By
From the Department of Laboratory Medicine, University of California, San Francisco, California 94143-0724
Lipopolysaccharide (LPS) stimulates immune responses by interacting with the membrane
receptor CD14 to induce the generation of cytokines such as tumor necrosis factor (TNF)-,
interleukin (IL)-1, and IL-6. The mechanism by which the LPS signal is transduced from the
extracellular environment to the nuclear compartment is not well defined. Recently, an increasing amount of evidence suggests that protein tyrosine kinases especially the Src-family kinases Hck, Fgr, and Lyn, play important roles in LPS signaling. To directly address the physiological function of Hck, Fgr and Lyn in LPS signaling, a genetic approach has been used to
generate null mutations of all three kinases in a single mouse strain. hck
/
fgr
/
lyn
/
mice are
moderately healthy and fertile; macrophages cultured from these mice express normal levels of
CD14 and no other Src-family kinases were detected. Although the total protein phosphotyrosine level is greatly reduced in macrophages derived from hck
/
fgr
/
lyn
/
mice, functional
analyses indicate that both elicited peritoneal (PEMs) and bone marrow-derived macrophages
(BMDMs) from triple mutant mice have no major defects in LPS-induced activation. Nitrite production and cytokine secretion (IL-1, IL-6, and TNF-
) are normal or even enhanced in
hck
/
fgr
/
lyn
/
macrophages after LPS stimulation. The development of tumor cell cytotoxicity is normal in triple mutant BMDMs and only partially impaired in PEMs after LPS stimulation. Furthermore, the activation of the ERK1/2 and JNK kinases, as well as the transcription
factor NF-
B, are the same in normal and mutant macrophages after LPS stimulation. The current study provides direct evidence that three Src-family kinases Hck, Fgr, and Lyn are not
obligatory for LPS-initiated signal transduction.
Lipopolysaccharide (LPS)1, an outer membrane component of Gram negative bacteria, is a potent activator of
monocytes and macrophages. LPS triggers the abundant secretion of many cytokines from macrophages including IL-1
(1), IL-6 (2), and TNF- Comparatively less is known about intracellular signaling
events after binding of LPS to CD14 (10). There is evidence to suggest that G proteins (11), phospholipase C
(12), protein kinase A, and protein kinase C (13) are involved in LPS responsiveness. Strong evidence indicates
that protein tyrosine kinases (PTKs) play critical roles in
LPS signaling. Within minutes of LPS stimulation, numerous proteins become tyrosine phosphorylated, in particular MAP kinases p42 (ERK2), p44 (ERK1), and p38, the mammalian homologue of the yeast MAPK-like kinase HOG1
(14). Synchronous with tyrosine phosphorylation these
MAPKs also become enzymatically activated. The c-Jun
kinase (JNK) also becomes enzymatically activated within minutes after LPS treatment of macrophages (15). Direct
stimulation of the ceramide-activated protein kinase by
LPS has also been suggested as a major signaling mechanism
(18). Pretreatment of cells with herbimycin A, a general
protein tyrosine kinase inhibitor, blocks phosphorylation of
the MAPKs and inhibits LPS-induced biological responses
(16, 17). More specific tyrosine kinase inhibitors of the tyrphostin family also block LPS-induced TNF- Three members of the Src-family of protein tyrosine kinases, Hck, Fgr, and Lyn are strong candidates for the primary signal transducers of LPS responses (20). All three
of these kinases are rapidly activated after LPS treatment.
A portion of intracellular Lyn directly co-associates with
CD14 (21). LPS-induced association of Lyn with PI 3-kinase
has also been observed (23). Moreover, expression in macrophages of a constitutively active mutant of Hck augments
TNF- To directly test the role of the Src-family kinases, Hck,
Fgr, and Lyn in LPS signal transduction, we used macrophages
from triple knockout mice that were generated by inter-crossing hck Generation of hck (3), which together contributes
to the pathophysiology of septic shock. The major cell surface receptor for LPS on macrophages is CD14, a 55-kD glycosyl-phosphatidylinositol-linked membrane protein (4). High-affinity binding of LPS to CD14 is greatly enhanced
by the presence of the serum protein LPS-binding protein
(5). The crucial role played by CD14 in initiating LPS responses has been demonstrated in animal models. Transgenic mice overexpressing CD14 are hypersensitive to LPS
(6), while CD14-deficient mice show no responses to lowdose LPS stimulation (7). At higher concentrations of LPS,
macrophages may be activated by a CD14-independent pathway since anti-CD14 mAbs do not block all biological
responses (8, 9).
production
and MAPK phosphorylation, and prevent septic shock in
mice (19).
production while antisense oligonucleotides to Hck
inhibit LPS-induced responses (26). Chronic exposure (24-
48 h) of macrophages to LPS induces increased synthesis of Hck and Lyn, which correlates with the ability of LPS to
prime macrophages for respiratory burst (22). All of these
observations suggest that protein tyrosine kinases, especially
Hck, Fgr, and Lyn play critical roles in LPS-initiated signaling pathways.
