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Originally published online as doi:10.1189/jlb.0506350 on September 7, 2006

Published online before print September 7, 2006
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(Journal of Leukocyte Biology. 2006;80:1522-1528.)
© 2006 by Society for Leukocyte Biology

The Fps/Fes kinase regulates the inflammatory response to endotoxin through down-regulation of TLR4, NF-{kappa}B activation, and TNF-{alpha} secretion in macrophages

Sean A. Parsons*,{dagger} and Peter A. Greer*,{dagger},{ddagger},1

* Division of Cancer Biology and Genetics,
{dagger} Department of Biochemistry, and
{ddagger} Department of Pathology and Molecular Medicine, Queen’s University Cancer Research Institute, Kingston, Ontario, Canada

1 Correspondence: Cancer Research Institute, Botterell Hall, Room A309, Queens University, Kingston, Ontario K7L 3N6, Canada. E-mail: greerp{at}post.queensu.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hypersensitive to systemic LPS challenge, and Fer-deficient mice displayed enhanced recruitment of leukocytes in response to local LPS challenge. This study identifies physiological, cellular, and molecular defects that contribute to the hyperinflammatory phenotype in Fps/Fes null mice. Plasma TNF-{alpha} levels were elevated in LPS challenged Fps/Fes null mice as compared with wild-type mice and cultured Fps/Fes null peritoneal macrophages treated with LPS showed increased TNF-{alpha} production. Cultured Fps/Fes null macrophages also displayed prolonged LPS-induced degradation of I{kappa}B-{alpha}, increased phosphorylation of the p65 subunit of NF-{kappa}B, and defective TLR4 internalization, compared with wild-type macrophages. Together, these observations provide a likely mechanistic basis for elevated proinflammatory cytokine secretion by Fps/Fes null macrophages and the increased sensitivity of Fps/Fes null mice to endotoxin. We posit that Fps/Fes modulates the innate immune response of macrophages to LPS, in part, by regulating internalization and down-regulation of the TLR4 receptor complex.

Key Words: knockout mouse • lipopolysaccharide • cytokine • endocytosis • tyrosine kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The fps/fes proto-oncogene, (hereafter referred to as fps), encodes a 92-kDa Fps protein, which belongs to subgroup IV of the nonreceptor protein tyrosine kinases [1 , 2 ]. The ubiquitously expressed 94-kDa Fer protein is the only other known member of this subgroup of kinases [2 3 4 ]. In the adult, Fps is expressed in hematopoietic cells of the myeloid lineage, including macrophages, neutrophils, mast cells, platelets, and red blood cells, as well as in certain neuronal and epithelial cells. In contrast, during development, Fps is expressed in all three germ layers [5 6 7 ] (reviewed in [8 ]).

The innate immune system defends against invading pathogens by initiating an inflammatory response, and this requires the activation of key cell types, including macrophages, neutrophils, and mast cells [9 ]. Mouse knockout models have provided evidence for the involvement of Fps [10 ] and Fer [11 ] in the regulation of innate immune responses. Fps null mice displayed increased mortality in response to intraperitoneal challenge with the endotoxin LPS [10 ], and Fer-deficient mice displayed increased leukocyte recruitment at sites of localized LPS challenge [11 ] and enhanced intestinal barrier dysfunction in response to LPS [12 ]. LPS is a component of the cell membrane of gram negative bacteria, which is recognized by cells of the innate immune system. The biological response to LPS is mediated by a receptor complex composed of CD14, MD2, LBP, and Toll-like receptor (TLR) 4. TLR4 is a transmembrane receptor, belonging to the TOLL/IL-1 receptor family [13 ], and when stimulated, this receptor initiates an intracellular signaling cascade that results in the activation of Erk, Jnk, p38, Akt, and NF-{kappa}B [14 ]. Activation of NF-{kappa}B is a two-pronged process, whereby phosphorylation-induced degradation of its bound inhibitor, I{kappa}B, uncovers an NF-{kappa}B nuclear localization sequence, freeing it to move into the nucleus. As well, post-translational modifications of NF-{kappa}B such as phosphorylation of the p65/RelA subunit, causes a more robust and prolonged transcription of its target genes (reviewed in [15 ]). Once activated, these signaling molecules, particularly NF-{kappa}B, induce the transcription of proinflammatory mediators such as TNF-{alpha} [16 ].

