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HEMATOPOIESIS
From the Department of Biomedical Engineering and
Cardiovascular Research Center, University of Virginia School of
Medicine, Charlottesville; and the Gene Therapy Program and Department
of Physiology, Louisiana State University Health Sciences Center, New
Orleans.
Many mutant mice deficient in leukocyte adhesion molecules display
altered hematopoiesis and neutrophilia. This study investigated whether
peripheral blood neutrophil concentrations in these mice are elevated
as a result of accumulation of neutrophils in the circulation or
altered hematopoiesis mediated by a disrupted regulatory feedback loop.
Chimeric mice were generated by transplanting various ratios of
CD18+/+ and CD18 Adhesion molecule-deficient mice have provided
valuable information in elucidating leukocyte recruitment mechanisms.
Many of the mice deficient in leukocyte adhesion molecules display secondary phenotypes, including altered hematopoiesis and neutrophilia, which have not been fully investigated. Mice lacking P-selectin, leukocyte function-associated antigen-1, intercellular adhesion molecule-1 (ICAM-1), core-2 glucosaminyltransferase, P- and
L-selectin, P-selectin and ICAM-1, or L-selectin and ICAM-1 are mildly
neutrophilic.1-7 Mice deficient in multiple leukocyte
adhesion molecules, including CD18 integrins; E- and P-selectin; E- and
P-selectin and ICAM-1; E-, P-, and L-selectin; E-, P-, and L-selectin
and ICAM-1; CD18 and E-selectin; and CD18 and P-selectin show more
severe neutrophilia.3,8-14 A few adhesion
molecule-deficient mice, including mice lacking Mac-1, E-selectin, or
both E- and L-selectin, have normal circulating neutrophil
concentrations.2,3,15
Although the existence of physiologic mechanisms controlling peripheral
neutrophil counts has been proposed as early as 1991,16 the reason for elevated neutrophil counts in adhesion molecule knockout
mice is not known. One candidate mechanism for neutrophilia is passive
accumulation of circulating neutrophils because of altered neutrophil
survival. Although mice lacking Mac-1 show defective apoptosis in
transmigrated neutrophils17 and P-selectin-deficient mice
show an increased neutrophil half-life,18 there is no
evidence that these defects cause elevated neutrophil numbers.
Candidate mediators have been implicated in the regulation of
circulating neutrophil levels in adhesion molecule-deficient mice.
Frenette et al9 showed elevated serum levels of
granulocyte-macrophage colony-stimulating factor (GM-CSF) and
interleukin-3 (IL-3) in mice lacking E- and P-selectin compared with
wild-type and P-selectin-deficient mice. CD18 null mice had increased
IL-3 and IL-6 serum levels compared with wild-type mice.8
However, no causality has been established between the elevated
cytokine levels and neutrophilia in these leukocyte adhesion
molecule-deficient mice.
Neutrophil survival, proliferation, differentiation, and function are
all regulated by granulocyte colony-stimulating factor (G-CSF).16,19 G-CSF is produced by monocytes, macrophages, endothelial cells, fibroblasts, mesothelial cells, and stromal cells in
response to lipopolysaccharide, tumor necrosis factor- IL-17 has been reported to be expressed predominantly by activated
CD4+ and CD8+ memory T
lymphocytes,23 although its receptor (IL-17R) is
ubiquitously expressed.24 In vitro, IL-17 stimulates the
production of proinflammatory and hematopoietic cytokines. Recombinant
hIL-17 induces the secretion of IL-6, IL-8, prostaglandin
E2, and G-CSF from rheumatoid synovial fibroblasts,
synoviocytes, endothelial cells, and epithelial cells.23 IL-17 released from skin-infiltrating T lymphocytes stimulated the
secretion of IL-6 and IL-8 from keratinocytes and also induced the
expression of ICAM-1.23 Synovial fibroblasts cultured in the presence of hIL-17 sustained the proliferation of CD34+
hematopoietic progenitors and their maturation into
neutrophils.20 Cai et al25 showed that IL-17
increased steady-state G-CSF mRNA levels in the murine 3T3 fibroblast
cell line. Schwarzenberger et al26 have recently shown that
adenovirus-mediated delivery of mIL-17 cDNA to the liver resulted in
drastically altered hematopoiesis and increased granulopoiesis in
wild-type mice. These mice displayed leukocytosis, splenomegaly,
increased cellularity of the spleen, and a rapid rise in serum
G-CSF levels. These data indicate that IL-17 mediates granulopoiesis,
at least in part through G-CSF stimulation.
