Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3148-3151
Serum Levels of Substance P Are Elevated in Patients With Sickle Cell
Disease and Increase Further During Vaso-Occlusive Crisis
By
Lisa A. Michaels,
Kwaku Ohene-Frempong,
Huaqing Zhao, and
Steven
D. Douglas
From the Divisions of Hematology, Immunologic and Infectious
Diseases, and Biostatistics and Epidemiology, The Children's Hospital
of Philadelphia, the Department of Pediatrics, and Joseph Stokes Jr
Research Institute, University of Pennsylvania School of Medicine,
Philadelphia.
 |
ABSTRACT |
As a mediator of neurogenic inflammation and pain, we hypothesized
that levels of the neuropeptide Substance P (SP) would be elevated in
patients with sickle cell disease (SCD) with vaso-occlusive pain
crisis. SP is a known stimulator of tumor necrosis factor-
(TNF-)
release and a promoter of interleukin-8 (IL-8), which are reported to
be increased in SCD. These cytokines enhance adhesion of leukocytes to
endothelium and may play a role in vaso-occlusive events. Serum levels
of IL-8, TNF, and SP were studied in three groups of children aged 2 to 18 years: 30 well children with SCD, 21 with SCD in pain crisis, and
20 healthy age-matched controls. Serum levels of SP were elevated in
all SCD patients and were highest in patients in pain crisis. The
percentage of sera with detectable levels of IL-8 (>5.0 pmol/L) was
increased in SCD patients as compared with the control group. IL-8
levels were similar for well SCD patients and those with pain. TNF
levels were not significantly different among the three groups. In
three children with SCD, SP was measured at baseline and again during
pain crisis. In each case, serum levels during pain crisis were higher
than they were when the patient was well. We conclude that levels of SP
are high in patients with SCD and increase during pain crisis. These
results imply that SP plays a prominent role in the pain and
inflammation of SCD and may be a measurable laboratory marker of
vaso-occlusive crisis. We speculate that neurokinin receptor
antagonists may have a therapeutic potential in the treatment of crisis
pain.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ALTHOUGH SICKLE CELL disease (SCD) was
the first inherited disease to be understood at the molecular level and
is one of the most common genetic diseases, the mechanisms underlying
its complications remain poorly understood. It remains a disease of
significant morbidity and mortality. The complications of SCD have
often been attributed to vascular occlusion,1,2 however,
there is growing evidence that inflammatory stresses within the
microvasculature may play a significant role. Further understanding of
the immune mediators involved may contribute to developing future
treatments and help identify patients at risk of severe complications
before they occur.
Vaso-occlusive pain crisis is one of the most common complications of
SCD3 with clinical manifestations similar to those seen in
other inflammatory disorders or with infection.4 Pain is
frequently severe enough to warrant the use of parenteral narcotics.
