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Gill Heart Institute, Division of Cardiovascular Medicine, University of Kentucky, Lexington, KY 40536;
Cardiovascular Division, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;
Department of Vascular and Cardiac Diseases, Parke-Davis, Ann Arbor, MI 48106; and
§
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02155
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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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12/15-LO expression in cultured monocyte-derived macrophages (MDMs) is
regulated by the presence of exogenously applied cytokines. Cultured
human MDMs do not express 15-LO unless they are incubated with IL-4 or
IL-13 (5). Both of these cytokines are released from the Th2 subtype of
CD4+ helper cells (6). IL-4- and IL-13-stimulated 15-LO
expression in cultured MDMs is inhibited by IFN-
that is secreted
from Th1 cells (7, 8). Unlike human MDMs, mouse peritoneal macrophages
express considerable 12/15-LO activity at the time of their isolation.
The expression of the enzyme in mouse peritoneal macrophages is not
altered in mice lacking IL-4; this lack of alteration is possibly due
to redundancy in the cytokine system, since the same effects are
observed with IL-13 (5). IFN- does not have a profound effect on the
12/15-LO activity that is present upon isolation in mouse peritoneal
macrophages, indicating that it probably exerts its inhibitory effects
through a transcriptional effect (5, 9). While 12/15-LO activity has
been detected in mouse peritoneal macrophages at the time of isolation,
the enzyme is not expressed uniformly in every cell (10).
Since it has been hypothesized that IL-4 and IL-13 released from activated lymphocytes regulate the expression of 12/15-LO, we hypothesized that the total absence of mature lymphocytes would influence the activity of this enzyme. To address this hypothesis, we quantified 12/15-LO expression in mice with specific immune deficiencies. One strain used was recombinase activator gene (RAG)-2-/- mice that are incapable of V(D)J recombination, leading to a total deficiency of mature T and B lymphocytes (11). This immune deficiency also leads to an absence of the TCR and ablates lymphocyte activation and its associated cytokine secretion. To determine whether 12/15-LO originated from nonlymphoid sources of IL-4 or IL-13, STAT6-/- mice that do not respond to either cytokine were used. These data demonstrate that IL-4 and IL-13 secretion is not a requirement for macrophage 12/15-LO synthesis in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strain-matched, 5- to 7-wk-old RAG-2+/+ and RAG-2-/- mice were purchased from Taconic (Germantown, NY); STAT6-/- mice and strain-matched controls have been described previously (12). All mice were fed a standard laboratory diet and given water ad libitum.
Isolation of resident peritoneal macrophages
Mice were anesthetized with metaphane and killed via cervical dislocation. Macrophages were harvested via peritoneal lavage using saline (5 ml). Cells were resuspended in DMEM (catalog no. 10-013-LV; Mediatech, Washington, DC) containing heat-inactivated FBS (10% v/v), penicillin (10 U/ml), streptomycin (10 µg/ml), and fungizone (25 ng/ml), and plated into 6-well plates (Corning Costar, Cambridge, MA). Cells were incubated at 37°C with 5% CO2, 95% air for the times indicated. After incubation, media was removed, and cells were prepared for protein characterization or immunocytochemistry. For protein characterization, cells were washed twice with cold PBS and scraped into PBS (1 ml); aliquots were recovered for cell counting and for the determination of protein concentration (13). The remaining cells used for the assessment of enzyme activity were centrifuged at 13,000 x g for 30 s at room temperature; supernatants were subsequently removed, and cell pellets were frozen in liquid nitrogen and stored at -80°C.
The macrophages used for immunocytochemistry were isolated as described above and plated at an equivalent density onto Lab-Tek 8-chamber glass slides (Nunc, Naperville, IL). After incubation and washing, cells were fixed with paraformaldehyde (4% w/v) and permeabilized with ethanol at 4°C.