/
, fgr
/
, and lyn
/
animals. Since these three kinases
are the primary Src-family kinases expressed in macrophages
(27), cells from hck
/
fgr
/
lyn
/
triple mutant mice have
little, if any, Src-family kinase activity. Resting bone marrow
macrophages and inflammatory thioglycolate-elicited peritoneal macrophages were used in this study. Surprisingly, LPSinduced cytokine secretion, nitrite production, and tumoricidal activity were all essentially normal in triple knockout
macrophages. Although overall tyrosine phosphorylation
was dramatically reduced in these cells, phosphorylation of
MAPKs occurred normally after LPS stimulation. These studies convincingly demonstrate that the major Src-family kinases
present in macrophages, Hck, Fgr, and Lyn are not obligatory in LPS-mediated signal transduction.
/
fgr
/
lyn
/
Mice.
hck
/
fgr
/
double mutant
mice and lyn
/
single mutant mice were generated as previously
described (Chan, V., F. Meng, A. DeFranco, and C. Lowell, manuscript submitted for publication and reference 28).
/
fgr
/
double mutant and lyn
/
single mutant were interbred to generate hck
/
fgr
/
lyn
/
triple deficient mice. Genotyping of offspring was performed by PCR using specific primers and
further confirmed by immunoblotting using specific polyclonal
antibodies against Hck, Fgr, and Lyn. Primer sequences used to
specifically amplify the wild-type hck gene were: hck forward 5
GCT CCA TAG ATC CGT CGT GCC ATT TCC 3
and hck
reverse 5
GTT GTT TGG TCC CAG CTT GCT GGA GG 3
.
To amplify the hck mutant allele, primers, hck forward and 3
neo, 5
GCA TCG CCT TCT ATC GCC TTC TTG ACG 3
, were
used. To specifically amplify the fgr wild-type allele, primers, fgr
forward, 5
CAA GGC CGG ACT TCG TCC GTC TTT CC
3
, and fgr reverse, 5
GAG AGC CTT ACT GGA ATC CCT
CTT TAG C 3
were used. The fgr mutant allele was detected
using primers, 5
neo, 5
CAG TCA TAG CCG AAT AGC CTC TCC ACC 3
, and fgr reverse. The lyn wild-type allele was amplified with lyn forward-2, 5
CAT AGC CTG AGT TAG
TTC CCT AGC 3
, and lyn reverse, 5
TCA CAT ATG AAC
ATG TGT GTG TAC ATG TC 3
. These primers amplify only
the active lyn wild-type gene and do not react with the pseudogene (Chan et al., manuscript submitted for publication). The
mutant lyn gene was amplified with the 3
neo and lyn 6.2. 5
AGC CAC CAT TGT CCA GAC TTC 3
. PCR reactions were carried out in a 20-µl vol containing 1× PCR buffer (Perkin-Elmer Cetus Instrs., Norwalk, CT), 200 µM dNTPs, 10%
DMSO, 1× readiload (Research Genetics, Huntsville, AL), 0.4 µM primers, 2 U Taq DNA polymerase, and ~100 ng tail biopsy
DNA. Reaction conditions were: 94°C 45
, 60°C 45
, 74°C
60
for 35 cycles. The lyn PCR was done at 94°C 45
, 55°C
45
, 74°C 60
. PCR products were resolved on a 2% agarose
gel (1% regular, 1% Nu-Sieve GTG agarose; FMC Corp., Rockland, ME).
Isolation and Culture of Macrophages.
PEMs and BMDMs were
obtained as described by Lowell et al. (28). BMDMs were cultured in a 37°C incubator with 5% CO2 in -MEM (GIBCO
BRL, Gaithersburg, MD) containing 20% L cell conditioned medium and 10% fetal calf serum for 7 d before use. PEMs were cultured in DMEM containing 10% fetal calf serum for 2 h and nonadherent cells were washed away before use. Macrophages were
stimulated with IFN-
(20 ng/ml) and different concentrations of
LPS for indicated times.
