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HEMATOPOIESIS
From the Departments of Medicine-Hematology/Oncology
and Surgery and the IST-Academic Computer Center, UMDNJ-New Jersey
Medical School, Newark, NJ.
Bone marrow (BM) fibrosis may occur in myeloproliferative diseases,
lymphoma, myelodysplastic syndrome, myeloma, and infectious diseases.
In this study, the role of substance P (SP), a peptide with pleiotropic
functions, was examined. Some of its functions Bone marrow (BM) fibrosis is characterized by
fibrosis, hypercellularity, excessive deposits of extracellular matrix
proteins, increased circulating levels of particular cytokines, and
neo-angiogenesis in the BM.1-7 BM fibrosis is common to
several hematologic disorders, particularly chronic myeloproliferative
disorders (MPD). With reference to the onset of BM fibrosis, there are
2 major categories within the various subgroups.1 The
first is idiopathic myelofibrosis (IMF) or chronic BM fibrosis, in
which the onset correlates with myeloid metaplasia in the BM, spleen,
and liver. Although the underlying cause of this subgroup is undefined,
fibrosis could develop as a secondary process. The second is BM
fibrosis, which occurs as a secondary process at various times after
the initial diagnosis of polycythemia vera, essential thrombocythemia,
chronic myeloid leukemia,2,8-10 myeloma,11
lymphoma, and, infrequently, other nonhematologic
disorders.12,13
The mechanism of BM fibrosis remains mostly undefined. Immune-mediated
mechanisms in the development of BM fibrosis have been suggested.9,14,15 Monocytes/macrophages and megakaryocytes are included in the types of cells that have roles in the
pathophysiology of BM fibrosis.6,16,17 Regardless of the
underlying cause, patients with BM fibrosis have excessive
proliferation of fibroblastic mesenchymal cells.18 The
fibroblasts are heterogeneous, and experimental evidence suggests that
their proliferation is secondary to a clonal disorder of the
hematopoietic stem cell.19-22 Despite hematopoietic
activity in extramedullary tissues of patients with BM
fibrosis,23 patients nonetheless have different degrees of cytopenia.2 This dysregulated hematopoiesis is not
surprising because in the adult, the population in whom BM fibrosis
generally develops, extramedullary hematopoiesis cannot revert to the
neonatal state of balanced hematopoiesis.24 Although there
have been modest improvements in treatment options and understanding,
the complex mechanisms of BM fibrosis are yet to be elucidated, thus deterring satisfactory treatments. A commonly used treatment for myelofibrosis, splenectomy,25,26 could negatively
influence and alter a patient's immunocompetence.27
Fine-tuned dissection in the development of BM fibrosis could provide
therapeutic leads to reverse or prevent BM fibrosis. In this study, we
examined a potential role for substance P (SP). Indeed, we found
significant increases in SP levels in the sera of patients with BM
fibrosis compared to patients with hematologic diseases without BM
fibrosis and to healthy controls. SP exerts functions that favor the
development of BM fibrosis SP is an undecapeptide and is the major peptide encoded by the
preprotachykinin-I (PPT-I) gene.29 Neural and
nonneural cells express PPT-I.29,33-36
Neural-derived SP is released in the BM and other lymphoid organs as a
neurotransmitter.37,38 Nonneural sources of SP include
immune, hematopoietic, and BM stromal cells.28 Other
pleiotropic functions of SP and the other related
tachykinin-neurokinin family of peptides include immune and
hematopoietic regulation.30,31,33,34
PPT-I peptides interact with different affinities to 3 cloned
receptors: NK-1, NK-2, and NK-3.39 In BM stromal cells,
the expression of NK-1 is induced by cytokines associated with
stimulatory hematopoiesis such as stem cell factor. In these same BM
cells, the other homologous receptor, NK-2, is constitutively
expressed.32,34 Immunoprecipitation has shown that the
increased SP levels in patients with BM fibrosis was complexed to
another molecule(s). Protection of SP by a larger molecule is
important because this peptide could be degraded by endogenous
endopeptidases.40 Therefore, we hypothesized that SP may
be complexed to its natural high-affinity receptor (NK-1) or to a
homologous molecule. To this end, we screened cDNA libraries
constructed from BM cells with NK-1 cDNA. Results showed that SP is
complexed to fibronectin (FN), suggesting that FN could protect SP and
provide chemical stability to the small peptide. This finding could be
relevant because FN is increased in patients with
myelofibrosis.41,42 The potential for molecular mimicry by
FN for NK-1 was examined by computer-assisted molecular modeling. The significance of these findings with previous reports is discussed.