Western blot analyses
Total cell proteins from mouse peritoneal macrophages were resolved under reducing conditions on 10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The immunoreactivity of the proteins on the membranes was assessed using sheep antiserum to rabbit reticulocyte 15-LO, which recognizes the murine homologue 12/15-LO, as described previously (5). Immunoblots were simultaneously incubated with a mAb to ß-actin (Sigma, St. Louis, MO). Horseradish peroxidase-conjugated Abs to sheep and mouse IgG were used for detection in combination with chemiluminescent reagents according to the manufacturer’s instructions (enhanced chemiluminescence, Amersham, Arlington Heights, IL). The intensity of the immunoreactive band for 12/15-LO was compared with that for ß-actin within each sample by image analysis (SigmaGel, Jandel Scientific, San Rafael, CA).
Measurement of 12/15-LO products
The activity of 12/15-LO in cells was assessed as described previously (5). Briefly, cell pellets were sonicated in PBS containing 0.2% sodium cholate. Aliquots (100 µl) of the sonicated cell suspension were incubated with 360 µM of linoleic acid that was dissolved in a small volume of ethanol. The reaction was allowed to proceed on ice for 10 min and was stopped by the addition of an equal volume of mobile phase (acetonitrile/water/methanol/acetic acid; 350:250:150:1). The hydroperoxides produced were reduced to their corresponding hydroxy acids by the addition of trimethyl-phosphite. Products were identified and quantified by reverse phase HPLC against known standards. Additional studies have demonstrated that 13-hydroxyoctadecadienoate (13-HODE) measured by this method is of the (S) configuration and therefore accurately measures enzymatic activity.
Elicitation of peritoneal macrophages and incubation with IL-4
Macrophages were elicited by i.p. injection of a suspension of Sephacryl beads (1 ml, 2% w/v; Pharmacia, Piscataway, NJ) or thioglycolate (1 ml, 3% w/v; Sigma). At 4 days after the administration of Sephacryl or thioglycolate, macrophages were isolated by peritoneal lavage and cultured onto glass slides for 3 h as described above. Some cells from Sephacryl-treated and nontreated mice were washed and cultured for 2 days with fresh media containing murine rIL-4 (10 ng/ml; R&D Systems, Minneapolis, MN).
Immunocytochemistry and cell size analyses
Immunocytochemistry was performed using Vector avidin-biotin complex Elite kits (Vector Laboratories, Burlingame, CA) as described previously (14). Immunoreactivity was visualized using the red chromagen, aminoethyl carbazole (Biomeda, Foster City, CA). Macrophages were detected using rabbit antiserum to murine macrophages (Accurate, Westbury, NY) and 12/15-LO with sheep antiserum to rabbit reticulocyte 15-LO. The immune serum was compared with the appropriate nonimmune serum in each case. Cell size was calculated with ImagePro (Media Cybernetics, Silver Spring, MD) software using images of cells that had been immunostained with the macrophage antiserum.
Statistical analyses
Analyses of data for the determination of statistical significance were performed with SigmaStat (Jandel Scientific). Data that met appropriate criteria of equivalent variances and normal distribution were analyzed with the Student t test (two-tailed), while those that did not were analyzed by the Mann-Whitney rank-sum test. Data are expressed as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mass of 12/15-LO and ß-actin protein in individual
peritoneal macrophage preparations from RAG-2-/- and
RAG-2+/+ mice was determined by Western blotting. 12/15-LO
and ß-actin were detected in each sample by the simultaneous use of
sheep antiserum to rabbit reticulocyte 15-LO and a mAb to ß-actin. In
each of five mice per group, 12/15-LO and ß-actin were detected at
71 and 42 kDa, respectively, which was consistent with the
expected molecular mass of these proteins (Fig. 1
, inset). The intensity of the 12/15-LO
band was compared with that of ß-actin within each sample using image
analysis. Peritoneal macrophage populations from RAG-2-/-
mice had a significantly higher 12/15-LO/ß-actin ratio per cell than
those from RAG-2+/+ mice (p <
0.006; Fig. 1).
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). A previous analysis of
product chirality from this assay revealed a predominance of the S
isomer, which is consistent with enzymatic activity.
