| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
,
*
Gene Therapy Program and Departments of
Medicine,
Pediatrics, and
§
Physiology, Louisiana State University Health Science Center, New Orleans, LA70112
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In a previous publication, we demonstrated that in vivo overexpression of murine IL-17 (mIL-17)3 stimulates hematopoiesis, particularly granulopoiesis (6). Furthermore, we demonstrated that mIL-17 stimulates the release of G-CSF. The release of G-CSF preceded the induction of granulopoiesis; however, the peak expression occurred several days before the peak increase in absolute neutrophil count (ANC). Based on these data, we hypothesized that other growth factors may be involved in IL-17-induced granulopoiesis. Specifically, we investigated G-CSF and stem cell factor (SCF) because SCF is known to strongly synergize with G-CSF in stimulating granulopoiesis (7, 8, 9, 10, 11). In this study, we show that IL-17 induces enhanced expression of the transmembrane form of SCF in the bone marrow (BM)-derived stroma cell lines BMS2, S17, and NIH3T3. Moreover, these cell lines secrete G-CSF in response to IL-17 in a dose-dependent fashion. To test the hypothesis that IL-17 induced increases in membrane-bound SCF are required for granulopoiesis in vivo, we investigated IL-17-mediated granulopoiesis in Steel-Dickie mice (Sl/Sld), which have a mutation where the transmembrane and cytoplasmic domains of SCF are deleted. A truncated soluble form of SCF is produced, but functional transmembrane SCF is absent in these animals (12, 13). To study a synergistic role of G-CSF with the transmembrane form of SCF, G-CSF was neutralized in Sl/Sld mice and littermate control mice overexpressing IL-17.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NIH3T3 cells were obtained from American Type Culture Collection (Manassas, VA). S17 cells were a generous gift from Dr. Kenneth Dorshkind (Los Angeles, CA) (14). BMS2 cells were a generous gift from Dr. Paul Kincade (Oklahoma City, OK) (15). All cells were grown in IMDM (Life Technologies, Gaithersburg, MD) with 10% FBS (Life Technologies) with penicillin/streptomycin (50 U/50 µg/ml; Life Technologies). Recombinant mIL-17 was purchased from R&D Systems (Minneapolis, MN).
Animals
WCB6F1/J MgfSl/MgfSl-d (Sl/Sld) mice and their littermate controls were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions in a dedicated specific pathogen-free room in the vivarium of Louisiana State University Health Science Center with filter top cages. Autoclaved food and water was provided ad libitum. Sterility was monitored using sentinel animals that were periodically bled and tested for common mouse pathogens. To achieve overexpression of IL-17, animals were injected in the internal jugular vein with 5 x 109 PFU of recombinant adenovirus encoding either mIL-17 (Ad-mIL17), the control gene luciferase (AdCMVLuc), or PBS and were sacrificed at 7 days, the time of maximal induction of granulopoiesis (6). Organs were harvested under sterile conditions. Blood was collected via cardiac puncture or retro-orbitally and transferred into heparinized tubes. Spleens were excised and ground between two slides and cells were transferred into medium. BM cells from both femurs were flushed into medium (DMEM/15% FBS; Life Technologies). RBC were lysed in splenic specimens with Tris-buffered NH4Cl. Nucleated cell counts were performed using a hematocytometer and trypan blue staining to account for the viability of cells. Smears from peripheral blood were prepared using standard techniques and stained with a modified Wright-Giemsa stain (DiffQuick; Baxter, Deerfield, IL). For G-CSF neutralization studies in vivo, mice were injected with anti-G-CSF Ab or preimmune (IgG) serum at 3 mg 1 h before and 3 days after adenovirus injection. With this dose of anti-G-CSF, no G-CSF could be detected at any time point in animals of any treatment group.
Anti-CSF Ab purification
Polyclonal rabbit anti-G-CSF serum was generated using an immunization kit purchased from Ribi (Hamilton, MT), following the instructions of the manufacturer. Briefly, 8- to 10-lb rabbits (Charles River Laboratories, Wilmington, MA) were injected with the immunization preparation reconstituted with recombinant murine G-CSF (Amgen, Thousand Oaks, CA). Administration was performed intradermally (0.3 ml), i.m. (0.4 ml), s.c. (0.1 ml), and i.p. (0.2 ml). Rabbits were boosted 4 and 8 wk after the initial immunization and bled 10–14 days after each boost. Before use, rabbit plasma was purified over a Sepharose A column (Bio-Rad, Hercules, CA). Activity of the Ab was tested each time by using a bioassay: G-CSF activity and anti-G-CSF neutralization capacity were measured using a bioassay previously described by Dale et al. (16) and our group (17). Using the standard tritiated thymidine incorporation assay, the proliferative response of G-CSF in NSF-60 cells was completely blocked with purified anti-G-CSF.