Study subjects
Cytokines, antibodies, and other reagents
Preparation of bone marrow stroma Bone marrow stroma was prepared as described.43 Briefly, BM aspirates were obtained from healthy donors, ages 20 to 35 years, according to guidelines from the institutional review board of UMDNJ-New Jersey Medical School. Unfractionated cells from BM aspirates were cultured at 33°C in -MEM (Life Technologies, Grand Island,
NY) with 12.5% fetal calf serum (Hyclone Laboratories, Logan, UT),
12.5% horse serum (Hyclone Laboratories), 0.1 µM hydrocortisone, 0.1 mM 2-ME, and 1.6 mM glutamine. On day 3 of culture, red blood cells and
granulocytes were removed from the nonadherent fraction by
Ficoll-Hypaque density gradient (Sigma). Mononuclear cells were
replaced in the respective cultures, which were re-incubated with
weekly replacement of 50% medium until confluence.
cDNA libraries Three different cDNA libraries were used to screen for NK-1-related clones. One library, prepared from unstimulated pooled human BM cells, was purchased from Clontech (Palo Alto, CA). Two other cDNA libraries were prepared with poly-A RNA isolated from BM stroma stimulated with 25 ng/mL IL-1 or 10 ng/mL SCF. Each library was
prepared with mRNA isolated from at least 9 healthy donors. Donor pool
was represented by sex and ethnic diversity. Libraries were constructed
with the cDNA synthesis kit and the Zap Express cDNA Gigapack III Gold
cloning kit (both purchased from Stratagene, La Jolla, CA). Preparation
of the library was according to the manufacturer's instructions.
Briefly, second-strand cDNA with XhoI and EcoRI
adapters was prepared with 5 µg mRNA and then ligated in pZap.
Titration of packaged pZap was approximately 106 to
107 pfu/mL. Each library was screened with 107
pfu by plating 5 × 104 pfu/150 mm bacteriologic grade
Petri dishes (Fisher Scientific, Springfield, NJ). Plaques were
hybridized with human NK-1 cDNA44 using different
hybridization and washing parameters.
Quantitation of SP-IR Competitive enzyme-linked immunosorbent assay (ELISA) quantitated SP-IR as described.43 Briefly, Immulon 96-well plates (Dynatech Laboratories, Chantilly, VA) were coated with 100 µL streptavidin at 5 µg/mL. Each well was incubated with 100 µL biotinylated SP (Chiron Mimotopes, Emeryville, CA) at 750 ng/mL. After this, equal volumes (50 µL) of unknown samples and optimum rabbit anti-SP were added to quadruplicate wells. Each sample was assayed as undiluted and 3-serial dilutions. Bound anti-SP was detected with Alk Phos-conjugated goat anti-rabbit IgG and Sigma 104 phosphatase substrate (Sigma). SP-IR levels were calculated from a standard curve developed with optical density at 405 nm versus 12 serial dilutions of known SP concentrations, ranging from 100 to 0.08 pg/mL. Optimization experiments indicated that the working concentrations of rabbit anti-SP and goat anti-rabbit IgG were 1/15 000 and 150 ng/mL, respectively.Immunoprecipitation and Western blot analysis Immunoprecipitation and Western blot analysis were performed as previously described.6 Briefly, SP was immunoprecipitated from sera by incubating with rabbit anti-SP (1/10 000) at 4°C overnight. After this, samples were incubated with protein A Sepharose CL 4B (Sigma) at 4°C for 6 hours and then centrifuged at 4°C, 10 000g for 30 minutes. Pellets were washed (1×) with phosphate-buffered saline, resuspended in sample buffer, and electrophoresed on 12% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA), and SP was detected by overnight incubation at room temperature with rabbit anti-SP (1/15 000). After this, membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1/5000) for 45 minutes. HRP was developed with ECL Western blot detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ).Membranes were stripped by washing for 1 hour at room temperature
