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*
Departments of Medicine and Genetics and
Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104; and
Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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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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Vitamin D-binding protein (DBP) is a highly expressed, polymorphic serum protein with functions ranging from the transport of vitamin D metabolites to roles in the immune system, host defense, and neutrophil chemotaxis (reviewed in Refs. 6, 7, 8). Recent studies from our laboratory have addressed central issues of DBP in the transport, metabolism, and function of vitamin D using a DBP-deficient (DBP-/-) mouse model (9). In addition to these well-documented vitamin D-related functions, a body of literature suggests that DBP also plays a pivotal role in innate immunity. In particular, DBP has been reported to augment chemotaxis of neutrophils, monocytes, and fibroblasts when studied ex vivo (10, 11, 12). This effect has been attributed to a C5a cochemotactic activity of DBP that requires binding of DBP to chondroitin sulfate proteoglycans on the cell surface of leukocytes (13, 14, 15, 16). In light of these reports, we set out to test the role of DBP in recruitment of inflammatory cells in vivo using the DBP-/- mouse model.
Localized injection of thioglycollate medium (TG) into the peritoneum of test animals has been established as a straightforward and effective approach in examining inflammatory cell recruitment and for isolation of peritoneal macrophages (17, 18) and neutrophils (19). When TG is injected into the peritoneal cavity of mice or rats it induces localized sterile inflammation with subsequent recruitment of leukocytes in a characteristic, cell-specific pattern. Thermal "aging" of TG has been reported to increase its inflammatory effect due to the formation of advanced glycation end products that can accentuate cell recruitment (20). In contrast to other inflammatory agents such as LPS, TG can recruit large numbers of peritoneal leukocytes without activating microbicidal activity (21). The cell recruitment triggered by TG appears to operate via a complement-independent pathway, although the exact mechanism(s) of this established model of innate immune response remain poorly defined.
In the following study, we have used the TG experimental model to assay the putative role of DBP as a neutrophil or macrophage recruitment factor. The data argue against a unique role for DBP in TG-elicited peritoneal neutrophil or macrophage recruitment in vivo, and instead they reveal an unanticipated and profound defect on cell recruitment that reflects complex genetic determinants and is specific to the commonly used 129X1/SvJ isogenic mouse strain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD-1 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA); all other strains where purchased from The Jackson Laboratory (JAX mice; Bar Harbor, ME). The animals were housed in Jag-75 plastic cages with wood shavings in a high efficiency particulate air-filtered barrier unit maintained at 25°C with alternating 12-h periods of light and dark. The mice had access to drinking water at all times and were fed ad libitum with an autoclaved pelleted rodent diet. The great majority of mice used were between 6 and 12 wk of age at the time of the experiment. Four strains of mice were used.
129X1/SvJ. The 129X1/SvJ inbred mouse strain originated from crosses established in 1928. It is the strain most commonly used to generate ES cells for gene knockout studies (22).
C57BL/6J. C57BL/6J is widely used, accounting for 14% of all occasions in which an inbred strain has been used in the literature (http://www.informatics.jax.org).
BALB/c. The BALB/c inbred mouse strain is commonly used in the field of immunology (23).
CD1. CD1 is a commonly used albino, outbred mouse strain originating from Switzerland.
Thioglycolate medium
Two variants of TG were tested: ready-to-use, aged TG from stored at 4°C (BD Biosciences, Franklin Lakes, NJ), and dehydrated Brewer TG medium (Difco, Detroit, MI). The latter was taken up as a 3% solution (30 g of TG powder in 1000 ml of deionized water) and was autoclaved 20 min at 15 lbs/psi (121°C). The sterile 3% TG was protected from light and was "aged" at room temperature for at least 1 mo to ensure formation of advanced glycation end products (20). Preprepared and laboratory-prepared TG were identical in their abilities to trigger peritoneal recruitment of macrophages in all four mouse strains studied (data not shown). All data presented in this report were obtained through the use of preprepared TG medium.