Construction of Ad-mIL-17
The construction of mIL-17 adenovirus has been described in detail by our group (6). Briefly, viruses were propagated on 911 cells using endotoxin-free conditions and CsCl density purification as described previously (18, 19). mIL-17 production was measured in 911 cell supernatants using a bioassay as previously reported and described below (2, 3). Virus preparations were screened for replication competent adenovirus by propagation on A549 cells. This assay has a sensitivity of 1 contaminant/108 PFU. All viral preparations had a PFU:particle ratio of <100:1. All lots of recombinant adenovirus contained less than 1 endotoxin unit/ml as measured by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
G-CSF ELISA
Plasma G-CSF protein concentrations were determined using a specific enzyme-linked immunoassay developed in our laboratory using described procedures previously reported in detail (6). Briefly, the ELISA was performed using native and biotinylated forms of the above described anti-G-CSF rabbit polyclonal Ab as the capture and detection Abs, respectively. G-CSF concentrations were calculated from a standard curve of recombinant G-CSF (Amgen) using log-log linear regression. The assay had an interassay coefficient of variation of 4.4% and the assay failed to detect 1200 pg/ml homologous mouse proteins (growth hormone, prolactin) or murine GM-CSF, IL-3, or IL-6, or Escherichia coli LPS.
Measurement of mIL-17 by bioassay
The biological assay previously described by Fossiez et al. (4) and our group (6) was used to validate mIL-17 expression in vivo. mIL-17 and mIL-6 concentrations were calculated from standard curves using log-log linear regression. One mIL-17 unit is defined as the amount that results in release of 1 pg/ml mIL-17-dependent mIL-6 secretion in this assay.
SCF ELISA
The ELISA was performed following instructions provided and outlined by R&D Systems. ImmunoModule plates (Nalge Nunc International, Naperville, IL) were coated overnight with a rat anti-mouse SCF mAb (Genzyme, Cambridge, MA) at 2 µg/ml in 100 µl of PBS. The plates were rinsed in PBS and blocked with 1% BSA for 2–4 h. The plates were rinsed, dried, and stored at 4°C. Culture supernatants (50 µl) were diluted in 50 µl of 0.1% BSA, PBS, and 0.05% Tween 20 in PBS (pH 7.4). After an overnight incubation (4°C), the wells were rinsed three times with PBS and 0.05% Tween 20 (wash buffer). A biotinylated goat anti-murine SCF (100 µl of 200 ng/ml; R&D Systems) was applied to the wells for 1–2 h at room temperature. The wells were rinsed three times with wash buffer, and a 1/5000 dilution of neutravidin/HRP (Pierce, Rockford, IL) was applied for 30 min at room temperature. The wells were rinsed four times with wash buffer and incubated with 100 µl of a 1:1 solution of HRP substrate (R&D Systems). After 5–20 min, the reaction was stopped with 25 µl of 0.18 N H2SO4, and the results were read on a Dynatech Platereader (Biotech Instruments, Winooski, VT) at 450 nm. A known standard of rmSCF (R&D Systems) was diluted in culture medium for establishing a standard curve. SCF concentrations were determined using log-log linear regression. Sensitivity of the assay was determined up to 20 pg/ml.
Colony-forming assays
Methylcellulose cultures for CFU-GM, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte (GEMM), and CFU-high proliferative potential (HPP) were performed with MethoCult GF 3434 from Stem Cell Technologies (Vancouver, Canada) as described previously (6) (components: 0.9% methylcellulose, 15% FBS, 1% albumin, 10 µg/ml bovine pancreatic insulin, 200 µg/ml iron-saturated human transferrin, 10-4 M 2-ME, 2 mM L-glutamine, 10 ng/ml rmIL-3, 10 ng/ml recombinant human IL-6, 50 ng/ml rmSCF, and 3 U/ml recombinant human erythropoietin). Briefly, 2 x 104 BM cells or 2.5 x 105 spleen cells were suspended in 1ml of methylcellulose medium and plated in 35-mm tissue culture dishes (Fisher, Pittsburgh, PA). The dishes were placed into a dedicated incubator and maintained there at 37°C under a 5% CO2 atmosphere and saturated humidified conditions. Analysis for colony formation was conducted at day 10 for CFU-GM and day 15 for CFU-GEMM using an inverted microscope with defining a colony consisting as an accumulation of at least 50 cells. CFU-HPP is defined as an accumulation of at least 200 cells.