with 62 mM Tris (pH 6.8) containing 100 mM Computer-assisted modeling of SP and docking to FN The structure of SP had to be modeled because it was not solved by crystallography owing to its high flexibility. We modeled the secondary structure of SP and used the partial crystal structure of human FN to explain possible interactions between SP and FN.45 The sequence of SP, an 11-amino acid peptide, gives it probable, yet not fixed, structure. Its 3-dimensional structure was generated using Sybyl 6.6 molecular modeling package (TRIPOS Associates, St Louis, MO), and energy was optimized with Discover.Statistical analysis Data were analyzed using the Student t test to determine the significance (P value) between experimental values.
SP-IR levels in the sera of patients with or without BM fibrosis Increased proliferation of fibroblasts in the BM of patients with fibrosis indicates that the BM microenvironment is amenable to the mitogenic effects on fibroblasts. The increased frequency of fibroblast in the BM is heterogeneous rather than clonal.1,2 This suggests that the fibroblasts are responding to factors with mitogenic potential. We, therefore, determined whether SP, a mitogen for fibroblast,28 could be a candidate molecule by quantitating its levels in the sera of patients with fibrosis and compared its levels to those in patients with other hematologic disorders and in age-matched healthy controls. SP-IR levels were quantitated in samples taken at the time of diagnosis (3-4+ reticulin fibers) and at approximately 4-month intervals for up to 2 years. SP-IR levels in the samples collected within this period were not significantly different and ranged from 2 to 5 pg/mL. Results shown for each patient in Figure 1 represent the mean of 4 to 8 samples. Data shown in Figure 1B indicate significant (P < .01) increase in SP-IR in patients with BM fibrosis (162 ± 15.5, ± SD; n = 44) compared to those in patients without fibrosis (26 ± 7, ± SD; n = 46). There was no significant difference (P > .5) in SP-IR levels among the 3 groups of patients with BM fibrosis (MF, IMF, or non-MPD) (Figure 1A). The common feature of these 3 groups of patients is the development of BM fibrosis, though the underlying causes are different: MPD and non-MPD, or reactive and nonreactive fibrosis. Data indicate that increased SP-IR level correlates with BM fibrosis rather than with the type of underlying disorder.
Immunoprecipitation of SP-IR in the sera of patients with BM fibrosis Because SP is a small peptide and is the substrate for several endogenous endopeptidases,40 we next determined whether its presence in the circulation could be due to protection from other complexed proteins. We verified this by determining the approximate molecular weight of the circulating SP-IR by immunoprecipitation. We incubated sera that contained more than 10 pg/mL SP-IR (Figure 1) with anti-SP and then performed Western blot analysis with the immune complexes in nonreducing conditions. Representative results are shown in Figure 2A, and the results of all samples are summarized in Table 2. Strong bands at approximately 225 kd were detected in the samples containing more than 25 pg/mL SP-IR (Figure 2A). We also observed another band at 160 kd, the predicted size for IgG (data not shown). The 160-kd band was confirmed as the precipitating IgG by electrophoresing the samples in the presence or absence of reducing agent ( -ME) and then blotting with the second antibody. In the presence of -ME, we observed bands
at 55 kd (not shown), which is equivalent to the predicted size of a
single heavy chain for IgG.