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To visualize the distribution of 12/15-LO protein in
peritoneal macrophage populations, immunocytochemical tests were
performed on cultured cells from RAG-2+/+ and
RAG-2-/- mice. Resident cells were harvested from the
peritoneal cavity and cultured for 3 h. Although all adherent
cells from RAG-2+/+ mice immunostained as macrophages (Fig. 3A), only the larger
population exhibited positive 12/15-LO immunostaining (Fig. 3B). The smaller population was defined as macrophages based
on immunostaining with antisera that did not react with the other cell
types present in the peritoneal exudate. Furthermore, with increased
duration of culture, these cells exhibited a spreading pattern that is
consistently seen with macrophages. In contrast to the results obtained
from RAG-2+/+ mice, all macrophages from
RAG-2-/- mice (Fig. 3D) immunostained for
12/15-LO (Fig. 3E). Equivalent dilutions of nonimmune serum
were not immunoreactive (Fig. 3, C and F).
Furthermore, upon visualization of immunostained cells, it became
obvious that the pattern of 12/15-LO immunostaining in resident
peritoneal macrophages corresponded to cell size. Macrophages from
RAG-2+/+ mice that did not immunostain for 12/15-LO were
smaller than those that were immunoreactive for this protein.
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Morphologic differences that corresponded to the pattern of 12/15-LO immunostaining were observed between peritoneal macrophages from the two strains of mice. Macrophages from each strain were characterized to determine whether heterogeneity of 12/15-LO expression in RAG-2+/+ mice was related to the maturation state of resident cells.
RAG-2-/- mice had significantly fewer resident peritoneal
macrophages than RAG-2+/+ mice (p
< 0.001; Fig. 4A).
Furthermore, macrophages harvested from RAG-2-/- mice had
more total protein per cell than those from RAG-2+/+ mice
(p < 0.02; Fig. 4B).
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). The larger cells in both
RAG-2-/- and RAG-2+/+ expressed 12/15-LO
extensively; immunostaining was absent in the smaller cells.
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To determine whether 12/15-LO synthesis occurred immediately upon
entry into the peritoneum, an i.p. injection of Sephacryl beads or
thioglycolate was used to elicit macrophages in RAG-2-/-
mice. These agents recruit macrophages that exhibit mild activation
compared with that exhibited by resident cells (15). Sephacryl
increased the number of resident peritoneal macrophages by 289 ±
44%, while thioglycolate increased this number by 113 ± 4% in
RAG-2-/- mice. In RAG-2-/- mice, an
injection of Sephacryl resulted in the recruitment of a considerable
number of macrophages to the peritoneum that did not express 12/15-LO,
in contrast with the uniform expression observed in resident cells
(Fig. 6). Size analysis showed a uniform
distribution of cells within an area range of 140 to 360
µm2 for both nonelicited and Sephacryl-elicited cells
from RAG-2-/- mice (Fig. 7). Similar results for immunostaining
and size distribution were obtained after an injection of thioglycolate
(data not shown), indicating that 12/15-LO synthesis is stimulated only
after prolonged residence in the peritoneal cavity.
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12/15-LO protein mass is unaltered in mice that are unable to respond to IL-4 or IL-13
There was no statistically significant difference in the abundance
of 12/15-LO in peritoneal macrophages from STAT6-/- and
STAT6+/+ mice according to the relative content of 12/15-LO
and ß-actin as determined by Western blotting (Fig. 8). The morphology of peritoneal
macrophages from STAT6-/- was the same as that of the
wild-type (wt) strain match control with two discernible populations.