Peritoneal cell recruitment
For recruiting peritoneal cells, 1 ml of sterile TG medium (warmed to room temperature) was injected into the peritoneal cavity of male or female mice using 23-gauge needles. For neutrophils, harvesting of peritoneal cells 4 h post-TG injection has been recommended for optimal yield (19). For macrophages, harvesting of peritoneal cells 3–5 days post-TG injection has been shown yield optimal numbers (20, 21, 24). After the appropriate amount of time, mice were CO2 asphyxiated and cells were harvested by intraperitoneal lavage. Seven milliliters of ice-cold Dulbecco’s PBS (DPBS) in a 10-ml syringe with a 23-gauge needle was injected into the lower left quadrant of the exposed peritoneal cavity, close to the abdominal fat pad. Injection was conducted through the fat pad because this approach seals the puncture hole, preventing loss of DPBS-containing cells. The peritoneal compartment was gently massaged and agitated for 30 s, then the injected fluid was slowly withdrawn into the same syringe with a 23-gauge needle via a puncture made in the upper right quadrant (avoiding the spleen). At the point when the needle became blocked, it was removed and any remaining fluid was withdrawn from the opposite side of the abdomen. If at any point the gut or membrane was ruptured, the sample was discarded. Using this method, recovery was consistently 6.7–6.9 ml (>95% of instilled volume). The harvested cells were transferred to 15-ml Falcon tubes and were centrifuged at 200 x g for 10 min at 4°C. To ensure an accurate white cell count, the small number of contaminating erythrocytes was selectively lysed in RBC lysing buffer (8.3 g/L ammonium chloride, 0.01 M Tris-HCl pH; catalog no. R7757; Sigma-Aldrich, St. Louis, MO). Cells were suspended in 1 ml of RBC lysing buffer and they were incubated at room temperature for 10 min. The buffer was diluted with 14 ml of DPBS and was centrifuged at 200 x g for 10 min at 4°C. The supernatant was discarded and the white blood cell pellet was resuspended in 1 ml of DPBS for immediate counting. Duplicate 10-µl samples were counted manually using a hemocytometer. The average of the two counts was recorded as the total number of cells harvested from that mouse. Based on the total cell count, 1 x 105 cells were suspended in 200 µl of DBPS containing 4% BSA. The entire 200 µl was then loaded into a cytospin chamber and centrifuged at 500 x g for 5 min at room temperature in a cytocentrifuge. Slides were Wright-Giemsa stained (Diff-Quick; Dade Behring, Newark, DE) and differential cell counts were performed.
Statistical analysis
Significance of differences were tested using Student’s paired t test. Probabilities were considered significant at the 5%, 1%, and 0.1% levels. Results are expressed as mean values ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A previously described DBP-null (DBP-/-)
mouse model (9) was used to evaluate the in vivo role of
DBP in an established leukocyte recruitment model. An established model
of cell recruitment to a site of chemical inflammation was used for
this analysis. Aged TG broth was injected into the peritoneal cavity of
the mouse and the subsequent recruitment of inflammatory cells was
quantified at defined time points. A comparison was conducted between
the responses in DBP-/- and DBP-positive
(DBP+/+ or wild-type (WT)) mice. The pilot
studies used DBP-/- and
DBP+/+ mice with mixed genetic backgrounds that
had been generated in the following manner. The DBP gene deletion was
generated by homologous recombination in 129X1/SvJ-derived ES cells
(R1; Ref. 25) and the selected ES clone was injected into
C57BL/6J blastocysts. The chimeric progeny of these injected
blastocysts were mated to CD1 mice to generate the
F1 generation.