The affected area may be tender, accompanied by localized edema,
erythema, and warmth, and the patient may have systemic signs of
inflammation such as leukocytosis and fever.5 Consistent
with these observations, the acute phase reactants C-reactive
protein,6 1-glycoprotein, and transferrin7
are significantly elevated during painful crisis. High circulating
levels of the inflammatory cytokines tumor necrosis factor-
(TNF-), interleukin-1 (IL-1), IL-6, histamine, and leukotriene
B4 have also been described.8-12
The neuropeptide Substance P (SP) is an attractive candidate to explain
both the clinical findings and the cytokine elevations present during
vaso-occlusive pain crisis. SP is considered a major mediator of
neurogenic pain and inflammation.13,14 It has been
demonstrated to induce vasodilatation and plasma
extravasation.15 SP induces release of histamine from mast
cells,16 promotes secretion of several cytokines including
IL-1, IL-6, IL-8, and TNF-,17-20 and is itself a potent
chemotactic factor.21 Each of these functions suggests that
SP levels might be elevated during vaso-occlusive pain crisis. TNF-
and IL-8 are of further interest because their effects may impair blood
flow and impede recovery from ischemic episodes by increasing adhesion
of sickle red blood cells to endothelium22 and
modulate neutrophil:endothelial cell interactions.23
| |
MATERIALS AND METHODS |
Patients were recruited for this study from the outpatient Hematology
Clinic and the Emergency Department of the Children's Hospital of
Philadelphia, which is the site of a federal- and state-funded
Comprehensive Sickle Cell Center. Patients were asked to participate if
their hemoglobin genotype, as documented by hemoglobin electrophoresis
was SS, SC, or S-
° thalassemia. Patients were excluded if they
had received transfusion within the previous 6 months, if they were
receiving hydroxyurea or medications other than penicillin or folate,
or if they had a second chronic disease or inflammatory disorder. Well
children with SCD were seen during routine clinic visits and asked to
participate if there was no history of fever, pain, or infection for at
least 4 weeks before the day of study. In the Emergency Department,
children with SCD were recruited during evaluation for pain crisis. In
the majority of cases, patients had been treated with oral
acetaminophen and/or codeine at home. Patients were enrolled in
the study if the pain was not controlled by these medications and was
considered severe enough to require parental narcotics and, in most
cases, hospital admission. Control subjects consisted of healthy,
age-matched, hematologically normal children who were having blood
drawn for other purposes. All aspects of this study were approved by
the Committee for Protection of Human Subjects Institutional Review Board of the Children's Hospital of Philadelphia.
After written consent was obtained from the patient, or their parent,
venous whole-blood samples were obtained by a trained phlebotomist.
Blood from patients in pain crisis was drawn before initiation of
treatment. For cytokine assays, 5 mL of blood was drawn into a serum
isolation tube and immediately placed on ice. After 1 hour, the chilled
specimens were centrifuged at 2,500g for 15 minutes at 4°C.
The serum was carefully withdrawn using a plastic pipette, aliquotted
to fresh tubes, labeled, and frozen at 
70°C until use.
Measurement of IL-8 and TNF-.
Samples were thawed at room temperature immediately before assay.
Thawed samples were not refrozen or reused. Each sample was tested and
results confirmed in duplicate. Serum TNF- and IL-8 concentrations
were measured by sandwich enzyme-linked immunoassay using commercially
available kits (T Cell Diagnostics, Inc, Cambridge, MA for IL-8, and
Endogen, Inc, Cambridge, MA for TNF-). The kits were used according
to the manufacturer's procedure. Results were determined by measuring
the optic density of each well using a microtiter plate reader set at
an absorbance of 450 nm with a LABREPCO Elx 800 microplate reader
(Bio-Tek Instrument Inc, Horsham, PA). The detection limit of the
TNF- test kit is 1.5 pg/mL, and that for IL-8 is 2 pg/mL.
Measurement of SP.
Each freshly thawed sample was diluted with 4 vol of 4% acetic acid
and passed through a C-18 reverse phase column (Varian Sample
Preparation Products, Harbor City, CA) by gravity. The column was
washed five times with the 4% acetic acid solution to remove all
unbound material. SP was then eluted by the addition of buffer
containing 60% acetonitrile and 1% trifluoroacetic acid. Eluted
samples were dried with liquid nitrogen then reconstituted in distilled
water. In control experiments, overall recovery of SP was greater than
90%.24 The SP was quantified by the use of an antigen
competition, enzyme-linked immunoassay (Cayman Chemical Company, Ann
Arbor, MI). Each sample was run and results confirmed in duplicate.
Optical density was read at an absorbance of 405 nm. The limit of
detection of this assay is 5 pg/mL.
Statistical analysis.
For cytokine measurements, values had skewed distributions. Therefore,
all analyses were done either on the natural logarithm of the raw scale
(t tests, Pearson correlation coefficient) or using
nonparametric statistics (Wilcoxon rank-sum test). Statistical significance was declared if the P value was <.05.