Preparation of Cell Lysates and Immunoblotting. After stimulation with LPS, PEMs and BMDMs were washed once with cold 1× PBS, then lysed in lysis buffer (150 mM NaCl, 10 mM Tris, pH 7.0, 10 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, 1 mM Na2VO4, 1 mM PMSF, and 1 µg/ml each of leupeptin/pepstatin/aprotinin). The protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Labs., Richmond, CA). Total protein extracts (30 µg) were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes. For immunoblotting with mAbs, antiphosphotyrosine mAb 4G10 (UBI, Lake Placid, NY), anti-Src mAb 327 (UBI), and anti-Yes mAb (gift from M. Sudol, Mount Sinai School of Medicine, NY), the nitrocellulose membrane was blocked with 2% BSA in TBST (150 mM NaCl, 25 mM Tris, pH 7.5, 0.2% Tween-20) for 1 h at room temperature. For immunoblotting with rabbit polyclonal antibodies, anti-Lck, anti-Fyn (both from Santa Cruz, Biotechnology, Santa Cruz, CA), anti-Hck, anti-Fgr, anti-Lyn, and antiBlk (produced against the unique domain of each protein; references 28, 29), the nitrocellulose membrane was blocked with 5% milk/10% goat serum in TBST for 1 h at room temperature. Membranes were incubated sequentially with primary antibodies followed by peroxidase-conjugated secondary antibodies for 1 h at room temperature with continuous shaking. Specific binding was visualized by the enhanced chemiluminescent detection system (ECL; Amersham Corp., Arlington Heights, IL).
Nitrite Assay.
PEMs and BMDMs were seeded on 96-well
plates at 1 × 105 cells per well and cultured in 120 µl of appropriate media in the presence or absence of LPS and IFN- for 12 h.
NO2
released into supernatants was measured with Greiss reagent as described (30).
Quantitation of Cytokines and Herbimycin A Treatment.
The levels
of IL-1 and TNF- in the supernatants were determined by
ELISA kits (IL-1; R&D Systems, Minneapolis, MN and TNF-
; Genzyme Corp., Cambridge, MA) using protocols described by
the manufacturers. IL-6 was measured by ELISA using specific
antibody pairs (PharMingen, San Diego, CA). In brief, 96-well
plates were coated with 4 µg/ml purified rat anti-mouse IL-6
overnight at 4°C and blocked with PBS/10% fetal calf serum for
1 h at room temperature. 50 µl of cell culture supernatants, which
were harvested from cells stimulated by LPS and IFN-
for 12 h,
were added to each well and incubated at room temperature for 2 h.
After washing with PBS/0.2% Tween-20, biotin-conjugated rat
anti-mouse IL-6 was added and incubated for 45 min. Wells
were washed and then incubated with avidin-peroxidase for 30 min. After washing, ABTS substrate (Zymed Labs., South San
Francisco, CA) diluted in 0.1 M citric acid buffer (pH 4.0) was
added to each well and incubated for 30 min. The optical density
at 405 nm was measured with a microtiter plate reader (Molecular
Devices Corp., Menlo Park, CA). For herbimycin A treatment,
adherent PEMs were treated with 10 µg/ml herbimycin A (Sigma
Chem. Co., St. Louis, MO) for 4 h before stimulating with LPS
and IFN-
.
Cytotoxicity Assay.
Different numbers of PEMs and BMDMs
were seeded on 96-flat well plates and stimulated with LPS (100 ng/ml) and IFN- (20 ng/ml) for 48 h. 104 P815 tumor cells
prelabeled by incubation with 10 µCi of [3H]thymidine for 12 h
were overlaid on the activated macrophages. After co-culture of
tumor cells and stimulated macrophages for 24 h, 100 µl supernatant was used to determine [3H]thymidine release by liquid scintillation counting. The specific release was calculated based on the
formula described by Jadus et al. (31).
ERK and JNK Kinase Assay.