Recently, it has been demonstrated that signaling by TLR4 is regulated in part by endocytosis [17 ]. More specifically, inhibition of TLR4 internalization leads to an increase in NF-{kappa}B activation, and this process is dependent on dynamin and clathrin [17 ].

We have previously shown evidence of a defective innate immune response to LPS in mice lacking either Fps [10 , 18 ], or the closely related Fer kinase [11 , 12 ]. However, the precise cellular and molecular bases for these defects have not yet been established. We now report an increase in circulating TNF-{alpha} levels in LPS-challenged Fps null mice. This increase in pro-inflammatory TNF-{alpha} strongly correlates with the hypersensitivity of Fps null mice to LPS [10 ], and therefore suggests that Fps might play an important role in regulating the innate immune response to endotoxin through controlling production of this key cytokine. We also describe an enhanced activation of the LPS-induced NF-{kappa}B signaling pathway in Fps null macrophages, which provides a likely mechanistic basis for the enhanced TNF-{alpha} secretion by macrophages. We go on to demonstrate that down-regulation of TLR4 is defective in Fps null macrophages, which may be the cause of the enhanced NF-{kappa}B signaling. This defect in TLR4 internalization may reflect a more general role for Fps in internalization, since Fps null macrophages also showed defects in internalization of transferrin and uptake of E. coli. Defective TLR4 internalization, causing enhanced LPS-induced activation of NF-{kappa}B and leading to elevated production of TNF-{alpha}, provides a highly plausible physiological explanation for the increased susceptibility of Fps null mice to endotoxic shock.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
ELISA assays
For in vivo assays, age-matched male mice were weighed one day before intraperitoneal (i.p.) injection with 7 mg/kg LPS (E. coli, serotype 055:B5, Sigma), and killed at different times by chloroform inhalation. Chest cavities were opened, and blood was removed by cardiac puncture with a 1-ml syringe fitted with a 26-gauge needle using 0.3% trisodium citrate as an anticoagulant. Blood was centrifuged for 1 min at 18,000 g. Plasma was collected and frozen at –80°C. For in vitro assays, conditioned media from LPS-stimulated peritoneal macrophages at the indicated times (see below, and Fig. 2 ), was collected and frozen at –20°C. ELISA assays were performed using OptEIA Mouse IL-10, and TNF-{alpha} sets from BD Biosciences, according to the manufacturer’s instructions.


Figure 2
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  		Figure 2. Resident peritoneal macrophages from Fps null mice secrete increased levels of TNF-{alpha} in response to stimulation with LPS. Resident peritoneal macrophages were isolated by lavage and allowed to adhere to tissue culture dishes. Cells were stimulated for the indicated time points with 270 ng/ml LPS. After stimulation, culture media were removed and in vitro TNF-{alpha} levels were analyzed by ELISA. For all time points, n = 3.

 
Peritoneal lavage
Mice were killed as described above, and peritoneal lavage was performed twice with 5 ml of prewarmed lavage media [RPMI 1640 with 10 mM HEPES, 5 mM EDTA, 10 U/ml heparin, 1% (vol/vol) antibiotic-antimycotic (aa) (GIBCO), 50 µM {alpha}-monothioglycerol]. Cells were pelleted and resuspended in 5 ml of erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3, 100 µM EDTA) for 5 min at 4°C to lyse red blood cells. The remaining cells were then pelleted and resuspended at 1 x 106 cells/ml, and 5 x 105 cells/ml (for assays done in 6- and 12-well plates, respectively) in culture media (RPMI 1640 with 5% FBS, 1% aa., 50 µm {alpha}-monothioglycerol, 1 mM HEPES, 2 mM glutamine).

