (susceptible) and B6 (resistant) phenotypes for these two infectious agents (34). These findings, and our own observations, strongly support a role for MSR1 in host defense and innate immunity. MSR1 has also been implicated in the pathological deposition of cholesterol during atherogenesis (35). Consistent with these results, A/J mice have been found to be more resistant than B6 mice in experimental models of atherosclerosis, diabetes, and obesity (34). These observations also suggest a possible link between MSR1 and IL-10 in many diseases besides sepsis.

MSR1 has also been implicated in the clearance of apoptotic cells (29), the appearance of which in immune organs (i.e., spleen and thymus), intestinal epithelial, and liver is a hallmark of sepsis (36). Therefore, it could be speculated that cells expressing different Msr1 genetic variants could clear apoptotic cells with different efficiencies. The prolonged presence of apoptotic cells may result in postapoptotic necrosis that induces a secondary inflammatory response, which may lead to the hyperinflammatory stage characteristic of septic shock. Recently, polymorphisms have been detected within the human MSRI gene and correlated with the incidence of prostate cancer. The role of MSR1 in cancer may be related to observations indicating the infiltration of macrophages within the tumor (37). Thus, MSR1 appears to be involved in the inflammatory response associated with cancer. The involvement of MSR1 in sepsis introduces a possible new pathway in the septic process and contributes to a better understanding of the molecular mechanisms involved in this condition. Future research into the pathway involving MSR1 may reveal innovative therapeutic approaches to ameliorate the detrimental consequences of sepsis. Moreover, Msr1 may emerge as a genetic marker for the incidence of endotoxemia, sepsis, and related conditions. Finally, our study demonstrates the feasibility of using QTL to identify new components (genes) that are involved in the inflammatory process, which may be used as genetic markers to recognize a human population at risk for developing sepsis after trauma and infection.


   Acknowledgments
 
We thank Dr. Mason Freeman for his generous provision of Msr1 knockout mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by grants from the National Institutes of Health (GM50878 and GM62599). Back

2 Address correspondence and reprint requests to the current address of Dr. Antonio De Maio, Department of Surgery, University of California, San Diego, 9500 Gilman Drive, #0739, La Jolla, CA 92093-0739. Email address: ademaio{at}ucsd.edu Back

3 Abbreviations used in this paper: QTL, quantitative trait loci; Chr, chromosome; Msr1, macrophage scavenger receptor 1; PM{phi}, peritoneal macrophage; LRS, likelihood ratio statistic; LOD, logarithm of the odds; RI, recombinant inbred; Milt, modifier of IL-10; PVDF, polyvinylidene difluoride. Back

Received for publication October 5, 2005. Accepted for publication December 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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