PEMs and BMDMs that were
prestimulated with LPS (10 ng/ml) and IFN- (20 ng/ml) were
lysed in lysis buffer. Total protein lysates prepared from LPS/
IFN-
-stimulated PEMs or BMDMs were immunoprecipitated
(250 µg/assay) with ERK1/2 (antibody cross-reacts with both
ERK isoforms) and JNK specific polyclonal antibodies, respectively (Santa Cruz Biotechnology), for 1 h at 4°C and incubated with 2% BSA preblocked protein A-Sepharose beads for another
hour at 4°C with continuous shaking. After washing twice in lysis
buffer and once in kinase buffer (20 mM Hepes, pH 7.0, 10 mM
MgCl2, 0.5% Triton X-100, 10 mM MnCl2, and 0.1 mM
Na2VO4), 10 µCi
-[33P]ATP and 25 µg of the exogenous substrates myelin basic protein (Sigma) and c-Jun fusion protein (gift
of J. Hambleton, University of California, San Francisco, CA) were
added to the immunocomplexes of ERK1/2 and JNK, respectively,
and incubated for 15 min at 30°C. The protein samples were
boiled in 2× Laemmli buffer for 5 min and separated by 12%
SDS-PAGE followed by autoradiography.
NF-B Assay.
PEMs and BMDMs (5 × 106) were seeded
onto 100-mm plates and stimulated with IFN-
(20 ng/ml) and
LPS (10 ng/ml) for 2 h. Preparation of nuclear extracts was performed as described by Kitchens et al. (32). Sense and antisense
oligonucleotides, containing the NF-
B recognition sequences
derived from the murine immunoglobulin
enhancer, were synthesized, annealed and labeled with
-[32P]dCTP using Klenow
fragment as described by Hambleton et al. (15). To assess for nonspecific binding, a double-stranded oligonucleotide containing a
point mutation in the NF-
B-binding site that was incapable of
binding NF-
B, was also prepared. Labeled oligonucleotides were incubated with nuclear extracts for 15 min, then NF-
B-bound oligonucleotides were resolved on a 4% TBE gel.
To generate the hck/
fgr
/
lyn
/
-deficient mice, hck
/
fgr
/
double mutant mice
and lyn
/
single mutant mice were crossed. Genotyping of
offspring was performed by PCR as shown in Fig. 1 A.
Mice from this cross were interbred for several generations
until triple mutant animals were obtained. The hck
/
fgr
/
lyn
/
mice are fertile and have no general physiologic
phenotypes beyond that seen for the lyn
/
single mutant
mice (Chan et al., manuscript submitted for publication and references 33, 34, 35). All single mutant animals used to generate the triple knockout line had been back-crossed
at least two generations to C57BL/6 mice. To verify each
gene disruption in the triple mutants, proteins corresponding to Hck, Fgr, and Lyn were examined using specific
polyclonal antibodies. Expression of Hck, Fgr, and Lyn was
only detected in wild-type macrophages but not in hck
/
fgr
/
lyn
/
homozygotes (Fig. 1 B). FACS® analysis of resident peritoneal macrophages, PEMs and BMDMs from hck
/
fgr
/
lyn
/
animals showed normal expression levels
of CD14, Mac-1, Mac-2, F4/80, MHC class I, and CD29
(
1 integrin) surface molecules (data not shown).
Other Src-family Kinases Are Not Upregulated in Macrophages Derived from hck
To address the possibility that other Src-family kinases might be upregulated or
aberrantly expressed in macrophages derived from hck/
fgr
/
lyn
/
mice, we examined the expression level of all other
Src-family members in these cells by immunoblotting. The
expression level of other Src-family kinases was examined
using a panel of mAbs or polyclonal Abs specific for each
family member. As shown in Fig. 2, no other Src-family
kinases were detectable in hck
/
fgr
/
lyn
/
macrophages
although each protein was easily seen in extracts prepared
from brain tissue or lymphocytes. We conclude that hck
/
fgr
/
lyn
/
macrophages have no significant expression of
any Src-family kinases.
Production of Nitrite and Cytokines in hck
Since there is
extreme heterogeneity of macrophages (36) and their responses to LPS, we performed all our experiments on both
inflammatory PEMs and BMDMs. To investigate the importance of Hck, Fgr, and Lyn in LPS-induced macrophage
activation, we evaluated the LPS concentration-dependent
induction of nitrite, IL-1, IL-6, and TNF- in both wildtype and mutant cells. Over a wide range of LPS concentrations, there was no impairment of nitrite production in
hck
/
fgr
/
lyn
/
macrophages compared to wild-type cells
(Fig. 3). BMDMs from wild-type and triple mutant mice
produced equivalent amounts of nitrite in response to LPS
stimulation alone; PEMs did not respond to LPS-alone
stimulation. To further prime macrophages for LPS responses, we stimulated cells with the combination of LPS
and IFN-
. With this costimulation, BMDMs produced
more nitrite and manifested a left-shift in the LPS dose response curve; again, both wild-type and mutant macrophages
responded equivalently. LPS/IFN-
costimulation of PEMs
induced abundant nitrite production in wild-type macrophages while triple mutant PEMs appeared hyperresponsive, especially at low doses of LPS (0.001-0.01 ng/ml),
compared to wild-type mice. In the absence of LPS, IFN-
alone failed to induce nitrite release from both PEMs and
BMDMs.