FACS analysis
Murine BM stroma cell lines (BMS2, S17, NIH3T3) were cultured in
6-well plates (Corning, Corning, NY). The cells were collected by
removing the medium and incubating the cells for 15–30 min with 5 mM
EDTA in PBS. The cells were spun down and 
106
cells were resuspended in PBS with 1% BSA (wash buffer) containing
normal goat IgG (10 µg/100 µl; Sigma, St. Louis, MO). After 15 min,
biotinylated goat anti-mouse SCF (0.5 µg/100 µl; R&D Systems)
was added to the solution and incubated at 4°C for 1 h. The
cells were washed and incubated for 30 min in wash buffer containing 10
µg/ml solution of streptavidin conjugated with PE (PharMingen, San
Diego, CA). The cells were washed with wash buffer and analyzed with a
FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Histograms
and mean channel fluorescence were generated and analyzed using
CellQuest Software (Becton Dickinson).
Data analysis
Comparisons between the means were analyzed by ANOVA using the statistical software package StatView (Calabasas, CA). A p < 0.05 was considered to be statistically significant. Kolmogorov-Smirnov statistics were performed for flow cytometric analysis, using CellQuest software (Becton Dickinson). A p < 0.05 was considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
rmIL-17 induced G-CSF secretion over 24 h in a dose-dependent
manner in several BM-derived stroma cell lines (BMS2, NIH3T3, and S17,
Fig. 1
A). Peak secretion of
G-CSF occurred at an IL-17 concentration of 20 ng/ml. Heat-inactivated
mIL-17 failed to result in any detectable G-CSF secretion (data not
shown). To investigate whether IL-17 increased SCF expression in these
cells, S17, BMS2, or NIH 3T3 cells were incubated for 24 h with 40
ng/ml recombinant murine IL-17 or heat-inactivated IL-17.
|
B). Similar results were seen in BMS2 and NIH3T3 cells
(data not shown). To confirm these findings with an independent assay,
cell extracts were analyzed for cell-associated SCF by ELISA 24 h
after mIL-17 incubation (40 ng/ml). Cell-associated SCF increased
35 ± 5% in S17 cells (750 ± 79 vs 1180 ± 140 pg/ml;
p < 0.0001, n = 4). Similar increases
in cell-associated SCF by ELISA were observed in BMS2 cells (32 ±
7%) and NIH3T3 cells (70 ± 12%). No soluble SCF was detectable
in cell supernatants 24 h after mIL-17 treatment (data not
shown). In vivo induction of G-CSF by IL-17
G-CSF was measured in the plasma of mice at 1 and 7 days.
Administration of Ad-mIL17 resulted in a significant increase in plasma
G-CSF within 24 h in both littermate controls and
Sl/Sld animals to similar levels (1025 ± 370 pg/ml vs
670 ± 290 pg/ml, respectively, p > 0.05,
n = 6). The administration of anti G-CSF effectively
neutralized circulating G-CSF since no G-CSF could be measured in these
animals (data not shown). Similar to our previous report
(6), AdCMVLuc-treated control animals had a small but
detectable G-CSF response at 24 h, constituting <5% than in the
AdIL-17-treated animals. No G-CSF could be measured in the littermate
control groups at day 7 (AdmIL-17 and AdCMVLuc treated). However, in
the AdmIL-17-treated Sl/Sld animals G-CSF levels were
significantly elevated at 220 ± 31 pg/ml 7 days after vector
administration. No G-CSF was detected in AdCMVLuc-treated
Sl/Sld mice (Fig. 2).
|
Overexpression of mIL-17 for 7 days by adenovirus-mediated gene
transfer in control animals resulted in a 6.5-fold increase in white
blood cell count (WBC) (AdCMVLuc vs AdmIL-17, 4.6 ± 0.6 vs
30.7 ± 2.4 x 107/ml) (Fig. 3A). The increase in WBC was
largely due to an increase in granulocytes as measured by the ANC. The
mean ANC value in AdCMVLuc-treated animals was 1.6 ± 0.32 x
107/ml. In comparison, in AdmIL-17-treated
animals, the ANC rose to 23.5 ± 2.9 x
107/ml or a 14.5-fold increase over the AdCMVLuc
group (p < 0.0001; Fig. 3B). There
was no statistical difference in ANC between AdCMVLuc and PBS controls
(data not shown). Overexpression of mIL-17 in Sl/Sld mice
resulted in a lesser but significant 2-fold increase of the total WBC
compared with the AdCMVLuc-treated animals (7.8 + 0.45 vs 3.5 +
0.5 x 107/ml, p < 0.01)
(Fig. 3A). Similar to littermate control animals, this was
mainly due to expansion of mature granulocytes. The ANC in
AdmIL-17-treated Sl/Sld animals rose to 4.2 ±
0.52 x 107/ml (or a 4-fold increase)
compared with AdCMVLuc treatment (1.1 ± 0.24 x
107/ml; Fig. 3B).