F1(DBP+/-) mice were then
intercrossed to generate DBP-/- mice. To
minimize the need for repeated genotyping, the
DBP-/- mice were generated by
DBP-/- x DBP-/-
matings to maintain a DBP-/- stock for
analysis. In parallel, DBP+/+ controls were
generated by parallel mating to CD1 outbred mice. Thus, the initial
series of studies was conducted with DBP-/- and
DBP+/+ mice that were on mixed genetic
backgrounds of undetermined relationship to each other due to this
period of segregating matings. Mice from the
DBP-/- and DBP+/+ groups
were injected with TG and the peritoneal cells were harvested 4 days
later (Materials and Methods). There was a 10-fold
difference (p < 0.001) in the cell recruitment
between the two groups (Fig. 1
A, Mixed). In
fact, there was no significant recruitment in the
DBP-/- mice at this time point; the total
numbers of cells in the peritoneal cavity of the TG- and
saline-injected DBP-/- mice at 4 days were
equivalent. In these pilot studies, the data thus demonstrated a total
and selective inability of the DBP-/- mice to
recruit cells to the peritoneum in response to TG.
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A, OX1-OX4). The
impact of the outcrosses on cell recruitment was striking. As noted
above, the pilot analysis, using mice with mixed genetic backgrounds,
showed a 10-fold lower recruitment by the
DBP-/- mice when compared with controls (Fig. 1, Mixed). Analysis of OX1 generation mice revealed that the
recruitment deficit in the DBP-/- mice was
maintained when compared with matched DBP+/+
mice, but to a diminished degree (3-fold; p < 0.005).
At OX2, the difference between the two groups was slight but not
significant; the DBP-/- and
DBP+/+ groups both increased their peritoneal
cell count 
4-fold over that seen with saline. This equivalence of
cell recruitment in the two groups continued with the OX3 and OX4
groups. Of additional interest was a steady increase in overall cell
recruitment at each successive outcrossed generation (OX1 through OX4)
in both the DBP-/- and
DBP+/+ groups. By OX4, the overall recruitment
potential in the DBP+/+ and the
DBP-/- groups increased 4- and 20-fold,
respectively, over the corresponding groups with the mixed genetic
backgrounds. Thus, the progressive outcrossing of the genetically mixed
DBP-/- mice to the C57BL/6J strain revealed a
progressive gain of function in peritoneal cell migration as the
breeding proceeded from OX1 through OX4. These data clearly
demonstrated that the recruitment defect observed in the initial
studies was not linked to the DBP locus.
The loss of the apparent DBP-mediated defect in inflammatory cell
recruitment during the outcrossing to the C57BL/6J strain and the
corresponding overall increase in cellular recruitment suggested that
the defect originally detected might be related to the 129X1/SvJ
genetic background. To confirm and extend this observation, we directly
compared the TG-stimulated recruitment potential in the 129X1/SvJ and
C57BL/6J isogenic strains (Fig. 1B). Four days after TG
stimulation, the C57BL/6J strain responded with a 12-fold increase in
peritoneal cells over saline-injected controls compared with a total
lack of increase in the 129X1/SvJ strain (p <
0.001). Thus, the 129X1/SvJ line demonstrated a marked defect in
inflammatory cell recruitment.
To formally determine whether the difference in the cell recruitment in the C57BL/6J strain and the 129X1/SvJ strain reflected the action of a single dominantly acting "cell recruiter" gene, the two isogenic strains were crossed and cell recruitment was measured in the F1 generation. These F1 mice, obligate heterozygotes for 129X1/SvJ and C57BL/6J alleles at all loci, demonstrated a recruitment level that was midway between the two parental strains (data not shown). Thus, the observed recruitment phenotypes cannot be explained solely on the simple basis of a dominantly acting cell recruiter gene. Such a situation would have resulted in all F1 mice having a recruitment phenotype equivalent to that of the C57BL/6J strain. Therefore, the difference in cell recruitment between the C57BL/6J and 129X1/SvJ strains must reflect codominant or more complex genetic mechanisms.