Similarly, both types of triple mutant macrophages produced abundant amounts of IL-1, IL-6, and TNF- at 12 h
after LPS stimulation (Fig. 4). As seen with nitrite production, cytokine release from PEMs only occurred with LPS/
IFN-
costimulation, while BMDMs showed a left-shift of
the LPS dose-response curve with the addition of IFN-
(data not shown). hck
/
fgr
/
lyn
/
PEMs demonstrated
dramatically increased production of IL-1 compared to wildtype PEMs; triple mutant BMDMs also showed slightly increased IL-1 production compared to wild-type cells. The
wild-type PEMs and BMDMs produced nearly the same
amount of IL-1 after LPS/IFN-
stimulation; note the
change in scale between PEMs and BMDMs. Triple mutant PEMs also showed increased IL-6 production compared to wild-type cells; responses of BMDMs were equivalent. There were no differences in TNF-
production
between triple mutant and wild-type PEMs or BMDMs.
These data strongly support the conclusion that macrophages
lacking Hck, Fgr, and Lyn can be readily activated by LPS.
Indeed, PEMs from the triple mutant mice showed enhanced responses to LPS/IFN-
stimulation compared to
wild-type cells.
Partially Impaired Tumor Cytotoxicity in PEMs Derived from hck
Another major functional
response of LPS-stimulated macrophages is the development of tumoricidal activity. To test the tumoricidal ability
of macrophages derived from the hck/
fgr
/
lyn
/
mice,
we assessed cytotoxicity against P815 mastocytoma cells after stimulation with LPS and IFN-
. As demonstrated in
Fig. 5, the stimulated macrophages killed tumor cells in a
fashion proportional to the macrophage/tumor cell ratio;
greater killing was seen at higher macrophage cell numbers
in both wild-type and triple mutant mice. The extent of
killing by PEMs derived from hck
/
fgr
/
lyn
/
mice was
reproducibly less than wild-type PEMs at higher macrophage/ tumor cell ratios. This was not observed in BMDMs. This
evidence demonstrates that tumoricidal activity could be
induced in hck
/
fgr
/
lyn
/
macrophages by stimulation
with LPS and IFN-
.
hck
To test the effects of loss of
Hck, Fgr, and Lyn on the overall level of protein tyrosine
phosphorylation, we assessed changes in tyrosine phosphorylation after LPS/IFN- stimulation, in both PEMs and
BMDMs, using antiphosphotyrosine immunoblotting. As
shown in Fig. 6, we observed that hck
/
fgr
/
lyn
/
macrophages showed dramatically decreased tyrosine phosphorylation of multiple proteins in both resting and LPS-stimulated cells. One protein with an apparent molecular mass of
~42 kD was rapidly tyrosine phosphorylated upon LPS/
IFN-
stimulation in both wild-type and triple mutant
PEMs and BMDMs. This protein was one or both of the
p42/p44 (ERK2/ ERK1) MAP kinase isoforms (confirmed
by re-probing this blot with anti-ERK1/2, data not shown),
as has been demonstrated previously (16, 17). Interestingly,
hck
/
fgr
/
lyn
/
PEMs show slightly higher and more sustained tyrosine phosphorylation of the MAPKs compared
to wild-type PEMs. In BMDMs, there was no difference in
p42 phosphorylation between wild-type and triple mutant
macrophages. p42 phosphorylation in PEMs was detectable after 15 min after LPS stimulation, reached a maximum by
60 min, and declined at 120 min.
Analysis of ERK1/2 and JNK Activity in LPS-Treated Macrophages.
To evaluate the role of Hck, Fgr, and Lyn in
LPS-induced activation of MAPKs, we determined ERK1/2
and JNK kinase activity using defined substrates. ERK1/2
kinase activity was determined by phosphorylation of MBP.
In both PEMs and BMDMs, ERK1/2 kinase activity was
activated after LPS/IFN- treatment in hck
/
fgr
/
lyn
/
cells compared to wild-type cells (Fig. 7). No consistent
difference in ERK1/2 activation was observed between
wild-type and mutant cells. The activation of ERK1/2 was
detected at 60 min and declined at 2 h after LPS and IFN-
stimulation.