As described above, only the large population of cells immunostained
for the presence of 12/15-LO.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4,
and IL-13 has a profound effect on the expression of 12/15-LO in
cultured MDMs (5, 7, 8). Therefore, we hypothesized that the total
absence of lymphocytes in vivo would abolish both stimulatory and
inhibitory effects, with a net result of markedly decreased 12/15-LO
expression in mouse peritoneal macrophages. However, contrary to our
expectations, there was a significant increase in both the mass and
activity of 12/15-LO in peritoneal macrophages derived from
RAG-2-/- mice compared with the mass and activity seen in
cells from strain-matched, wt controls. This increase was due to the
uniform expression of 12/15-LO in all macrophages from
RAG-2-/- mice. In contrast, there was a subpopulation of
macrophages recruited from strain-matched RAG-2+/+ mice
that did not express the enzyme. Furthermore, there was no attenuation
of 12/15-LO abundance in STAT6-/- mice that were unable
to respond to IL-4 and IL-13. Although IL-4 was the first cytokine shown to increase 15-LO activity in cultured macrophages, we have demonstrated previously that the expression of the enzyme in vivo is not influenced by a deficiency of IL-4 (5). The subsequent finding that IL-13 mimics the effect of IL-4 on 15-LO expression in cultured macrophages may account for the lack of effect of IL-4 deficiency in vivo. Lymphocytes from RAG-2-/- mice cannot be activated to secrete IL-4 or IL-13. Therefore, the finding that 12/15-LO was present in peritoneal macrophages from RAG-2-/- mice may be indicative of other as yet undefined activators of synthesis. Alternatively, the stimulation of 12/15-LO synthesis may be restricted to IL-4 and IL-13, but these cytokines may originate from nonlymphoid sources. The most likely nonlymphocyte source of IL-4 and IL-13 is mast cells, which are present in the peritoneum. However, the enzyme is still abundant in peritoneal macrophages from STAT6-/- mice, which are unresponsive to both IL-4 and IL-13 due to an ablation of the common signaling pathway (12).
A possible cause of the increase in 12/15-LO expression in peritoneal
macrophages from RAG-2-/- mice could be the lack of the
inhibitory effect of IFN- (7). Indeed, even in T and B
lymphocyte-deficient animals, there can be abundant IFN- secreted
from NK cells (16). However, an effect of IFN- is unlikely, since
STAT6-/- mice have no known defect with regard to the
secretion of this cytokine; however, the strain-matched wt control had
an equivalent abundance of 12/15-LO.
A surprising result was the marked difference in the macrophage phenotypes observed in RAG-2-/- and RAG-2+/+ mice. Incubating cultured macrophages with exogenously applied lymphocyte-derived cytokines has profound effects on the properties of these cells. Since there are no known effects of RAG-2 deficiency that would directly result in changes in macrophage behavior, it is assumed that the changes in morphology were the result of an influence of lymphocytes. There is as yet no obvious mechanism by which lymphocytes affect macrophage morphology in the peritoneum of noninfected mice.
Although fewer macrophages were harvested from RAG-2-/- than from RAG-2+/+ mice, these cells adhered and spread rapidly and relatively uniformly. In contrast, macrophages from wt mice were heterogeneous in their spreading characteristics at 3 h after plating. Changes in macrophage phenotype could be due to changes in recruitment, activation, or residence time. Fewer resident macrophages in the peritoneum from RAG-2-/- mice, the uniform spreading, and the expression of 12/15-LO in every cell are consistent with a decreased recruitment of cells to the peritoneum of this strain. Unfortunately, all of the common modes of stimulating macrophage recruitment, including the administration of thioglycolate and Sephacryl, result in some degree of activation. Both of these agents greatly increased the number of peritoneal macrophages harvested. Unlike resident cells from RAG-2+/+ mice, macrophage populations from Sephacryl-injected RAG-2-/- mice spread on culture dishes to an extent that was similar to that seen with cells from untreated mice. However, Sephacryl converted the expression of 12/15-LO in RAG-2-/- mice to a heterogenous pattern, as seen in the wt mice. These data indicate that a deficiency of RAG-2 influences recruitment to the peritoneum and consequently the pattern of 12/15-LO expression.
A more precise definition of 12/15-LO regulation in macrophages may provide information regarding the mechanisms of several disease processes. At present, the local release of lymphocyte-derived cytokines is the only proposed mode of modulation of 12/15-LO expression in macrophages. For example, atherosclerosis is associated with a marked heterogeneity in the expression of 15-LO within lesion macrophages from both experimental animals and humans (17, 18). Atherosclerotic lesions contain many activated T lymphocytes, which may synthesize cytokines intramurally to modulate 15-LO expression (19). Although the enzyme is not expressed in all lesion macrophages, the recent demonstration of the pronounced decrease in the extent of atherosclerosis after the administration of a novel specific inhibitor of 15-LO is consistent with the importance of the enzyme to the disease process (20).