Calculations were performed using Stata Statistical Software, Release
4.0 (Stata Corp, College Station, TX).
| |
RESULTS |
A total of 51 samples was obtained on 20 male and 28 female patients
with SCD, aged 2 to 18 years. Of the 51 samples, 30 were obtained
during a routine visit for comprehensive sickle cell care and 21 samples from children in pain crisis. Three children were studied both
during a routine evaluation and again before admission for pain crisis.
One sickle cell patient was white, the remainder were black. Control
subjects consisted of 20 healthy children aged 2 to 19 years. Of these
subjects, 16 were white and 4 were black.
Possible associations among serum levels of IL-8, SP, and TNF- were
examined, as well as relationship to demographics. Data which could not
be confirmed in duplicate because of an insufficient volume of sample
was excluded from analysis. No correlation was found for either age or
sex with any of the measured cytokine levels. To control for
differences in race between the control subjects and the patients with
SCD, comparisons were repeated and confirmed after excluding all white
subjects from data analysis. High SP levels correlated with high levels
of IL-8 (P < .001); however, no such correlation was found
for SP and TNF-, or TNF- and IL-8.
Figure 1 compares the data for controls and
patients with SCD. There was no significant difference in TNF-
levels across all groups. IL-8 levels were higher for all SCD patients
when compared with controls (P < .001), but no difference was
seen between well SCD patients and those in pain crisis (P = .37). Levels of SP were elevated in both groups with SCD when compared with normal controls. Well SCD patients had higher levels than controls
(P < .001) and levels were higher in SCD patients in crisis
than in those who were well (P = .003).
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| Fig 1.
Serum cytokine levels of IL-8, SP, and TNF
in healthy controls and patients with SCD. The measured cytokine levels
box-plots are shown by SCD status. ( ) Indicate outliers within each
serum cytokine; X, extremes.
|
|
In the three children on whom serum samples were measured at baseline
and again during pain crisis, in each case, the serum levels of SP
during pain crisis were higher than they were when the patient was
well. IL-8 levels increased in two patients, but fell in one. TNF-
did not markedly change in any of the three patients. These data are
shown in Fig 2.
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| Fig 2.
Serum cytokine levels of SP and TNF in three patients
with SCD when well and in vaso-occlusive crisis. Dotted
line indicates patient 1; dashes, patient 2; solid line, patient 3.
|
|
| |
DISCUSSION |
SP is the most extensively studied and characterized tachykinin, a
family of bioactive neuropeptides defined by their common pharmacologic
properties and conserved carboxyl-terminal sequences.25 Released by sensory nerves and by a variety of nonneuronal sources, such as endothelial cells, macrophages, and eosinophils,18
SP has an important function in the modulation of
nociception.15 Furthermore, SP and the other tachykinins
mediate the events of neurogenic inflammation, hyperemia, plasma
extravasation, and leukocyte adhesion to the vascular
endothelium.26 SP also induces bronchoconstriction and
mucus secretion.27 Because of these effects, neurokinin
receptor antagonists have been actively investigated as
analgesics28 and for the treatment of asthma.29
Sickle cell crisis pain is typically nociceptive in nature, and it is
likely that SP plays a significant role. The sequence of events in SCD
that lead to the sensation of pain are not understood, but are thought
to develop secondary to vascular occlusion. Sickle erythrocytes adhere
strongly to endothelium,30,31 suggesting that endothelial
cell injury and subsequent neutrophil activation play a role. The
resulting mediators of inflammation, such as histamine, bradykinin, and
prostaglandins activate or sensitize nociceptive afferent nerve
fibers.32 SP is released by afferent nerves after
appropriate stimulation and enhances the inflammatory response by
exerting direct effects on polymorphonuclear neutrophils and monocytes
and by promoting the release of other inflammatory cytokines.