Activation of JNK kinase activity was carried out using a
c-Jun/GST fusion protein as a exogenous substrate. JNK
activity was detectable after 1 h stimulation with LPS and
IFN- in both wild-type and mutant PEMs and BMDMs
(Fig. 7). There was no consistent difference in JNK kinase
activation between LPS-treated wild-type and hck
/
fgr
/
lyn
/
macrophages. In separate experiments, both JNK
and ERK1/2 activation was observed within 15 min of
LPS stimulation in wild-type and mutant cells (data not
shown).
Another major signaling pathway that is initiated in macrophages after LPS
treatment is activation of NF-B DNA binding activity by
dissociation from I
-B (37). To assess whether the loss of Hck, Fgr, and Lyn affected NF-
B activation after LPS
stimulation, we performed electrophoretic mobility shift
assays (EMSA) using nuclear extracts from untreated and
LPS-treated cells incubated with a double-stranded oligonucleotide containing an NF-
B recognition sequence
(38). After LPS/IFN-
stimulation for 2 h, we detected inducible NF-
B DNA binding activity in PEMs and BMDMs
derived from both wild-type and hck
/
fgr
/
lyn
/
macrophages (Fig. 8). No binding was observed using an oligonucleotide containing point mutations in the NF-
B recognition sequence. The results demonstrate that the deficiency
of Hck, Fgr, and Lyn did not effect NF-
B activation by LPS.
Herbimycin A Inhibits MAPK Phosphorylation and Nitrite Production in hck
To investigate
whether macrophage activation by LPS remained dependent
on tyrosine kinase activity in hck/
fgr
/
lyn
/
macrophages,
we examined the effects of the protein tyrosine kinase
inhibitor herbimycin A on LPS-induced tyrosine phosphorylation and secretion of nitrite. As shown in Fig. 9 A, pretreatment with herbimycin A significantly reduced LPSinduced tyrosine phosphorylation of the p42 (ERK1/2)
protein in both wild-type and mutant cells. Similarly, nitrite
production (Fig. 9 B) was reduced to the same extent in
both wild-type and hck
/
fgr
/
lyn
/
BMDMs. This evidence demonstrates despite the lack of Hck, Fgr, and Lyn,
the triple mutant macrophages remain sensitive to inhibition of LPS-induced activation by the protein tyrosine kinase inhibitor herbimycin A.
Exposure of macrophages to bacterial LPS initiates a signal transduction cascade that leads to increased production
of nitrite, secretion of proinflammatory cytokines, and acquisition of enhanced bactericidal/tumoricidal activity, the
hallmarks of an activated macrophage. Therefore, regulation of this signaling pathway is critical for controlling the
initial phases of the immune response to foreign organisms.
A number of signaling pathways have been implicated in
LPS responses in macrophages; however the most rapidly induced signaling responses are likely to involve tyrosine
phosphorylation events. The predominant Src-family kinases expressed in macrophages, Hck, Fgr, and Lyn, have
been implicated in a number of studies as being the initial
signal transducers for tyrosine phosphorylation events after
LPS stimulation (20). To directly test the role of these
kinases in LPS signaling in macrophages, we generated hck/
fgr
/
lyn
/
triple mutant mice by intercrossing single mutant animals, then tested LPS-induced functional responses and signaling properties in peritoneal exudate and
BMDM from these mice. Much to our surprise, both triple
mutant macrophage types showed no significant impairments in nitrite secretion, cytokine production, tumoricidal
activation, or signal transduction after treatment with LPS.
These data conclusively demonstrate that these Src-family
kinases are not required for LPS-induced signal transduction or macrophage activation.