Stimulation of resident peritoneal macrophages
Cells collected by peritoneal lavage were plated in 12-well plates (5 x 105 cells per well), and 2 to 3 h later, nonadherent cells were washed off using sterile PBS. Cells were then allowed to incubate overnight, and the next day adherent cells were stimulated with LPS at the indicated concentrations and times. To stop reactions, media were removed, and plates were placed on ice with 1 ml of TBS containing 100-µm sodium orthovanadate (TBS-V) per well. Soluble cell lysates were prepared by aspirating TBS-V and scraping cells into 250 µl of kinase lysis buffer (KLB), containing protease and phosphatase inhibitors (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% (vol/vol) Nonidet P-40, 0.5% (vol/vol) sodium deoxycholic acid, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 µM sodium orthovanadate, 100 µM phenylmethylsulfonyl fluoride) using a rubber policeman. Lysates were then spun for 10 min at 4°C and 14,000 g, soluble material added to clean tubes containing 6x SDS protein sample buffer, heated to 100°C for 5 min, passed through a P-200 pipette tip several times to shear high molecular weight DNA, and spun briefly at 14,000 g. Lysates were either frozen at –20°C, or run immediately on 7.5% or 11% SDS-polyacrylamide gels. Whole cell lysates were obtained by scraping cells into 1x SDS protein sample buffer directly and frozen without centrifuging. Proteins were transferred by semi-dry blotting to Immobilon-P membrane (Millipore), blocked with either 5% milk powder in TBS-Tween, or 5% BSA in TBS-Tween, and probed with the following primary antibodies: rabbit anti-phospho-p44/42 (pERK1/2), rabbit anti-phospho-p38, rabbit anti-p38, rabbit anti-I{kappa}B{alpha}, rabbit anti-phospho-p65 (Ser536) NF-{kappa}B (Cell Signaling Technology, Danvers, MA), rabbit anti-p44/42 (ERK1/2) (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-Fps/Fer antibody [5 ].

Flow cytometry
For Fig. 4 and 5 A, adherent cells’ Fc{gamma}II/III receptors were blocked by incubation with conditioned media from 2.4G2 hybridoma cells (ATCC# HB-197) for 5 min on ice. Cells were then incubated with either 2 µg/ml PE-conjugated rat anti-mouse TLR4/MD2 (eBioscience, San Diego CA) for 15 min on ice, post-LPS stimulation (Fig. 4) , or 40 µg/ml Alexa594 transferrin (Molecular Probes, Eugene, Oregon), for 30 min. at 37°C (Fig. 5A) . Cells were then washed with ice-cold PBS, fixed for 15 min with formaldehyde/zinc fixative (Electron Microscopy Sciences, Fort Washington, PA), and scraped into ice-cold PAB [PBS containing 0.3% bovine serum albumin (wt/vol) and 0.1% sodium azide (wt/vol)] for analysis by flow cytometry. For Fig. 5B , cells from the peritoneal cavity were collected by lavage. After lysing erythrocytes, cells were resuspended at 1 x 106 cells/ml in RPMI and incubated with 5 x 105/ml GFP-expressing E. coli, for 30 min at 37°C. Cells were then washed, resuspended in PAB, placed on ice, and incubated with 0.1 µg/ml PE-conjugated rat anti-mouse F4/80 antibody to stain for macrophages. Cells were then washed, fixed as above, and analyzed by flow cytometry.


Figure 4
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  		Figure 4. Resident peritoneal macrophages from Fps null mice display a defect in down-regulation of TLR4/MD2 in response to LPS stimulation. Resident peritoneal macrophages were isolated by lavage and allowed to adhere to tissue culture dishes. Cells were stimulated for the indicated time points with 270 ng/ml LPS. Following stimulation, cells were washed with ice-cold TBS-V and labeled with a PE-tagged {alpha}-TLR4/MD2 antibody. Cells were then scraped and analyzed by flow cytometry to measure the relative surface expression of TLR4/MD2. For all time points, n = 6.

 

Figure 5
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  		Figure 5. Resident peritoneal macrophages from Fps null mice display a reduced internalization of transferrin, and reduced uptake of E. coli. Resident peritoneal macrophages were isolated by lavage and either allowed to adhere to tissue culture dishes (A) or used directly (B). (A) Cells were stimulated with 40 µg/ml Alexa594-labeled transferrin for 30 min. Transferrin-incubated cells were then either surface-stripped or not, using a solution of 0.5 M NaCl and 0.2 M acetic acid. Cells were then scraped and analyzed by flow cytometry. For all time points, n = 4. (B) Cells were incubated with GFP-expressing bacteria, for 30 min. Cells were then incubated with {alpha}-F4/80 antibody to detect macrophages, washed, and analyzed by flow cytometry. For all time points n = 3; for bold quadrants, n = 0.071 WT vs. Fps null.