|
As previously reported by our laboratory in C57BL/6 mice, no
changes were observed in BM cellularity 7 days after AdmIL-17 or
AdCMVLuc administration in littermate controls or Sl/Sld
animals (6). However, as previously observed with C57BL/6
mice, AdmIL-17-treated littermate controls demonstrated a significant
increase in absolute CFU (-GM and -GEMM, 458 ± 60 vs 811 ±
33, p < 0.005) (Fig. 4).
No statistically significant effect was observed on CFU-HPP (data not
shown). In contrast to littermate controls, Sl/Sld mice
failed to show an increase in CFU (-GM and -GEMM) after AdmIL-17
administration compared AdCMVLuc controls (399 ± 48 vs 425
± 53). Furthermore, no IL-17-induced changes were seen with BM CFU-HPP
(data not shown).
|
AdmIL-17 administration in littermate controls resulted in a
significant increase in splenic cellularity by 1.4-fold compared with
AdCMVLuc-treated animals (p <
0.05). Moreover, AdmIL-17 administration resulted in a total splenic
CFU (-GM and -GEMM) increase from 1336 ± 60 to 4086 ± 188,
p < 0.0001)compared with the AdCMVLuc group (3-fold
increase) (Fig. 5A). Absolute
splenic CFU-HPP demonstrated a more dramatic 5-fold increase from 211
± 17 to 1022 ± 110 in AdmIL-17-treated animals over AdCMVLuc controls
(Fig. 5B, p < 0.0001). AdmIL-17
administration in Sl/Sld mice resulted in a significant
3.4-fold increase in splenic CFU (-GM and -GEMM) from 840 ± 136 to
2846 ± 99 (p < 0.001) compared with AdCMVLuc
controls (Fig. 5A). There was also a significant 3.6-fold
increase in CFU-HPP in Sl/Sld mice after AdmIL-17 treatment
from 176 ± 17 to 638 ± 144 (p < 0.001, Fig. 5B).
|
The studies performed in Sl/Sld mice suggested a
critical role of the membrane-bound form of SCF in IL-17-mediated
granulopoiesis. However, in Sl/Sld mice, there remained a
significant increase in mature neutrophils as well as neutrophil
precursors. As G-CSF has been shown to synergize with SCF in inducing
granulopoiesis, we investigated the role of endogenous G-CSF in
IL-17-mediated granulopoiesis. Control or Sl/Sld mice were
injected with anti-GCSF or nonimmune IgG before administration of
PBS, AdCMVLuc, or AdmIL-17. Statistically significant differences were
not found between the AdCMVLuc group compared with the PBS controls in
any of the experimental outcomes; therefore, only AdCMVLuc control data
are shown as controls. Dale and colleagues. (20)
previously reported neutropenia in dogs with the development of
neutralizing anti-G-CSF Abs . Similar to their report, over the
7-day experiment, anti-G-CSF administration resulted in mild but
statistically significant neutropenia in both AdCMVLuc-treated
Sl/Sld and littermate control animals (Fig. 6B). Anti-G-CSF also
significantly attenuated AdmIL-17-mediated increases in ANC (Fig. 6B). The greatest attenuation by anti-G-CSF, in
IL-17-mediated increases in WBC and ANC, was seen in the
AdmIL-17-treated control mice (Fig. 6), suggesting that there is
synergism between G-CSF and SCF in IL-17-induced granulopoiesis. This
is further supported by the fact that AdmIL-17 induced only modest
increases in WBC and ANC in Sl/Sld mice (Fig. 6) despite the
fact that these mice release G-CSF in response to IL-17.
|
). Thus,
IL-17 protected both control and Sl/Sld mice from
anti-G-CSF-induced neutropenia (Fig. 6).
One week of G-CSF neutralization did not have a significant effect on
BM cellularity or BM precursor frequency (CFU-GM, -GEMM, and -HPP) in
AdCMVLuc-treated control animals (AdCMVLuc vs AdCMVLuc anti-G-CSF:
440 ± 50 vs 458 ± 60) or Sl/Sld animals (356 ± 43 vs 349
± 48). However, anti-G-CSF treatment completely reversed the
AdmIL-17-induced BM precursor expansion seen in control animals
(CFU-GM, CFU-GEMM, AdmIL-17 vs AdmIL-17 anti-G-CSF, 811 ± 33 vs
528 ± 63) (Fig. 7A).