Our experiments were designed to test the hypothesis that adhesion
molecule-deficient mice with high neutrophil counts have defective
neutrophil trafficking, which alters hematopoiesis, neutrophil
half-life, and/or survival. We tested whether peripheral neutrophil
concentrations in leukocyte adhesion molecule-deficient mice are
elevated as a result of (1) accumulation of neutrophils in the
circulation because of enhanced neutrophil survival and/or the
inability of neutrophils to transmigrate out of the vessels; or (2)
altered hematopoiesis mediated by a disrupted regulatory feedback loop,
causing the bone marrow to increase neutrophil production. To
investigate the mechanism(s) underlying the high neutrophil levels in
leukocyte adhesion molecule-deficient mice, we generated chimeric mice
reconstituted with varying ratios of CD18+/+ and
CD18 Animals
Generation of chimeric mice
Blood sampling and flow cytometry Serum or plasma samples were obtained from peripheral blood collected by tail vein bleeding. Total leukocyte counts and leukocyte differentials were obtained from Kimura-stained blood samples. The percentages of CD18+/+ and CD18 /
neutrophils in the peripheral blood of chimeric mice were determined by
direct immunofluorescence using a laser flow cytometer (FACScan; Becton
Dickinson, San Jose, CA), as described previously.13 Whole
blood was incubated with fluorescein isothiocyanate-labeled monoclonal
antibody (mAb) Gr-1 (Pharmingen, San Diego, CA; 0.5 µg/106 cells) to identify granulocytes
(Gr-1hi) and monocytes (Gr-1low), and
phycoerythrin-labeled mAb C71/16 to label CD18 (Pharmingen; 0.5 µg/106 cells) or isotype control (R35-95, Pharmingen; 0.5 µg/106 cells). Samples were incubated for 30 minutes on
ice. Unlabeled antibody was removed by aspiration after centrifugation.
Peripheral blood was resuspended in 150 mM NH4Cl, 10 mM
NaHCO3, 1 mM Na2 EDTA in deionized distilled water to lyse
red blood cells. Neutrophils were identified and gated by expression of
Gr-1 antigen. Data are presented as fluorescence histograms of CD18
expression of Gr-1-positive cells on a 4-decade log scale.
AdIL-17RFc To inhibit IL-17-mediated signaling in vivo, the extracellular domain of the mouse IL-17R was amplified using the primers IL-17R A, 5'-TGGTACCCGGGCTATGGCGATTCGGC-3'; and IL-17R B-TCS, 5'-GGATCCACGCGGAACCAGCCACAGGGGAATGTAGTC-3'; and KlenTaq (Clontech, Palo Alto, CA). The IL-17R B primer incorporated amino acids encoding a thrombin-sensitive cleavage site that serves as a bridge between the IL-17R extracellular domain and immunoglobulin (Ig) G1, as described previously.28,29 A 1680-bp fusion product of IL-17R and mIgG1 CH2 and CH3 was obtained and cloned into PCR2.1 (Invitrogen, Carlsbad, CA), the sequence of the fusion was verified by dideoxynucleotide sequencing, and the product was subcloned into pACCMV PLA.30 To verify protein expression of this construct in vitro, we performed transient transfections with the resultant plasmid pCCMVIL17RFc in 293 cells using lipofectamine (Life Technologies, Gaithersburg, MD). Western blotting of transfected 293 cells revealed a 140-kd product on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis that reacted with both antimouse IgG1 or antimouse IL-17R (R&D Systems, Minneapolis, MN). IL-17RFc-containing media showed a significant, dose-dependent suppression of IL-6 production in a bioassay, as described.31IL-17RFc was subcloned into pAd5MCSloxP (provided by Dr Blake Roessler, University of Michigan, Ann Arbor, MI), and a recombinant adenoviral genome was generated by in vitro recombination with Cre recombinase, as described.32 Adenovirus expressing IL-17 receptor-Fc construct (AdIL-17RFc) clones were further screened by polymerase chain reaction, and protein production was confirmed by Western blotting of cell supernatants using an antimouse IgG antibody (Bio-Rad, Hercules, CA). Viruses were propagated in 911 cells using endotoxin-free conditions and purified by CsCl. Virus preparations were screened for replication-competent adenovirus by propagation in A549 cells. This assay has a sensitivity of one contaminant per 108 plaque-forming units (PFUs). All viral propagations had a PFU-to-particle ratio of less than 100:1. All lots of recombinant AdIL-17RFc contained less than 1 endotoxin unit/mL, as measured by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). mIL-17 and G-CSF assays The biologic assay previously described by Fossiez et al20 and Schwarzenberger et al26 was used to validate mIL-17 expression in vivo. The concentration of mIL-17 was 