 
Mice
All animals used in this study were inbred SvJ/129 mice, between 7 and 12 wk old, and all experiments were carried out according to the guidelines of the Canadian Council on Animal Care and with the approval of the institutional animal care committee.

Statistics
All error bars represent means ± SE. All reported statistical values were calculated using the Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Elevated plasma levels of TNF-{alpha} in Fps null mice challenged with LPS
We have previously shown that Fps null mice experience a higher rate of mortality when challenged with an i.p. injection of LPS corresponding to the LD50 for wild-type mice [10 ]. Since production of inflammatory and anti-inflammatory cytokines plays a key role in regulating the endotoxin response, we examined plasma TNF-{alpha} and IL-10 levels in mice challenged with an i.p. injection of LPS. ELISA analysis demonstrated that Fps null mice had statistically significant increases in circulating TNF-{alpha} at 1 h post-LPS injection (5277 pg/ml vs. 8025 pg/ml, P = 0.014, Fig. 1A ). At 2 h postinjection, Fps null mice also displayed an apparent decrease in IL-10, (1432 pg/ml vs. 875 pg/ml, P = 0.092, Fig. 1B ), although this difference did not reach statistical significance. These results suggest a skewing in the balance of pro- and anti-inflammatory cytokines toward a heightened inflammatory state in Fps null mice, which correlates with the increased mortality observed previously [10 ].


Figure 1
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  		Figure 1. Fps null mice display a hyper-inflammatory phenotype as assessed by measuring plasma cytokine levels post-challenge with LPS. Mice were injected intraperitoneally with 7 mg/kg LPS. At the indicated time points afterward, mice were killed, and whole blood was collected by cardiac puncture. Blood was centrifuged, and plasma collected and analyzed by ELISA for TNF-{alpha} (A) and IL-10 (B). For all time points, n = 4 or 5.

 
Resident peritoneal macrophages from Fps null mice secrete increased amounts of TNF-{alpha} in response to LPS stimulation ex vivo
We next sought to examine whether macrophages could be one of the cell types responsible for the increased levels of circulating TNF-{alpha} in LPS-challenged Fps null mice. To address this possibility, resident peritoneal macrophages were harvested, stimulated with LPS, and TNF-{alpha} levels in the culture media were measured by ELISA (Fig. 2 ). At 1, 2, and 4 h post-stimulation, the level of TNF-{alpha} in the media of Fps null cultures was higher than in the wild-type cultures and by 6 h post-stimulation, it was 2.5 times higher in Fps null compared with wild type (Fig. 2 ; P<0.05 for 1, 4, and 6 h time points). This suggested that macrophages were at least partly responsible for the in vivo differences observed between Fps null and wild-type mice subsequent to an LPS challenge.

Prolonged I{kappa}B-{alpha} degradation and enhanced NF-{kappa}B phosphorylation following LPS stimulation of Fps null macrophages
We next looked for differences in downstream signaling. Immunoblotting analysis of a number of signaling molecules was performed on peritoneal macrophages after in vitro challenge with LPS. This analysis showed a prolonged period of degradation of I{kappa}B-{alpha} in Fps null macrophages, as compared with wild-type counterparts (Fig. 3A ). I{kappa}B-{alpha} levels returned to near basal levels between 1 and 2 h after LPS challenge in wild-type cells, whereas I{kappa}B-{alpha} levels remained depleted at 2 h in Fps null cells. We also examined the phosphorylation status of ERK and p38, two other kinases known to be activated downstream of LPS signaling; however, no difference in the phosphorylation status of either kinase was found between WT and Fps null macrophages (Fig. 3A) .