Similarly, 1 week of G-CSF neutralization did not affect splenic
cellularity or precursor frequency in either control or
Sl/Sld mice (AdCMVLuc vs AdCMVLuc anti-G-CSF, littermate
control: 33.4 ± 1.5 vs 32.2 ± 1.9 for CFU-GM/CFU-GEMM, and 5.3 + 0.4
vs 6 + 0.9 for CFU- HPP; Sl/Sld mice: 21 ± 3 vs 26.9 ± 3.4
for CFU-GM/CFU- GEMM, and 4.4 ± 0.5 vs 5.4 ± 0.8 for CFU-HPP).
Furthermore, anti-GCSF treatment also attenuated the
AdmIL-17-induced precursor expansion in the spleen in both littermate
control and Sl/Sld mice (littermate control: AdmIL-17 vs
AdmIL-17 anti-G-CSF 102 ± 4.7 vs 69.4 ± 4.3 for CFU-GM/CFU-GEMM,
and 25.6 ± 2.8 vs 10.5 ± 0.9 for CFU-HPP; Sl/Sld: AdmIL-17
vs AdmIL-17anti G-CSF 71.2 ± 4.7 vs 44.5 ± 4.3 for CFU-GM/CFU-GEMM,
and 16 ± 2.8 vs 11.5 ± 0.9 for CFU-HPP). In this study, in contrast
to BM, splenic precursors remained statistically significantly elevated
in both strains over AdCMVLuc-treated controls (Fig. 7, B
and C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We hypothesized that IL-17 acts on BM stroma cells by inducing the release of G-CSF and SCF. Furthermore, we hypothesized that these cytokines mediate proliferation and differentiation of myelopoietic progenitors.
Our in vitro results show that mIL-17 induces the release of G-CSF from
several BM stroma cell lines and the induction of the
membrane-associated form of SCF on stroma cells. We have previously
demonstrated that in C57BL/6 mice, the in vivo IL-17-induced increase
in plasma G-CSF levels was transient and that G-CSF became undetectable
at 48 h in the blood, which coincided with the rising ANC. This
was in spite of the continuous systemic overexpression of bioactive
mIL-17, which was detected for over 2 wk (6). Results in
this series of experiments were consistent with our previous report,
and at day 7 we could not detect G-CSF in normal Sl/Sld
littermate controls (Fig. 2). Interestingly, we observed reduced but
significant levels of G-CSF in the plasma of Sl/Sld mice 7
days after AdmIL-17 administration. This persistence of G-CSF in the
plasma was also associated with a significantly attenuated
IL-17-induced increase in ANC (Fig. 3B). Layton et al.
(21) observed that despite the continuous infusion of
G-CSF into animals, G-CSF protein could no longer be detected in plasma
as neutrophilia developed. The authors demonstrated that the expanded
population of neutrophils could more effectively absorb and bind G-CSF
(21). These data suggest that IL-17-induced increase in
granulocytes contribute to the clearance of G-CSF in this model.
Our data demonstrate three key points of IL-17-induced granulopoiesis. First, membrane SCF is required for optimal IL-17-induced granulopoiesis as Sl/Sld mice have a reduced ANC, an absent increase in BM CFU, and reduced absolute splenic precursors after IL-17 overexpression compared with littermate controls. Second, endogenous G-CSF is also required and synergizes with SCF to effect optimal IL-17-induced granulopoiesis. Evidence to support this is the fact that anti-G-CSF blocks IL-17 increases in ANC by 82% in littermate control mice and by 28% in Sl/Sld mice. Moreover, anti-G-CSF blocked IL-17 increases in BM CFU completely as well as it attenuated splenic CFU-GEMM and -HPP responses. These data indicate that G-CSF secretion is predominantly responsible for IL-17-mediated stimulation of BM hematopoiesis and largely responsible for splenic hematopoiesis. However, the presence of the transmembrane form of SCF amplifies this response synergistically and Sl/Sld mice are able to secrete G-CSF in response to IL17, yet they lack efficient IL-17-induced granulopoiesis. These studies support the conclusion that IL-17-mediated effects on the progenitor compartments spleen and BM are to a large extent the result of G-CSF secretion which is augmented by the induction or presence of transmembrane SCF. In fact, Ulich et al. (11) combined exogenous administration of SCF and G-CSF and observed a dramatic synergistic effect in stimulation of both medullary and extramedullary hematopoiesis and neutrophilia. Third, there is a SCF-/G-CSF-independent effect of IL-17 on increasing the peripheral blood ANC since IL-17 can increase and stabilize the ANC in the presence of anti-G-CSF. The IL-17-mediated neutrophil increase was similar by 6-fold in both anti-G-CSF-treated Sl/Sld and littermate control mice.