Increased serum concentrations of SP during a painful event is an
expected finding, but the high baseline levels are not as easily
explained. This finding is consistent with data from prior studies,
which have concluded that there is a persistent inflammatory process in
patients with SCD.33 It is also likely that chronic asymptomatic endothelial injury is a contributing factor. Vascular intimal hyperplasia is frequent in these patients and is accepted as
the most important cause of stroke in SCD.34 Another
possible interpretation of this finding is the role of SP in the
control of hematopoiesis. SP receptors are present on hematopoietic
cells and SP enhances myeloid and erythroid colony formation via
induction of IL-1, IL-3, IL-6, and granulocyte-macrophage
colony-stimulating factor (GM-CSF).18,35 In SCD, patients
with low levels of fetal hemoglobin (HbF) (< 9%) have
high levels of GM-CSF and IL-3,11 and an inverse
relationship was observed between HbF and serum values of
TNF-.9 Such observations suggest that SP may be produced
in response to hematopoietic stress.18,35
Although SP promotes secretion of TNF-, by monocytes in
vitro,20 we were unable to reproduce the results of
previous studies, which reported elevated levels of TNF- in patients
with SCD.9,10 Several factors may account for this
discrepancy. In one of these studies,9 TNF- was measured
in plasma, whereas we used serum. We do not believe that this is a
contributing factor, as results in our laboratory showed nearly
equivalent levels of this cytokine in both plasma and serum. Assay
sensitivity may differ between our enzyme-linked immunosorbent assay
(ELISA) and those used in the other two studies, however, our assay was
sufficiently sensitive to detect TNF- in nearly all of the patients
and healthy controls studied. Our results are consistent with other
reports, which did not find significantly detectable levels of TNF-
in patients with the acute complications of SCD.4,36
Another possible confounding factor is that the age of our study
population was significantly younger than those in the previous
studies.9,10 Although our data does not demonstrate a
correlation between cytokine measurements and age in children under 20 years, there is evidence that cytokine levels do differ with age.
Abboud et al37 demonstrated high concentrations of IL-8 and
TNF- in adults with SCD, but neither was detected in serum from
children with this disease. We did detect increased levels of IL-8 in
our SCD patients as compared with healthy control subjects in whom IL-8
was not measurable.
Our results support the hypothesis that SP is an important mediator of
both the pain and the inflammation observed in sickle cell
vaso-occlusive crisis and demonstrates that measurement of serum SP
level is a useful laboratory marker of ongoing sickle cell crisis.
Furthermore, these findings suggest that neurokinin receptor
antagonists may have a therapeutic potential in the treatment of SCD
patients in pain crisis.
| |
FOOTNOTES |
Submitted February 3, 1998;
accepted June 30, 1998.
Supported by Grant No. MH49981 from the National Institutes of Mental
Health, National Institutes of Health, Bethesda, MD (to
S.D.D.).
Address reprint requests to Steven D. Douglas, MD, Children's Hospital
of Philadelphia, 34th and Civic Center Blvd, Room 1211 Abramson
Research Building, Philadelphia, PA 19194; e-mail:
douglas{at}email.chop.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
REFERENCES |
1.
Steinberg MH,
Darling BJ,
Lovell WJ:
Sickle cell anemia: Erythrokinetics, blood volumes and a study of possible determinants of severity.
Am J Hematol
2:17,
1977[Medline]
[Order article via Infotrieve]
2.
Francis RB,
Johnson CS:
Vascular occlusion in sickle cell disease: Current concepts and unanswered questions.
Blood
77:1405,
1990[Free Full Text]
3.
Plat OS,
Thorington BD,
Brambilla DJ,
Milner PF,
Rosse WF,
Vinchinsky E,
Kinney TR:
Pain in sickle cell disease: Rates and risk factors.
N Engl J Med
325:11,
1991[Abstract]
4.
Griffin TC,
McIntire D,
Buchanan GR:
High-dose intravenous methylprenisolone therapy for pain in children and adolescents with sickle cell disease.
N Engl J Med
330:733,
1994[Abstract/Free Full Text]
5.