Complex genetic regulation of the 129X1/SvJ recruitment defect: distinct levels of peritoneal cell recruitment in four commonly used mouse strains
To further define the relationship of cell recruitment to mouse
strain, we analyzed the recruitment response to TG in four of the most
commonly used mouse strains: isogenic strains C57BL/6J, BALB/c, and
129X1/SvJ, and outbred CD1. Mice from each strain were injected with 1
ml of 3% TG, and peritoneal cells were harvested after 4 days. The
total cell recruitment in each strain was strikingly different (Fig. 2A); C57BL/6J was the most
responsive, followed by BALB/c, CD1, and the least responsive was the
129X1/SvJ strain. All pairwise comparisons revealed highly significant
differences (p < 0.001). Differential cell
counts were established on the recruited cells in each strain (Fig. 2B). These data revealed that macrophages formed the
majority of the cells recruited to the peritoneum after 4 days
(70–90%). Macrophage numbers closely paralleled the total cell
counts.
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To characterize the difference in recruitment phenotypes in
greater detail, a time course of cell recruitment was conducted in the
C57BL/6J and 129X1/SvJ strains (Fig. 3, A–C). Twenty-eight mice of each strain were
injected with TG, and peritoneal cells from four mice of each strain
were harvested at 0 and 4 h postinjection and on days 1, 2, 3, 4,
and 5. Total and differential cell counts were determined at each time
point. Before injection with TG, resident cell counts were similar in
the two strains. At 4 h post-TG injection, the total cell counts
increased 2- to 3-fold in both strains. Of note, the majority of cells
in both groups at 4 h were neutrophils. Macrophages did not appear
in significant numbers in either group until day 1. In the 129X1/SvJ
strain, the total cell counts were at their highest 4 h post-TG
injection, and fell progressively at subsequent time points. In
contrast, the total cell count in the C57BL/6J strain rose steadily
from 4 h through 2 days, plateaued from 2 to 4 days, and then
began to decrease on day 5. During this period, the differential cell
count of the cell population in the C57BL/6J peritoneal fluid shifted
from predominantly neutrophils at 4 h and at 1 day post-TG
injection to an increasingly predominant proportion of macrophages at
2, 3, 4, and 5 days (70%, 85%, 97%, and 99%). A similar shift from
neutrophils to macrophages was seen in the 129X1/SvJ mice, although
overall cell numbers were much lower and the levels dropped earlier.
During the entire time course, a small number of lymphocytes were also
seen; the absolute number of lymphocytes was low at all times and
showed a higher degree of variability (data not shown). Thus, there was
an equivalent recruitment of neutrophils in both groups in response to
TG at early time points postinjection. The major and sustained
macrophage response seen in the C57BL/6J mice did not occur in the
129X1/SvJ mice. Thus, the defect in inflammatory cell recruitment is
not global, but appears to be limited to the macrophage/monocyte
lineage.