There exist several possibilities to explain this finding. IL-17 could induce myeloid progenitor differentiation, irrespective of the presence of G-CSF or SCF, although these growth factors appear to amplify the mIL-17-mediated response in synergism. This effect of mIL-17 could be a direct effect or an effect that is caused by release of other cytokines such as IL-3 induction (22, 23), GM-CSF (24, 25), or IL-6 (26). Another possibility is that IL-17 makes the mature neutrophil more resistant to apoptosis. G-CSF has been reported to protect against neutrophil apoptosis (27, 28, 29, 30). Thus, IL-17 may protect against neutrophil apoptosis in the absence of G-CSF.
The significantly decreased IL-17-mediated response on granulopoiesis in Sl/Sld mice indicates the requirement of transmembrane SCF for its optimal effect. For instance, expansion of BM HPP progenitor cells was observed in control mice but not in the BM of Sl/Sld mice. Although IL-17 caused substantially enhanced absolute splenic precursor expansion in controls compared with Sl/Sld mice, the differences for the relative or fold increases were less pronounced between strains. Splenic precursors increased by 3.6-fold vs 4.9-fold for CFU-HPP and 3.4-fold vs 3.1-fold for CFU-GM/-GEMM in Sl/Sld vs control mice, respectively.
The data presented suggest a differential effect of IL-17 on different compartments of granulopoiesis. G-CSF neutralization completely reversed the IL-17-mediated effect on BM precursor expansion. Although spleen precursor frequency was also reversed completely with anti-G-CSF treatment, the absolute splenic precursor number remained significantly elevated in both control and Sl/Sld mice. Thus, IL-17 is capable of expanding the myeloid progenitor pool both via G-CSF and SCF induction as well as by a G-CSF- and SCF-independent effect.
The IL-17-mediated, G-CSF- and SCF-independent effects were most pronounced on peripheral neutrophils: Although 1 wk of G-CSF neutralization substantially reduced ANC in both control and Sl/Sld animals, IL-17 expression increased granulocytes equally by 6-fold in both animal strains. Thus, IL-17 can independently increase neutrophils in the absence of known key stimulators of granulopoiesis, G-CSF, and SCF.
G-CSF was recently found to be a response cytokine for emergency situations securing uninterrupted supply of neutrophils and macrophages by "BM priming" (31, 32). Little is known about cross-talk between immune mediating cells and as to how an organism can optimize its defense mechanisms against microbial invaders. IL-17 is a cytokine found exclusively in activated T cells. It is possible that IL-17 functions as a cross-talk cytokine between the cellular and innate immune system by inducing proliferation and differentiation of myeloid progenitors in the hematopoietic organs and the periphery. Furthermore, at a site of inflammation, activated T cells could potentially accelerate neutrophil differentiation via IL-17 secretion.
Although the role of T cells in regulation of hematopoiesis is unclear, it is believed their regulatory function occurs locally within the hematopoietic microenvironment (5). The data presented could identify a potential mechanism through which T cells might regulate hematopoiesis (4). For instance, T cells were found to enhance engraftment in BM transplantation while decreased numbers of T cells in donor marrow result in failure to engraft (33). This implies that interactions occur between T cells and other hematopoietic precursor cells. These interactions occur either directly via cell to cell contact, or via cytokines as messengers, or through combined effects.
Ongoing experiments in our laboratory examine the functionality of IL-17-differentiated granulocytes. We are also examining the role of IL-17 in BM transplantation. The data presented in this work demonstrate that mIL-17 induces G-CSF and SCF in BM stroma cells. In vivo effects of mIL-17 on hematopoiesis and secondary granulopoiesis are predominantly related to the release of G-CSF and the presence of functional SCF for optimal action. Moreover, mIL-17 independently induces and accelerates neutrophil maturation from myeloid precursors in control and Sl/Sld mice treated with neutralizing anti G-CSF Ab. These results can potentially be utilized to develop treatment strategies for patients with a functional deficit in myelopoiesis or in patients that need a rapid increase in mature granulocytes. Such conditions include serious bacterial or fungal infections, patients with compromised hematopoiesis as seen in aplastic anemia, cancer treatment induced or AIDS related.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Paul Schwarzenberger, Hematology/Oncology, Louisiana State University Health Science Center, 1542 Tulane Avenue, Suite 604K, New Orleans, LA 70112.
3 Abbreviations used in this paper: mIL-17, murine IL-17; ANC, absolute neutrophil count; SCF, stem cell factor; Ad-mIL-17, adenovirus-encoding mIL-17; AdCMVLuc, adenovirus-encoding luciferase; rm, recombinant murine; BM, bone marrow; WBC, white blood cell count; GEMM, granulocyte-erythrocyte-megakaryocyte-monocyte; HPP, high proliferative potential.