Figure 3
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  		Figure 3. Resident peritoneal macrophages from Fps null mice show prolonged degradation of I{kappa}B-{alpha} and increased phosphorylation of NF-{kappa}B, in response to stimulation with LPS. Resident peritoneal macrophages were isolated by lavage and allowed to adhere to tissue culture dishes. Cells were stimulated for the indicated time points with 270 ng/ml LPS (+) or vehicle control (–). After removing media for ELISA analysis, cells were lysed, and soluble cell lysates (SCLs) were resolved on either 7.5% or 11% SDS-PAGE gels, transferred to membranes and probed with the indicated antibodies.

 
The observed difference in LPS-induced I{kappa}B-{alpha} degradation/recovery kinetics (Fig. 3A) suggested prolonged or enhanced NF-{kappa}B activation in Fps null macrophages. This was assessed by immunoblotting analysis using a phospho-specific antibody against serine 536 of the p65/RelA subunit of NF-{kappa}B. These analyses also indicated enhanced LPS-induced NF-{kappa}B activation in Fps null macrophages as compared with wild-type. Phosphorylation of p65 was increased in Fps null relative to wild type macrophages at 15 min post LPS stimulation. The levels of p65 phosphorylation declined between 15 and 30 min in both genotypes, and continued to decline in WT cells back to pre-LPS challenged levels by 120 min. However, in Fps null macrophages, p65 phosphorylation levels persisted at the same level between 30 and 120 min (Fig. 3B) . Interestingly, this correlated with the previously observed kinetics of I{kappa}B-{alpha} degradation/recovery (Fig. 3A) . Taken together, these observa-tions suggest a role for Fps in both LPS-induced NF-{kappa}B activation and inactivation.

Prolonged surface expression of TLR4/MD2 following LPS stimulation of Fps null macrophages
In attempts to explain the molecular basis for the observed difference in LPS signaling, we next assessed surface expression of the LPS receptor, TLR4, before and after LPS stimulation of cultured peritoneal macrophages. Flow cytometry analysis using an antibody specific for TLR4/MD2 showed rapid loss of surface-accessible receptor in LPS-treated wild-type cells, with ~25% remaining after 5 min (Fig. 4 ). In contrast, greater than 60% remained on the surface of Fps null macrophages after 5 min (27% WT vs. 63% FN; P = 0.029), and there was little apparent further reduction at 15 min (28% WT vs. 52% FN; P = 0.030). These data suggested that Fps is participating in the process by which TLR4 is internalized from the surface of macrophages after LPS exposure.

Fps null macrophages show reduced uptake of both transferrin and E. coli
Internalization of growth factor and cytokine receptors [19 ], reorganization of cell-cell and cell-matrix receptors [20 ], and phagocytosis [21 ], all involve dynamic cytoskeletal reorganization. A number of studies have suggested that Fps [22 23 24 25 ] and the related Fer kinase [26 27 28 29 30 31 32 ] might play roles in cytoskeletal remodeling. We therefore considered the possibility of a more general receptor internalization defect in Fps null macrophages. Indeed, flow cytometry analysis of Alexa594-labeled transferrin showed that uptake of transferrin by wild-type macrophages was 3.5 times higher than in Fps null cells (Fig. 5A middle, P<0.001). To differentiate between transferrin on the cell surface and that which had been internalized, a stripping method was used to remove any transferrin bound to the cell surface. This approach revealed a defect in transferrin internalization in Fps null macrophages, with wild-type cells internalizing 2.3 times more than their Fps null counterparts (Fig. 5A , right, P=0.018).

Transferrin [33 ] and TLR4 [17 ] are both internalized by receptor-mediated mechanisms. Therefore, we explored the possibility that the defects in receptor internalization in Fps null cells might represent a broader defect that was not restricted to receptor-mediated endocytosis. Wild-type and Fps null macrophages were incubated with E. coli expressing EGFP for 30 min and then examined by flow cytometry. In this analysis, 41.6% of wild-type cells stained positive for both F4/80 antigen and GFP, while in the Fps null cells, only 27.1% were double positive (Fig. 5B , P=0.071, wild type vs. Fps null). This was consistent with Fps having a broader role in internalization, and not specifically in receptor-mediated endocytosis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have used knockout mice to establish a role for Fps in the regulation of innate immunity and inflammation [10 , 34 ]; however, they fail to provide a mechanistic basis for Fps’s participation in these processes. Here, we further the understanding of Fps’s role in inflammation and innate immunity by showing that Fps null mice displayed an increase in plasma TNF-{alpha} in response to an in vivo LPS challenge. This effect was also observed ex vivo in isolated Fps null peritoneal macrophages treated with LPS. In addition, we provided evidence that the heightened TNF-{alpha} secretion was likely due to an enhanced activation of the NF-{kappa}B pathway in response to LPS stimulation, which might be a consequence of the failure of Fps null macrophages to internalize TLR4. Finally, we showed a role for Fps in internalization of both transferrin and E. coli, suggesting a more general role for Fps in internalization.