The primary surface receptor for LPS is CD14, which
binds LPS with high affinity in the presence of LPS-binding protein. At low doses of LPS (<1 ng/ml) treatment of
macrophages with anti-CD14 mAb completely blocks all
LPS-induced signaling and macrophage functional activation
(8, 16). At higher doses (>10 ng/ml) of LPS, anti-CD14
mAbs do not completely block LPS signaling or functional
activation, indicating that CD14-independent mechanisms of LPS stimulation can occur. Moreover, activation of macrophages derived from CD14-deficient mice has also been
observed at high LPS doses (7). The experiments done in
this work were all conducted in the low dose range of LPS,
hence the majority of signaling we examined was CD14
dependent. Since CD14 is a GPI-linked protein, and hence
has no intracellular protein domains, it has been postulated that it serves only as a binding protein and that the LPS-
CD14 complex then associates with another transmembrane
protein that serves as the primary signal transduction unit
(39). In analogy to many cytokine receptors, this heterodimeric LPS receptor complex would then activate the
appropriate membrane associated tyrosine kinases to initiate
the signaling cascade. Clearly, this process occurs normally
in the absence of Hck, Fgr, and Lyn. Since triple mutant
macrophages expressed normal levels of CD14, high-affinity binding of LPS is presumably not altered in the hck/
fgr
/
lyn
/
cells. However, it is possible that the putative
signal transducing subunit may have specificity for a wide
range of kinases, or that CD14 can associate with a variety
of signal transducing molecules, so that other non-Src-family kinases may be recruited to the membrane. Another
possible explanation for the normal LPS responses in hck
/
fgr
/
lyn
/
mutants would be compensation by other Srcfamily kinases that become aberrantly expressed in the triple mutant cells; however, immunoblot analysis revealed
that no other Src-family kinases were expressed in the triple
mutant cells (Fig. 2). Src has been reported to be expressed
in human monocytes (40), however we did not detect it in
our immunoblots. Moreover, LPS-induced nitrite secretion
and cytokine production occur normally in src
/
macrophages (Lowell, C.A., unpublished observations). Nevertheless, we can not rule out that very low expression of
known Src-family kinases, or the presence of other undescribed Src-family kinases, may be contributing to LPS-
induced responses in our hck
/
fgr
/
lyn
/
macrophages.
One such kinase may be the Yrk kinase; a Src-family kinase
cloned from avian cells for which a mammalian homologue has not yet been described (41).
Based on previous studies, macrophage activation by
LPS is closely related to activation of Src-family kinases in
macrophages, in particular Hck and Lyn, and correlates
with the association of Lyn with CD14 and PI3-kinase (21,
23). However, as shown in Figs. 3 and 4, a deficiency of
these kinases does not impair nitrite secretion or cytokine
release over a wide range of LPS concentrations. Indeed,
with thioglycolate-elicited PEM, we observed just the opposite, the triple mutant macrophages tended to be hyperresponsive to LPS activation. The hyperresponsiveness of hck/
fgr
/
lyn
/
PEMs was correlated with the observations that LPS-induced tyrosine phosphorylation of the
p42/p44 ERK proteins was slightly higher and more prolonged in triple mutant cells compared to wild-type (Fig.
6), although we were unable to demonstrate increased enzymatic activation of these kinases. Analysis of hck
/
fgr
/
double mutants and lyn
/
single mutant mice has shown
that the hyperresponsiveness of PEMs is the result of the
loss of Lyn activity (data not shown and reference 28). Thus,
it appears that rather than serving as a positive regulator of
LPS signaling, it is more likely that the Lyn kinase can serve
as a negative regulator of macrophage activation. A negative regulatory function of Lyn is also observed in lyn
/
B-cells (Chan et al., manuscript submitted for publication
and reference 34). Cross-linking of the B cell antigen receptor on lyn
/
cells produces a hyperproliferative response
that is correlated with enhanced activation of ERK1/
ERK2. The hyperresponsiveness of immune cells from lyn
/
mice may contribute to the development of autoimmunity
in these animals (33, 35). lyn
/
B-cells also failed to show
normal downregulation of proliferation after co-cross-linking of the antigen receptor with the Fc
RIIB receptor
(Chan et al., manuscript submitted for publication and 34).
The Fc
RIIB receptor is known to downregulate antigen
receptor signaling by recruiting the tyrosine phosphatase,
SHP-1, into the receptor complex (42), suggesting that
Lyn may be required for activation of this tyrosine phosphatase. Alternatively, Lyn may be required for activation
of the p145SHIP inositol phosphatase, which has also been
implicated in downregulation B cell and mast cell activation through the Fc
RIIB receptor (43). Whether Lyn is
acting to regulate these tyrosine phosphatases and whether
they, in turn, function to downregulate LPS-induced macrophage activation remains to be determined. Of the cytokines we tested, it appeared that hck
/
fgr
/
lyn
/
macrophages showed the greatest overproduction of IL-1.
Indeed, we found that PEMs from the triple mutant mice
spontaneously secreted IL-1 in the presence of IFN-
alone
and when cultured without any stimuli at all (Fig. 4 and
data not shown). Since IL-1 production has been shown to
induce expression of IL-6 by inflammatory macrophages elicited with turpentine injection (44), it is possible that the
higher levels of IL-6 production may be secondary to continuous IL-1 secretion. In contrast to PEMs, triple mutant
BMDMs do not spontaneously secrete IL-1; hence some
aspect of the inflammatory stimulus caused by the peritoneal injection of thioglycolate used to elicit macrophage influx into the abdominal cavity is contributing to the hyperresponsiveness of these cells.