Buchanan GR,
Glader BE:
Leukocyte counts in children with sickle cell disease.
Am J Dis Child
132:396,
1978[Abstract/Free Full Text]
6.
Singhal A,
Doherty JF,
Raynes JG,
McAdam KP,
Thomas PW,
Serjeant BE,
Serjeant GR:
Is there an acute-phase response in steady-state sickle cell disease?
Lancet
341:651,
1993[Medline]
[Order article via Infotrieve]
7.
Monnet D,
Diallo I,
Sangare A,
Yapo AE:
Interet clinique du dosage de la proteine C-reactive, de l'alpha 1-glycoproteine acide et de la transferrine au cours de la drepanocytoses homozygote.
Bull Soc Pathol Exot
86:282,
1993[Medline]
[Order article via Infotrieve]
8.
Ibe BO,
Kurantsin-Mills J,
Usha Raj J,
Lessin LS:
Plasma and urinary leukotrienes in sickle cell disease: Possible role in the inflammatory process.
Eur J Clin Invest
24:57,
1994[Medline]
[Order article via Infotrieve]
9.
Malave I,
Perdomo Y,
Escalona E,
Rodriguez E,
Anchustegui M,
Malave H,
Arends T:
Levels of tumor necrosis factor /cachetin in sera from patients with sickle cell disease.
Acta Haematol
90:172,
1993[Medline]
[Order article via Infotrieve]
10.
Francis R,
Haywood LJ:
Elevated immunoreactive tumor necrosis factor and interleukin-1 in sickle cell disease.
J Natl Med Assoc
84:611,
1992[Medline]
[Order article via Infotrieve]
11.
Croizat H:
Circulating cytokines in sickle cell patients during steady state.
Br J Haematol
87:592,
1994[Medline]
[Order article via Infotrieve]
12.
Enwonwu CO,
Lu M:
Elevated plasma histamine in sickle cell anemia.
Clin Chem Acta
203:363,
1991[Medline]
[Order article via Infotrieve]
13.
Frenk M:
The role of neuropeptides in pain
, in Nemeroff CD
(ed):
Neuropeptides in Psychiatric and Neurological Disorders.
Baltimore, MD, The Johns Hopkins University Press
, 1988
, p 199
14.
Moussaoui S,
Carruette A,
Garret C:
Further evidence that substance P is a mediator of both neurogenic inflammation and pain: Two phenomena inhibited either by postsynaptic blockade of NK1 receptors or by presynaptic action of opioid receptor agonists.
Regul Pept
46:424,
1993[Medline]
[Order article via Infotrieve]
15.
Pernow B:
Substance P.
Pharm Rev
35:85,
1983[Medline]
[Order article via Infotrieve]
16.
Okayama Y,
el-Lati SG,
Leiferman KM,
Church MK:
Eosinophil granule proteins inhibit substance P-induced histamine release from human skin mast cells.
J Allergy Clin Immunol
93:900,
1994[Medline]
[Order article via Infotrieve]
17.
Lotz M,
Vaughan JH,
Carson DA:
Effect of neuropeptides on production of inflammatory cytokines by human monocytes.
Science
241:1218,
1988[Abstract/Free Full Text]
18.
Rameshwar P,
Ganea D,
Gascon P:
In vitro stimulatory effect of Substance P on hematopoiesis.
Blood
81:391,
1993[Abstract/Free Full Text]
19.
Serra MC,
Calzetti F,
Ceska M,
Cassatella MA:
Effect of substance P on superoxide anion and IL-8 production by human PMNL.
Immunology
82:63,
1994[Medline]
[Order article via Infotrieve]
20.
Lee H,
Ho WZ,
Douglas SD:
Substance P augments tumor necrosis factor release in human monocyte-derived macrophages.
Clin Diagn Lab Immunol
1:419,
1994[Abstract/Free Full Text]
21.