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There was no defect in peritoneal monocyte/macrophage recruitment
attributable to DBP because no significant differences in cell counts
at 4 days postinjection in OX2 through OX4 generations were observed
(Fig. 1A). Although strain-related leukocyte recruitment
kinetics differed markedly, the absolute level of neutrophil
recruitment was not significantly different in C57BL/6J and 129X1/SvJ
mice at 4 h after TG injection (Fig. 3C). Therefore, to
determine whether the DBP allele contributed to peritoneal neutrophil
recruitment, cells from DBP-/- and
DBP+/+ mice from OX3 were studied at 4 h
post-TG injection. Mean and SEM neutrophil counts in
DBP+/+ and DBP-/- groups
were (11.4 ± 1.47) x 106 and
(8.8 ± 0.98) x 106 cells/ml,
respectively (n = 5). These differences were not
significant (p = 0.163). Thus, no significant
role could be attributed to DBP in either the macrophage or neutrophil
recruitment in this experimental model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observed defect in cell recruitment in the 129X1/SvJ strain was unanticipated and would predict an increased susceptibility to certain infectious diseases. This prediction is supported by two observations. In a study by others of the resistance of different mouse strains to Mycobacterium tuberculosis infection, 129X1/SvJ mice were found to be highly susceptible, whereas C57BL/6 mice were highly resistant to infection (27). Significantly, clearance and killing of M. tuberculosis is a macrophage-dependent process, and the observed defect in resistance to M. tuberculosis infection parallels the defect in macrophage recruitment to TG-induced inflammation detected in the present study. A set of data from our own laboratory further supports the significance of the current study. We have observed a marked decrease in the rate of in vivo clearance of the intracellular parasite Leishmania donovani in "mixed" genetic background DBP-/- mice (P. White, M. Murphy, S. Liebhaber, J. Farrell, and N. Cooke, manuscript in preparation). Remarkably, this defect in L. donovani clearance is lost upon outcrossing the DBP- allele to the C57BL/6J strain. Thus, observations regarding resistance to M. tuberculosis and L. donovani suggest that the cell recruitment defect identified in the 129/SvJ strain using the TG-stimulated inflammation model reflects physiologically relevant defects in macrophage recruitment and/or function.
The observed contribution of the 129X1/SvJ genome to the phenotype of
the DBP-/- mice in the initial analysis (Fig. 1A, Mixed) can be traced back to the fact that the DBP gene
mutation was originally introduced in 129X1/SvJ ES cells. These cells
were then injected into a C57BL/6J blastocyst. The subsequent mating of
the chimeric founder mouse to a C57BL/6J mouse resulted in
DBP+/- progeny that carried one set of 129X1/SvJ
genes and one set of C57BL/6J genes. Interbreeding these
DBP+/- mice was conducted to generate progeny
that were homozygous null, heterozygous null, and WT at the DBP locus.
These mice had recombinant genotypes from the two parental strains;
certain loci (including the disrupted DBP allele) were derived from
129X1/SvJ strain and others from C57BL/6J strain. Thus, the WT
littermates did not constitute an optimal control population for the
DBP-/- mice because the non-DBP alleles in each
mouse differed. Whereas expanding the population size under study would
provide statistical power and minimize this problem, this approach is
not able to fully randomize the alleles. Furthermore, genes closely
linked to the DBP locus would be unlikely to segregate from the
disrupted DBP allele and would therefore remain largely of 129X1/SvJ
origin. This was of particular relevance in the present situation
because peritoneal leukocyte recruitment is considered to be dependent
upon local chemokine gradients (28), and the human DBP
gene is closely linked to a cluster of nine CXC chemokine genes,
including IL8 and the GRO1, GRO2, and
GRO3 genes (29, 30). Significantly, the defect
in cell recruitment in the DBP-/- mice in the
initial analysis was indistinguishable from that seen in the 129X1/SvJ
strain (Fig. 2A). The recruitment profiles in the
DBP+/+ and DBP-/- groups
were indistinguishable from each other after several rounds of
outcrossing of these DBP-/- mice to the
C57BL/6J strain. These data are fully consistent with the conclusion
that the initial recruitment defect in the
DBP-/- mice reflected their 129X1/SvJ genetic
content and not DBP or a DBP-linked CXC chemokine gene.
What is the genetic basis for the recruitment defect in the 129X1/SvJ
mice? The intermediate level of recruitment in the
F1 progeny of the 129X1/SvJ and C57BL/6J
intercross (see Results) demonstrated that the inflammatory
response was not controlled by a single dominantly acting "recruiter
locus." Three inbred lines have three distinct levels of recruitment
(Fig. 2), allowing us to further conclude that the trait is controlled
by three or more alleles at a single locus or that the trait is
polygenic. The graded levels of recruitment with each outcross of the
initial mouse line to the C57BL/6J strain (Fig. 1A) further
supports the conclusion that the trait is the product of a complex set
of determinants that, in sum, result in a net recruitment level.