Most of the experiments shown use macrophages that
are stimulated with LPS plus IFN-. We found that PEMs
failed to respond to LPS-alone stimulation (as assessed by
nitrite and cytokine release) while BMDMs showed less robust responses. Induction of macrophage tumor cell cytotoxicity also requires costimulation of cells with LPS plus
IFN-
(31). The mechanism by which IFN-
primes macrophages for augmented responses to LPS is unknown, however it is unlikely to be mediated by increased signaling
through Src-family kinases since the triple mutant BMDMs
showed equivalent enhancement of LPS responses with
IFN-
costimulation compared to wild-type cells.
The induction of tumor cell cytotoxicity in hck/
fgr
/
lyn
/
macrophages by LPS/IFN-
was completely normal
in BMDMs but reproducibly showed a 50% decrease in
PEMs (Fig. 5). Given that other aspects of macrophage activation and signal transduction caused by LPS were increased in triple mutant PEMs, the cause for the modest impairment in tumoricidal capability is unclear. Previous
work has suggested that hck
/
fgr
/
neutrophils and macrophages have defects related to integrin-mediated adhesion
(45). Since integrin-dependent adhesion also contributes to
macrophage cytotoxicity (46), it is possible that impairments in cell adhesion may account for the slightly diminished tumoricidal capacity of triple mutant PEMs.
Activation of the pleiotropic transcription factor NF-B
is also a well-described signaling pathway initiated by LPS
treatment of macrophages (10). Activation of NF-
B DNA
binding activity requires phosphorylation of serine residues
of both the p65 and p50 subunits (47). Although the signaling pathway from membrane CD14 that leads to NF-
B
activation is not clearly defined, tyrosine phosphorylation
events may be important, since herbimycin A will block
LPS-induced NF-
B activation (48). An indirect effect of
herbimycin A on inhibition of NF-
B activation has also
been suggested (49). Activation of NF-
B binding activity
occurred normally in LPS/IFN-
-stimulated triple mutant
macrophages (Fig. 8), demonstrating that these Src-family
kinases are not essential in initiating this LPS-induced signal
transduction pathway.
The major conclusion from this work is that despite the
biochemical evidence suggesting that Hck, Fgr, and Lyn
are involved in LPS signal transduction, macrophages that
are devoid of these kinases, and express no other detectable
Src-family kinases, have normal (or even enhanced) LPS
functional responses. Activation of the major mediators of
LPS signal transductionMAP kinases, JNK, and NF
B
occurs essentially normally in the absence of Src-family kinases. This begs the question of what is (are) the other
kinase(s) responsible for in CD14-mediated LPS signal
transduction. Based on the observation that herbimycin A
suppresses nitrite production and p42/44 activation equivalently in both wild-type and triple mutant macrophages
(Fig. 9 A), a tyrosine kinase is likely to be involved in some
step of the pathway, though not necessarily at the initial
step. It is unlikely that the p72syk kinase is involved, since
LPS responses are also normal in syk
/
mutant macrophages
(Crowley, M., and A. DeFranco, unpublished observations). An attractive candidate for the kinase responsible for
initiating LPS signaling may be the ceramide activated protein kinase, since LPS has been shown to activate this enzyme (50). Moreover, ceramide responses are defective in
the LPS non-responder macrophages cultured from C3H/
HeJ mice (18). Use of the hck
/
fgr
/
lyn
/
macrophages
may dramatically facilitate biochemical and/or genetic approaches to isolation of the responsible kinases involved in LPS signaling since Src-family members are not expressed
in these cells. Finally, this work also has major implications
in designing therapeutics for endotoxic shock; it is very
likely that compounds that inhibit Src-family kinase function will not serve as successful drugs for septic shock.
Address correspondence to Clifford A. Lowell, Department of Laboratory Medicine, University of California, San Francisco, California 94143-0724.
Received for publication 26 December 1996 and in revised form 22 January 1997.
1 Abbreviations used in this paper: BMDM, bone marrow-derived macrophage; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PEM, thioglycolate-elicited peritoneal macrophage; PTK, protein tyrosine kinase.We wish to thank J. Hambleton, V. Chan, S. Weinstein, M. Crowley, and A. DeFranco for help with reagents, assays, and critical review of this work.
This work was supported by grants from the National Institutes of Health (T32 A107334-09 to F. Meng and DK50267 and HL54476 to C.A. Lowell).
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