Because the developed bands shown in Figure 2A were not at the predicted size of SP, we next determined the specificity of the reaction. Western blot analysis was repeated with anti-SP that was pre-absorbed with SP (undiluted antibody incubated with 0.1 mg SP for 2 days at 4°C). Previously developed bands disappeared with the pre-incubated antibody indicating specificity for SP. Representative results are shown in Figure 2B. Results described in this section showed that SP-IR is detected in the sera of patients with BM fibrosis, though at a high molecular weight. These results led to the hypothesis that the increased level of circulating SP-IR in patients with BM fibrosis is complexed to one or more molecules. Screening of cDNA libraries for structural analogues of NK-1 The predicted molecular weight of SP and its natural receptor is approximately 80 kd in nonreducing conditions.46 However, because we consistently observed bands heavier than 200 kd, we investigated whether SP could be bound to a homologue of NK-1 or to a molecule that shares homology with NK-1. To address this, we screened 3 cDNA libraries IL-1-stimulated BM stroma, SCF-stimulated BM
stroma, and pooled unstimulated BM mononuclear cells. DNA from clone 1 was sequenced in the forward and reverse directions and determined to
be 97% homologous with the ED-A region of FN.47 BESTFIT
of clone 1 with NK-1 cDNA44 indicated alignment with exon
5 of NK-1. Sequences spanning nt 1045 to 1061 shared 81% homology with
clone 1 and 71% with nt 951 to 992 (Figure
3). DNA analyses were performed with the
Wisconsin Genetics Computer Group (Madison, WI) package of DNA-protein
sequence analysis programs (version 10). Results indicated that 2 areas
of NK-1 cDNA shared significant homology with the ED-A region
of FN.
Co-precipitation of FN and SP in the sera of patients with BM fibrosis We next determined whether FN could be the carrier protein complexed to SP in the sera of patients with BM fibrosis (Figure 2). Immunoprecipitation with anti-SP was performed, and the complex was determined for the co-migration of SP and FN in Western blots. Indeed, it was shown by consecutive blotting for SP and FN that the developed bands could be superimposed at the same molecular weight. Representative blots are shown in Figure 4, and the results of all experiments are summarized in Table 2. Twenty-five patients with myeloproliferative disorders and BM fibrosis showed complexes of SP and FN. Although the total number of patients was low (n = 6) for non-MPD with BM fibrosis, all showed significantly high levels of SP-IR (Figure 1, Table 2). However, only 3 were complexed to FN, and the other 3 migrated to the predicted size (Table 2). Because immunoprecipitation was performed with anti-SP, the zero values in the numerator in Table 2 indicate that SP was not complexed to FN. These results indicate that for most of the patients with BM fibrosis, elevated circulating SP-IR is complexed to FN.
Possible interactions between SP and fibronectin We assigned the N-terminal region of SP structure, SP 1-4, as the head and the C-terminal, SP 5-11, as the tail. Both phenyl rings of the adjacent Phe residues are optimal when they are in trans, and their position lowers the probability of folding between the head and tail ends. Both prolines create a kink in the head, and the electrostatic interaction between the positive and negative residues helps to stabilize the kink. We used SYBYL to dock SP to the crystal structure of repeats 3 to 10 of FN (Protein Data Bank, 1FNF).45 This complex was based on the structural, electrostatic, and secondary structural characteristics of both SP and FN. Figure 5B shows the electrostatic potential of SP, outlined in white, to be relatively positive, which is complimentary to the electronegative potential of FN in the binding cleft. The amino terminal head region is shown at the top of SP (marked in white outline). The area of interactions is within repeat 7 of FN, thus leaving the RGD region exposed outside and available for interactions with the family of integrins.45
In this study, we describe a potentially novel mechanism for the
development of BM fibrosis. Data show a strong correlation between
increased levels of SP-IR in the sera and advanced BM fibrosis (Figure
1). This correlation is irrespective of the underlying cause of BM
fibrosis. However, because the grade of BM fibrosis was greater than 3 in each of the subject groups with fibrosis, a correlate between the