Definitive assignment of the genetic basis for the distinct cell
recruitment phenotypes will of course be based on subsequent genetic
and biochemical mapping studies.
Our examination of cell recruitment over a 5-day period revealed
strain-specific effects in the kinetics of leukocyte recruitment.
Previous reports indicated that peak recruitment of peritoneal
neutrophils was observed 4 h post-TG injection (19).
We found this to be true for 129X1/SvJ, but the more robust neutrophil
response in C57BL/6J occurred later (24 h). Because the neutrophil
recruitment remained essentially intact except for this kinetic shift,
it is likely that generally acting, receptor-linked chemoattractant
pathways and the actin cytoskeletal structures remain intact. Mutant
genes in such pathways would be expected to result in global defects in
the inflammatory cell recruitment phenotype, and the defect observed
was remarkably limited to the macrophage/monocyte lineage. For
macrophages, the peak response has typically been reported to occur
3–5 days post-TG injection in studies using Sprague Dawley rats, Swiss
mice, or C3H/HeN mice (20, 21, 24). In contrast, the few
macrophages that were recruited to the peritoneum in the 129X1/SvJ
strain appeared much earlier (2 days). The kinetic differences for
these two cell types, as well as the distinct quantitative responses in
the various inbred lines, are consistent with a complex genetic basis
of the overall inflammatory cell recruitment process.
The results of this current study highlight the need for careful
outcrossing to establish reliable control groups for targeted knockout
lines before phenotypic characterization. This is of particular
importance in dealing with complex genetic phenotypes such as those
involving the immune system. The present study emphasizes that this
need is of specific importance in the analysis of peritoneal leukocyte
recruitment phenotypes. In a survey of the literature, we have
identified a set of published papers in which a peritoneal recruitment
defect in response to TG has been attributed to the targeted
interruption at each of four specific loci. These loci include the
CCR-2 gene (31, 32), the monocyte chemoattractant
protein-1 gene (33), the plasminogen gene
(34), and the Duffy gene (35). It is
noteworthy that in each case, the gene disruption was introduced by
homologous recombination into 129 ES cells that were implanted in a
C57BL/6 blastocyst, as in the current study. In addition, in each case,
the 129X1/SvJ x C57BL/6 mixed genetic background mice homozygous
for the null allele were demonstrated to have 2- to 10-fold lower cell
counts than WT controls 3–4 days after i.p. TG injection, whereas the
4-h neutrophil counts were unaffected. These results are remarkably
similar to those observed in our initial analyses of the
DBP-/- mice obtained on mixed genetic
backgrounds before outcrossing to the C57BL/6J (Figs. 1 and 3C). Of note, in none of these studies were backcrosses into
a purebred strain undertaken to be certain that the recruitment
phenotype reflected the biologic effect of the gene knockout as opposed
to a genetic background effect of the 129X1/SvJ strain. In light of our
present finding that the 129X1/SvJ strain has a profound deficit in
macrophage recruitment, it is reasonable to suggest that
genotype-phenotype conclusions based on observations in these studies,
and others like them, will require careful reconsideration. In
addition, the demonstrated strain-dependent differences in the cell
recruitment process may be used to advantage in future studies to
dissect the various genetic determinants of this innate immune
function.
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Acknowledgments |
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Footnotes |
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2 Current address: RHeoGene LLC, P.O. Box 949, Spring House, PA 19477-0949.
3 Address correspondence and reprint requests to Dr. Nancy E. Cooke, Division of Endocrinology, Diabetes and Metabolism, Departments of Medicine and Genetics, University of Pennsylvania School of Medicine, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104-6149. E-mail address: necooke{at}mail.med.upenn.edu
4 Abbreviations used in this paper: ES, embryo stem; DBP, vitamin D-binding protein; TG, thioglycolate medium; DPBS, Dulbecco’s PBS; WT, wild type.
Received for publication August 29, 2001. Accepted for publication November 6, 2001.
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