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. Serum G-CSF protein concentrations were determined using a specific enzyme-linked immunoassay with antibody pairs purchased from R&D Systems. For this purpose, the wells of a 96-well plate (Nunc Immunoplate Maxisorb, Neptune, NJ) were coated with 2 µg/mL capture antibody and incubated overnight at 4°C. The plates were then washed 5 times with wash buffer (0.05% Tween 20 in phosphate-buffered saline) and blocked with 200 µL 2% bovine serum albumin in wash buffer for 2 hours at room temperature. G-CSF standards and samples were diluted in wash buffer containing 2% FCS. Both standards (31.25 to 1000 pg/mL) and samples (50 µL) were added to wells, and the plates were incubated for 1 hour at 37°C. After washing, 50 µL biotinylated anti-G-CSF (0.1 µg/mL in dilution buffer) was added, and the plates were incubated for 1 hour at 37°C. Wells were then washed and incubated for 1 hour after adding 100 µL of 0.1 µg/mL peroxidase-conjugated streptavidin (Jackson Laboratories, West Grove, PA) in dilution buffer. After washing the plate, we added 100 µL tetramethylbenzidine (TMB) (Sigma) as substrate and allowed color to develop for 30 minutes in the dark. After the reaction was stopped with 50 µL of 3 M H2SO4, the plates were read at 450 nm. G-CSF concentrations were calculated from the standard curve using log-log linear regression.Neutralization of IL-17 and G-CSF function in vivo To block IL-17 function, we performed adenovirus-mediated cytokine delivery of the cDNA encoding for soluble murine IL-17 receptor (AdIL-17R), delivered intravenously (5 × 109 PFU/mouse). Expression of smIL-17R serves as a decoy, binding IL-17 and preventing it from reaching its cellular receptor. Control mice were injected with adenovirus encoding enhanced green fluorescent protein (AdEGFP) intravenously (5 × 109 PFU/mouse). Blood samples were collected for systemic leukocyte counts and serum samples on days 0, 3, 5, and 10. For in vivo G-CSF neutralization studies, mice were injected with 1 mg anti-G-CSF antibody or 1 mg preimmune (IgG) serum. Blood counts and serum samples were collected on days 0, 1, 6, and 10.Statistical analysis Systemic leukocyte counts, serum G-CSF levels, and plasma IL-17 levels were compared using one-way analysis of variance and the Tukey or Dunn method multiple-comparison test by SigmaStat 2.03 (SPSS, Chicago, IL).
Distribution of CD18+/+ and CD18 / bone marrow. This
resulted in ratios of CD18+/+ and CD18/
neutrophils in the circulation that corresponded to the ratios at which
bone marrow cells were transplanted (wild-type/CD18/ at
10:1, 1:1, and 1:10) (Figure 1A). We
hypothesized that neutrophils derived from CD18/ bone
marrow cells would show high counts if the neutrophilia was
accumulation dependent because these cells, but not the
CD18+/+ neutrophils, would remain in the
circulation. Remarkably, despite the presence of CD18/
neutrophils in the circulation (approximately 10%, 50%, or 90% of
systemic neutrophils in 10:1, 1:1, or 1:10 reconstituted mice, respectively; Figure 1A), neutrophil counts were not significantly different from those in mice reconstituted with 100%
CD18+/+ bone marrow cells (Figure 1B). The presence of only
10% CD18+/+ neutrophils was sufficient to prevent the
severe neutrophilia seen in mice reconstituted with
CD18/ bone marrow cells (Figure 1B) or in
CD18/ mice.8 These data indicate that the
neutrophilia is not driven by the accumulation of neutrophils in the
circulation and suggest that the high neutrophil levels result from
altered hematopoiesis that may be due to defective neutrophil
trafficking.
Increased G-CSF and IL-17 serum levels in adhesion molecule-deficient mice We next tested whether neutrophilia in adhesion molecule-deficient mice may be caused by a common mechanism that increases hematopoiesis. On the basis of previous data showing the necessity of G-CSF for granulopoiesis21 and the induction of granulopoiesis in vivo through IL-17 administration,26 we investigated the role of these prohematopoietic cytokines in CD18/ mice. Serum G-CSF and plasma IL-17 levels were
significantly elevated (P < .05) in CD18/
mice (514 ± 109 pg/mL and 159 ± 28 pg/mL, respectively) compared with wild-type mice (12 ± 2 pg/mL and 20 ± 6 pg/mL, respectively) (Figure 2A,D). Therefore, G-CSF and IL-17
levels were examined in mice lacking single or multiple leukocyte
adhesion molecules, including E-selectin (E/), E- and
P-selectin (EP/), E- and P-selectin and ICAM-1
(EPI/), CD18 integrins (CD18/), CD18
and E-selectin (CD18/E/), and CD18 and
P-selectin (CD18/P/).