In innate immune responses to infection with gram negative bacteria, recognition of LPS by macrophages and other cells results in the release of proinflammatory cytokines such as TNF-{alpha}, followed by the release of anti-inflammatory cytokines such as IL-10. Balanced production of these and other key mediators of inflammation is essential to a controlled innate immune response. Fps null mice displayed significantly higher levels of TNF-{alpha} in their plasma after LPS challenge (Fig. 1 ; P=0.014). Previous work has shown that administration of a TNF-{alpha} antibody 6 h prior to LPS injection in mice reduces mortality by ~50% [35 ] and that mice injected with recombinant TNF-{alpha} display many of the same pathophysiological symptoms as those injected with LPS [36 ], thus establishing TNF-{alpha} as a pivotal mediator of the effects of endotoxin in vivo. Therefore, our observation that plasma TNF-{alpha} levels induced by LPS challenge was nearly twice as much in Fps null mice than wild-type mice provided a highly plausible physiological explanation for the differences in LPS-induced mortality described previously in these animals [10 ]. We also observed a 64% decrease in the peak levels of IL-10 in the plasma of Fps null mice (Fig. 1) . Although this difference did not reach statistical significance, it was consistent with a general defect in the balance of pro- and anti-inflammatory cytokine levels. Taken together, these results suggest that defective regulation of cytokine release by macrophages is a major factor in the higher susceptibility of Fps null mice to challenge with LPS.

Among known Fps-expressing cell types, macrophages represented an excellent candidate for the cell type responsible for the increased production of TNF-{alpha} during inflammation. Furthermore, they are also an important source of IL-10 during the resolution phase [37 ]. This was substantiated by the observation that cultured peritoneal Fps null macrophages produced significantly increased LPS-induced TNF-{alpha} levels at 1, 4, and 6 h in vitro (Fig. 2) , suggesting that this cell type was at least partially responsible for the increased in vivo levels of TNF-{alpha} in LPS-challenged Fps null animals. Since Fps is also expressed in other innate immune cells, including mast cells and neutrophils, it should be appreciated that these might also contribute to the observed in vivo hyperinflammatory phenotype; however, because of the observed role for Fps in TNF-{alpha} secretion in cultured peritoneal macrophages (Fig. 2) , it was clear that the macrophage response was important to the phenotype observed in vivo.

The observed differences in I{kappa}B-{alpha} degradation suggested a prolonged time over which NF-{kappa}B would be localized to the nucleus in Fps null macrophages. This would lead to enhanced TNF-{alpha} transcription, and eventually an increase in secretion of TNF-{alpha} into the culture media. Indeed, ELISA analysis provided direct evidence for accumulation of greater TNF-{alpha} levels in Fps null cultures after LPS challenge (Fig. 2) . However, an optimal NF-{kappa}B response also involves post-translational modifications, including phosphorylation the p65/RelA subunit on Ser529 and Ser536 (reviewed in [15 ]). Interestingly, we observed a more robust phosphorylation of p65/RelA on Ser536 in Fps null macrophages at 15 min after LPS stimulation compared with wild-type cells (Fig. 3B) . This provided additional evidence for an increase in the transcriptional activation status of NF-{kappa}B in Fps null macrophages. An intriguing possibility is that this increase in NF-{kappa}B signaling might also exist downstream of TNF-{alpha} stimulation. This is intriguing as TNF-{alpha} can act back on these cells in an autocrine manner [38 ] and cause degradation of I{kappa}B-{alpha} (reviewed in [39 ]); therefore the increased TNF-{alpha} secretion by Fps null macrophages (Fig. 2) could have contributed to the prolonged degradation of I{kappa}B-{alpha} seen in these cells (Fig. 3A) , and participation in TNF-{alpha} signaling would give Fps a two-tiered role in the LPS response.