degree of fibrosis and SP-IR levels cannot be determined. Results in
this study add to the complex mechanism reported for patients with MPD,
with or without BM fibrosis.1 The major question that
arises from this study is the anatomic and cellular source of SP, which
could be produced by neural and nonneural cells.28 One
likely source is the macrophage, increased in MPD and activated in BM
fibrosis.6,17,48 Because macrophages express
NK-1,49-51 our findings might explain a potential
mechanism for the activation of circulating monocytes reported for
patients with myelofibrosis.6,52 Interactions between
circulating SP and NK-1 could induce cytokines.28 Released
cytokines could stimulate cells through an autocrine mechanism to
activate NF- The role of SP as an activating signal for monocytes in patients with BM fibrosis is a subject of current investigation in our laboratory. In particular, we are trying to determine whether monocyte activation or cytokine production by monocytes in patients with myelofibrosis6,52 could be partly mediated by the increased levels of circulating SP. We recently cloned the human PPT-I promoter and found that the activation of BM cells leads to autostimulation, similar to the concept of autoreceptors, described for neurotransmitter release.54,55 We are now studying whether similar autostimulation could occur in the macrophages of patients with myelofibrosis. The findings of this study could help unravel an alternative or synergistic pathway with other identified mechanisms for the activation of monocytes in myelofibrosis.6,52 Despite the angiogenic function of SP,29 this study does not prove that SP is directly involved in the increased angiogenesis and osteosclerosis seen in patients with BM fibrosis. Therefore, further studies are required to show a cause-and-effect relation between the pathophysiology of myelofibrosis and SP levels. Identification of FN as the carrier molecule for SP provides another
mechanism in which the ubiquitous extracellular matrix proteins could
be involved in BM fibrosis.14,16 Furthermore, this study
also demonstrates a function for the increased FN demonstrated in the
BM of patients with myelofibrosis.41 Interestingly, Reilly et al41 showed that enhanced distribution of FN correlated
with hypercellularity and vascularity of the BM, both of which could be
partly attributed to functions exerted by SP.28-30 As
entities, SP and FN are mitogenic to fibroblast. However, a biologic
response by the FN-SP complex cannot be eliminated because other
authors have shown enhanced chemotactic functions of the FN-TNF- TGF- If future studies verify a role for PPT-I in BM fibrosis, it would not be unique because dysregulation of this gene is implicated in the pathophysiology of several diseases and infections.43,49,60-62 Current treatments for myelofibrosis have shown modest promise, but there is an obvious need for better treatments.1,25,26 This report, which points to the necessity for further research, might very well add to the understanding of the complex mechanism by which BM fibrosis occurs. If substantiated, such insights could begin to provide a direction toward targets that may be useful in cytoreduction and ultimately restoration of the BM microenvironment. This is particularly important in light of the proliferative effects of SP on the 2 BM cell populations relevant to fibrosis: fibroblasts and hematopoietic progenitors.28
Submitted October 2, 2000; accepted January 9, 2001.
Supported by grants from the National Institutes of Health (HL-54973, HL-57675) and the National Cancer Institute (CA89868).
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: Pranela Rameshwar, Dept of Medicine-Hematology/Oncology, UMDNJ-New Jersey Medical School, MSB, Rm E-579, 185 South Orange Ave, Newark, NJ 07103; e-mail: rameshwa{at}umdnj.edu.
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Top
Abstract
Introduction
70°C until analysis. Participants, summarized in Table
, other MPD, n = 19; 
, IMF, n = 15)
or without BM fibrosis (
, n = 19). Non-MPD with fibrosis
(
, n = 10) or without fibrosis (
, n = 27). NC,
healthy controls (n = 30). (B) Grouped data from patients, shown in
panel A: BM fibrosis (3-4+ reticulin fibers) versus no fibrosis.
Results are expressed as mean ± SD. *P < .01 versus
no fibrosis.
B, a transcriptional factor activated in the monocytes
of patients with myelofibrosis.