E Neutralization of IL-17 or G-CSF in vivo On the basis of these data, we hypothesized that IL-17 may increase neutrophil counts in adhesion molecule-deficient mice by stimulating G-CSF release. To directly test this hypothesis, we blocked IL-17 function in CD18/E/ mice, which
have high neutrophil counts and high levels of IL-17 (Figure 2).
CD18/E/ mice were injected
intravenously with 5 × 109 PFU AdIL-17R (IL-17 receptor)
or a control virus (AdEGFP). Neutrophil counts showed a peak reduction
(approximately 50%, P < .05) 5 days after AdIL-17R
administration (Figure 3A), but not after AdEGFP administration (Figure 3A). Neutrophil counts in individual mice
administered AdIL-17R began to decrease after day 3 and remained significantly reduced through day 10 (Figure 3C). The delayed reduction
in neutrophil counts with AdIL-17R treatment is probably caused by delayed protein expression in this adenoviral system. Blocking IL-17 function reduced the serum G-CSF levels in
CD18/E/ mice by 53%. G-CSF levels
began to decrease after day 3 and reached a minimum on day 7 (Figure 3D).
Similarly, CD18
Previous data17,18 have suggested that neutrophilia in
adhesion molecule-deficient mice possibly results from passive
accumulation of neutrophils in the systemic circulation. This theory is
refuted by normal neutrophil counts in chimeric mice reconstituted with mixtures of CD18 Instead, neutrophilia in leukocyte adhesion molecule-deficient mice
appears to result from increased hematopoiesis through an altered
regulatory feedback loop. CD18-null mice showed a 20-fold increase in
IL-3 and IL-6 levels compared with wild-type mice.8 Similarly, EP G-CSF is a potent stimulator of granulopoiesis16; however,
G-CSF has been investigated so far in only one adhesion
molecule-deficient mouse, the CD18 Many cytokines have been shown to stimulate granulopoiesis, including GM-CSF, stem cell factor, IL-3, IL-6, IL-11, and flt3/flk2 ligand, most with efficiencies less than that of G-CSF.34-44 These cytokines act synergistically with G-CSF to stimulate maximum granulopoiesis.34-40,45-47 It is possible that G-CSF-independent granulopoiesis pathways are up-regulated in leukocyte adhesion molecule-deficient mice to stimulate granulopoiesis in the absence of G-CSF, which is suggested by the incomplete effect of blocking G-CSF. IL-17 levels significantly correlated with neutrophil levels in all
genotypes. Blocking IL-17 function reduced neutrophil numbers by
approximately 50% in CD18 The loss of important leukocyte adhesion molecules critically impairs
the host defense. It is possible that neutrophilia partially results
from enhanced (or "emergency") hematopoiesis in response to
subclinical normal flora infections. Uncleared subclinical infections
might constantly signal for more neutrophils. Both CD18
Our findings establish that restoring physiologic neutrophil functions, such as trafficking, in adhesion molecule-deficient mice prevents the development of neutrophilia. Disrupting the feedback loop at 3 different checkpoints shows that the transmigration-IL-17-G-CSF axis appears to be the major regulating mechanism of neutrophil homeostasis in leukocyte adhesion molecule-deficient mice in vivo. Additional modifiers and parallel pathways are likely to participate in regulating neutrophil numbers.
We thank Drs A. L. Beaudet (Baylor College of Medicine,
Houston, TX) and D. C. Bullard (University of Alabama at
Birmingham) for E
Submitted May 11, 2001; accepted July 31, 2001.
Supported by National Institutes of Health grants R01-HL54136 to K.L.; NRSA-HL10447 to S.B.F.; R01-CA81125-01 to P.S.S.; R29-AA10384, R01-HL61271, and R01-HL62052 to J.K.K.; and AA09803 to G.J.B.; and by Leukemia Society of America Translational Research Award 6191 to P.O.S.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Klaus Ley, Department of Biomedical Engineering, University of Virginia, Health Sciences Center Box 800759, Charlottesville, VA 22908; e-mail: klausley{at}virginia.edu.
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Top
Abstract
Introduction
(TNF-
(IFN-
2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation.
Immunity.
1996;5:653-666