Received for publication September 24, 1999. Accepted for publication February 16, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. Smith, S. von Vietinghoff, M. A. Stark, A. Zarbock, J. M. Sanders, A. Duley, J. Rivera-Nieves, T. P. Bender, and K. Ley T-Lineage Cells Require the Thymus but Not V(D)J Recombination to Produce IL-17A and Regulate Granulopoiesis In Vivo J. Immunol., November 1, 2009; 183(9): 5685 - 5693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Bai, J. Cheng, X. Gao, A. G. Joyee, Y. Fan, S. Wang, L. Jiao, Z. Yao, and X. Yang IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function J. Immunol., November 1, 2009; 183(9): 5886 - 5895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. R. Crowe, K. Chen, D. A. Pociask, J. F. Alcorn, C. Krivich, R. I. Enelow, T. M. Ross, J. L. Witztum, and J. K. Kolls Critical Role of IL-17RA in Immunopathology of Influenza Infection J. Immunol., October 15, 2009; 183(8): 5301 - 5310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Gounaris, N. R. Blatner, K. Dennis, F. Magnusson, M. F. Gurish, T. B. Strom, P. Beckhove, F. Gounari, and K. Khazaie T-Regulatory Cells Shift from a Protective Anti-Inflammatory to a Cancer-Promoting Proinflammatory Phenotype in Polyposis Cancer Res., July 1, 2009; 69(13): 5490 - 5497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Freitas, J. C. Alves-Filho, T. Victoni, T. Secher, H. P. Lemos, F. Sonego, F. Q. Cunha, and B. Ryffel IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis J. Immunol., June 15, 2009; 182(12): 7846 - 7854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ke, K. Liu, G.-Q. Huang, Y. Cui, H. J. Kaplan, H. Shao, and D. Sun Anti-Inflammatory Role of IL-17 in Experimental Autoimmune Uveitis J. Immunol., March 1, 2009; 182(5): 3183 - 3190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Horiuchi, T. Kimura, T. Miyamoto, K. Miyamoto, H. Akiyama, H. Takaishi, H. Morioka, T. Nakamura, Y. Okada, C. P. Blobel, et al. Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF J. Immunol., February 15, 2009; 182(4): 2093 - 2101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Liu, Y. Yuan, Y. Li, J. Zhang, G. Xiao, Y. Vodovotz, T. R. Billiar, M. A. Wilson, and J. Fan Interacting Neuroendocrine and Innate and Acquired Immune Pathways Regulate Neutrophil Mobilization from Bone Marrow following Hemorrhagic Shock J. Immunol., January 1, 2009; 182(1): 572 - 580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Park, K. Staehling-Hampton, M. W. Appleby, M. E. Brunkow, T. Habib, Y. Zhang, F. Ramsdell, H. D. Liggitt, B. Freie, M. Tsang, et al. A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic Protein 1 in lymphopoiesis and innate immunity J. Exp. Med., November 24, 2008; 205(12): 2899 - 2913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Smith, M. A. Stark, A. Zarbock, T. L. Burcin, A. C. Bruce, D. Vaswani, P. Foley, and K. Ley IL-17A Inhibits the Expansion of IL-17A-Producing T Cells in Mice through "Short-Loop" Inhibition via IL-17 Receptor J. Immunol., July 15, 2008; 181(2): 1357 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Smith, A. Zarbock, M. A. Stark, T. L. Burcin, A. C. Bruce, P. Foley, and K. Ley IL-23 Is Required for Neutrophil Homeostasis in Normal and Neutrophilic Mice J. Immunol., December 15, 2007; 179(12): 8274 - 8279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Umemura, A. Yahagi, S. Hamada, M. D. Begum, H. Watanabe, K. Kawakami, T. Suda, K. Sudo, S. Nakae, Y. Iwakura, et al. IL-17-Mediated Regulation of Innate and Acquired Immune Response against Pulmonary Mycobacterium bovis Bacille Calmette-Guerin Infection J. Immunol., March 15, 2007; 178(6): 3786 - 3796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Schnyder-Candrian, D. Togbe, I. Couillin, I. Mercier, F. Brombacher, V. Quesniaux, F. Fossiez, B. Ryffel, and B. Schnyder Interleukin-17 is a negative regulator of established allergic asthma J. Exp. Med., November 27, 2006; 203(12): 2715 - 2725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Chen, A. Laurence, Y. Kanno, M. Pacher-Zavisin, B.