At first glance, there seem to be discrepancies between the present results and those that were described in a previous paper [10 ], since in the previous paper, we reported no observed differences between wild-type and Fps null cells with respect to I{kappa}B degradation. However, there are two key differences in how the experiments were conducted, which might account for these differences. First of all, the previous paper used bone marrow-derived macrophages, while this study used resident peritoneal macrophages. Differences in the production of cytokines such as TNF-{alpha}, between various subtypes of macrophages is well established [40 ]. We therefore speculate that some differences in signaling might also be due to the use of two different subtypes of macrophages. Second, in the previous paper, we stimulate bone marrow-derived macrophages at an LPS concentration of 1 µg/ml, which is 4 times higher than the 270 ng/ml used here. We observed that 270 ng/ml LPS corresponds to a concentration that induces near maximal stimulation of macrophages (data not shown); therefore, the use of a LPS concentration, which is nearly 4 times higher in the previous paper might have masked some of the differences we describe here.

Macrophages isolated from Fps null mice did not internalize TLR4 to the same extent as their wild-type counterparts, at both 5 and 15 min poststimulation with LPS. This observation was consistent with our other data, since major defects in the NF-{kappa}B pathway were observed at 15 min post-LPS challenge (Fig. 3) . Furthermore, our results agree with the observations that TLR4 has been shown to generate inflammatory signals from the cell surface [41 ] and that endocytosis of this receptor is necessary in limiting LPS-induced NF-{kappa}B activation [17 ]. However, as is shown in Fig. 3A , the observed defect in endocytosis of TLR4 does not appear to affect phosphorylation of either p38 or ERK. This result is consistent with other reports describing an effect of endocytosis on some, but not all signaling pathways downstream of the same stimulus [42 43 44 ].

Interestingly, the internalization defect observed in Fps null macrophages was not specific for TLR4, but also extended to internalization of transferrin and uptake of E. coli (Fig. 5A 5B) . We therefore propose that Fps’ role in LPS signaling might be at the level of receptor internalization and/or intracellular receptor-complex trafficking, possibly by regulating cytoskeleton reorganization processes required for these events. This is an attractive hypothesis for a number of reasons. First, TLR4 signaling is not necessarily connected to the internalization of bacteria (reviewed in [45 ]), and therefore, the observed participation of Fps in both suggests a less specific role. However, both processes are dependent on actin cytoskeleton reorganization, and the Fps-related Fer kinase has been shown to be connected to actin cytoskeleton function through regulation of cortactin phosphorylation [27 , 29 , 46 , 47 ]. Because Fps and Fer are highly homologous and share the same domain structure [4 ], it is thought that these kinases may have similar or redundant roles within the cell, and so Fps might also contribute to regulation of the actin cytoskeleton. Second, recent work by Laurent et al. showed a role for human Fps in the regulation of the tubulin cytoskeleton [22 ], which is also important for both phagocytosis [48 ] and endocytosis (reviewed in [49 ]). Also, Fps partially colocalized with Rab5B, Rab7, and a marker of the trans-golgi network, suggesting a role in vesicular trafficking [50 ], and TLR4 has been shown to traffic to lysosomes for degradation [17 ].

In summary, this study establishes a biological role for Fps in the regulation of innate immunity through control of TNF-{alpha} production by macrophages, which might explain why it’s absence negatively affects the survival of mice challenged with LPS [10 ]. Although the precise molecular basis of this function is still unknown, we propose that it is due to a defect in internalization of TLR4, which leads to a more pronounced and sustained activation of NF-{kappa}B. Finally, since internalization of transferrin and uptake of E. coli are also affected by the absence of Fps, this might suggest a more general role for Fps in modulation of the cytoskeleton.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr. Andrew Craig and Dr. Waheed Sangrar for their insightful suggestions regarding the manuscript, and Matthew Gordon and Jeffrey Mewburn for their assistance in flow cytometry. This research was funded by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society.

Received May 24, 2006; revised June 30, 2006; accepted July 25, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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