Haines KA,
Kolanski SL,
Cronstein BN,
Reibman J,
Gold LI,
Weissmann G:
Chemoattraction of neutrophils by Substance P and transforming growth factor-1 is inadequately explained by current models of lipid remodeling.
J Immunol
151:1491,
1993[Abstract]
22.
Hebbel RP,
Moldow CF,
Steinberg MH:
Erythrocyte adherence to endothelium in sickle cell anemia: A possible determinant of disease severity.
N Engl J Med
302:992,
1980[Abstract]
23.
Lipowski HH,
Chien S:
Role of leukocyte-endothelium adhesion in affecting recovery from ischemic episodes.
Ann N Y Acad Sci
565:308,
1989[Medline]
[Order article via Infotrieve]
24.
Fehder WP,
Ho W-Z,
Campbell DE,
Tourtellotte WW,
Michaels LA,
Cutilli JR,
Uvaydova M,
Douglas SD:
The development and evaluation of a chromatographic procedure for the partial purification of substance P with quantitation by an enzyme immunoassay.
Clin Diagn Lab Immunol
5:303,
1998[Abstract/Free Full Text]
25.
Nakanishi S:
Substance P precursor and kininogen: Their structures, gene organizations, and regulation.
Physiol Rev
67:1117,
1987[Free Full Text]
26.
Lundberg JM:
Tachykinins, sensory nerves, and asthma 
an overview.
Can J Physiol Pharm
73:908,
1995[Medline]
[Order article via Infotrieve]
27.
Colten HR,
Krause JE:
Pulmonary inflammation a balancing act.
N Engl J Med
336:1094,
1997[Free Full Text]
28.
Henry JL:
Substance P and inflammatory pain: Potential of substance P antagonists as analgesics.
Agents Actions
41:65,
1993
29.
Joos GF,
Germonpre PR,
Kips JC,
Peleman RA,
Pauwels RA:
Sensory neuropeptides and the human lower airways: Present state and future directions.
Eur Respir J
7:1161,
1994[Abstract]
30.
Hoover R,
Rubin R,
Wise G,
Warren R:
Adhesion of normal and sickle erythrocytes to endothelial cells derived from umbilical veins.
J Clin Invest
54:872,
1979
31.
Vordermeier S,
Singh S,
Biggerstaff J,
Harrison P,
Grech H,
Pearson TC,
Dumonde DC,
Brown KA:
Red blood cells from patients with sickle cell disease exhibit an increased adherence to cultured endothelium pretreated with tumor necrosis factor.
Br J Haematol
81:59,
1992
32.
Ballas SK,
Mohandas N:
Pathophysiology of vaso-occlusion.
Hematol Oncol Clin
10:1221,
1996[Medline]
[Order article via Infotrieve]
33.
Kurantsin-Mills J,
Ibe BO,
Natta C,
Raj JU,
Siegel RS,
Lessin LS:
Elevated urinary levels of thromboxane and prostacyclin metabolities in sickle cell disease reflects activated platelets in the circulation.
Br J Haematol
87:580,
1994[Medline]
[Order article via Infotrieve]
34.
Francis RB:
Large-vessel occlusion in sickle cell disease: Pathogenesis, clinical consequences, and therapeutic implications.
Med Hypotheses
35:88,
1991[Medline]
[Order article via Infotrieve]
35.
Rameshwar P,
Gascon P:
Substance P mediates production of stem cell factor and interleukin-1 in bone marrow stroma: Potential autoregulatory role for these cytokines in SP receptor expression and induction.
Blood
86:482,
1995[Abstract/Free Full Text]
36.
Little FM,
Hess B,
Moore RB:
Changes in inflammatory cytokines and 1-acid glycoprotein in sickle cell subjects with acute chest syndrome.
Blood
88:12a,
1996(abstr)
37.
Abboud MR,
Ballas SK,
Habib D,
Xu F,
Laver J:
Levels of interleukin 8 are elevated in patients with sickle cell disease.
Blood
88:12a,
1996(abstr)
Abstract
Introduction
Abstract