-M. Zhu, C. Tato, A. Yoshimura, L. Hennighausen, and J. J. O'Shea Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells PNAS, May 23, 2006; 103(21): 8137 - 8142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Tan, W. Huang, Q. Zhong, and P. Schwarzenberger IL-17 Receptor Knockout Mice Have Enhanced Myelotoxicity and Impaired Hemopoietic Recovery Following Gamma Irradiation J. Immunol., May 15, 2006; 176(10): 6186 - 6193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lubberts, P. Schwarzenberger, W. Huang, J. R. Schurr, J. J. Peschon, W. B. van den Berg, and J. K. Kolls Requirement of IL-17 Receptor Signaling in Radiation-Resistant Cells in the Joint for Full Progression of Destructive Synovitis J. Immunol., September 1, 2005; 175(5): 3360 - 3368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ning, A. M. K. Choi, and C. Li Carbon monoxide inhibits IL-17-induced IL-6 production through the MAPK pathway in human pulmonary epithelial cells Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L268 - L273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. McAllister, A. Henry, J. L. Kreindler, P. J. Dubin, L. Ulrich, C. Steele, J. D. Finder, J. M. Pilewski, B. M. Carreno, S. J. Goldman, et al. Role of IL-17A, IL-17F, and the IL-17 Receptor in Regulating Growth-Related Oncogene-{alpha} and Granulocyte Colony-Stimulating Factor in Bronchial Epithelium: Implications for Airway Inflammation in Cystic Fibrosis J. Immunol., July 1, 2005; 175(1): 404 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-H. Chen, R. A. McDonald, J. C. Wells, G. B. Huffnagle, N. W. Lukacs, and G. B. Toews The Gamma Interferon Receptor Is Required for the Protective Pulmonary Inflammatory Response to Cryptococcus neoformans Infect. Immun., March 1, 2005; 73(3): 1788 - 1796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Linden, M. Laan, and G. P. Anderson Neutrophils, interleukin-17A and lung disease Eur. Respir. J., January 1, 2005; 25(1): 159 - 172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Miyamoto, M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, and S. H. E. Kaufmann Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17 J. Immunol., May 15, 2003; 170(10): 5228 - 5234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakae, S. Saijo, R. Horai, K. Sudo, S. Mori, and Y. Iwakura IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist PNAS, May 13, 2003; 100(10): 5986 - 5990. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Kolls, S. T. Kanaly, and A. J. Ramsay Interleukin-17: An Emerging Role in Lung Inflammation Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 9 - 11. [Full Text] [PDF]
|
|
|
|
 |
|
 F. Benchetrit, A. Ciree, V. Vives, G. Warnier, A. Gey, C. Sautes-Fridman, F. Fossiez, N. Haicheur, W. H. Fridman, and E. Tartour Interleukin-17 inhibits tumor cell growth by means of a T-cell-dependent mechanism Blood, March 15, 2002; 99(6): 2114 - 2121. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Aggarwal and A. L. Gurney IL-17: prototype member of an emerging cytokine family J. Leukoc. Biol., January 1, 2002; 71(1): 1 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Pan, D. French, W. Mao, M. Maruoka, P. Risser, J. Lee, J. Foster, S. Aggarwal, K. Nicholes, S. Guillet, et al. Forced Expression of Murine IL-17E Induces Growth Retardation, Jaundice, a Th2-Biased Response, and Multiorgan Inflammation in Mice J. Immunol., December 1, 2001; 167(11): 6559 - 6567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Forlow, J. R. Schurr, J. K. Kolls, G. J. Bagby, P. O. Schwarzenberger, and K. Ley Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice Blood, December 1, 2001; 98(12): 3309 - 3314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ye, P. B. Garvey, P. Zhang, S. Nelson, G. Bagby, W. R. Summer, P. Schwarzenberger, J. E. Shellito, and J. K. Kolls Interleukin-17 and Lung Host Defense against Klebsiella pneumoniae Infection Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 335 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Ye, F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr, P. Schwarzenberger, P. Oliver, W. Huang, P. Zhang, J. Zhang, et al. Requirement of Interleukin 17 Receptor Signaling for Lung Cxc Chemokine and Granulocyte Colony-Stimulating Factor Expression, Neutrophil Recruitment, and Host Defense J. Exp. Med., August 20, 2001; 194(4): 519 - 528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Schwarzenberger, W. Huang, P. Oliver, P. Byrne, V. La Russa, Z. Zhang, and J. K. Kolls IL-17 Mobilizes Peripheral Blood Stem Cells with Short- and Long-Term Repopulating Ability in Mice J. Immunol., August 15, 2001; 167(4): 2081 - 2086. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