In summary, we have demonstrated that in two mouse strains, one that does not secrete IL-4 or IL-13 from lymphocytes and one that fails to respond to either of these cytokines, there is no decrease in the abundance of macrophage 12/15-LO as predicted from cell culture studies. However, the absence of 12/15-LO expression in newly recruited peritoneal macrophages and its presence in resident cells demonstrates that an unidentified factor that is unrelated to T lymphocytes has the ability to stimulate the synthesis of this enzyme.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan Daugherty, Gill Heart Institute, Division of Cardiovascular Medicine, Room L-543 KY Clinic, University of Kentucky, Lexington, KY 40536-0284. E-mail address:
3 Abbreviations used in this paper: LO, lipoxygenase; MDM, monocyte-derived macrophage; RAG, recombinase activator gene; wt, wild-type; 13-HODE, 13-hydroxyoctadecadienoate.
Received for publication January 13, 1998. Accepted for publication April 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Lipid Res. 35:893.[Abstract]
2-macroglobulin receptors in macrophages of atherosclerotic lesions from cholesterol-fed New Zealand and heterozygous Watanabe heritable hyperlipidemic rabbits. Arterioscler. Thromb. 14:2017. are costimulators of IFN- production by NK cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725.This article has been cited by other articles:
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  A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
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 A. Daugherty, N. R. Webb, D. L. Rateri, and V. L. King Thematic review series: The Immune System and Atherogenesis. Cytokine regulation of macrophage functions in atherogenesis J. Lipid Res., September 1, 2005; 46(9): 1812 - 1822. [Abstract] [Full Text] [PDF]
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A. Daugherty, N. Kosswig, J. A. Cornicelli, S. C. Whitman, S. Wolle, and D. L. Rateri Macrophage-specific expression of class A scavenger receptors enhances granuloma formation in the absence of increased lipid deposition J. Lipid Res., July 1, 2001; 42(7): 1049 - 1055. [Abstract] [Full Text] [PDF]
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A. Daugherty, S. C. Whitman, A. E. Block, and D. L. Rateri Polymorphism of class A scavenger receptors in C57BL/6 mice J. Lipid Res., October 1, 2000; 41(10): 1568 - 1577. [Abstract] [Full Text]
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J. A. Cornicelli, D. Butteiger, D. L. Rateri, K. Welch, and A. Daugherty Interleukin-4 augments acetylated LDL-induced cholesterol esterification in macrophages J. Lipid Res., March 1, 2000; 41(3): 376 - 383. [Abstract] [Full Text]
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 D. J. Conrad and M. Lu Regulation of Human 12/15-Lipoxygenase by Stat6-Dependent Transcription Am. J. Respir. Cell Mol. Biol., February 1, 2000; 22(2): 226 - 234. [Abstract] [Full Text]
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 S. Herblot, P. Chastagner, L. Samady, J.-L. Moreau, C. Demaison, P. Froussard, X. Liu, J. Bonnet, and J. Theze IL-2-Dependent Expression of Genes Involved in Cytoskeleton Organization, Oncogene Regulation, and Transcriptional Control J. Immunol., March 15, 1999; 162(6): 3280 - 3288. [Abstract] [Full Text] [PDF]
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 Y. I. Miller, M.-K. Chang, C. D. Funk, J. R. Feramisco, and J. L. Witztum 12/15-Lipoxygenase Translocation Enhances Site-specific Actin Polymerization in Macrophages Phagocytosing Apoptotic Cells J. Biol. Chem., May 25, 2001; 276(22): 19431 - 19439. [Abstract] [Full Text] [PDF]
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) had a
significantly higher cellular ratio of 12/15-LO than cells from
RAG-2+/+ mice (
). Histobars represent the mean of five
observations per group, and bars represent the SEM
(*p < 0.006).
