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Prepublished online as a Blood First Edition Paper on July 25, 2002; DOI 10.1182/blood-2002-04-1270.
RED CELLS
From the Department of Genetics, Biology and
Biochemistry, University of Turin and the San Giovanni Battista
Hospital, Experimental Medicine Research Center, Turin,
Italy; the Department of Biological Sciences, University
of South Carolina, Columbia; and the Department of Molecular and
Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY.
Intravascular hemolysis is associated with several pathologic
conditions that include hemoglobinopathies, trauma, malaria, and
bacterial infections. Among plasma-protective proteins against oxidative damage caused by red blood cell rupture, haptoglobin and
hemopexin are thought to play a crucial role. Haptoglobin and
hemopexin, by binding with high-affinity hemoglobin and heme, respectively, exert an antioxidant action by preventing heme-catalyzed free radical production. Moreover, these proteins prevent iron loss by
inhibiting glomerular filtration of hemoglobin and heme diffusion
through plasma membranes. Analysis of single-null mice demonstrated the
antioxidant action of haptoglobin and hemopexin in vivo and suggests
that the 2 proteins cooperate in the resolution of hemolytic stress. To
evaluate the physiological relevance of the haptoglobin-hemopexin
system and the principal targets of its action, we generated
haptoglobin-hemopexin double-knockout mice and analyzed them under
basal conditions and after acute hemolysis. Whereas
haptoglobin-hemopexin double-null mice displayed no obvious alteration
in phenotype under basal conditions, nonlethal hemolytic stress in
these animals led to pronounced splenomegaly as well as liver
inflammation and fibrosis. These data demonstrate that haptoglobin and
hemopexin together are essential for protection from splenomegaly and
liver fibrosis resulting from intravascular hemolysis.
(Blood. 2002;100:4201-4208) Haptoglobin (Hp) and hemopexin (Hx) are plasma
proteins with the highest binding affinity for hemoglobin (Hb)
(Kd Hb is the most abundant and functionally important protein in
erythrocytes. However, once released from red blood cells, it becomes
highly toxic because of the oxidative properties of heme (protoporphyrin IX and iron), which participates in the Fenton reaction
to produce reactive oxygen species causing cell injury.3 The toxicity of heme is increased by heme hydrophobicity, which enables
it to intercalate into lipid membranes and other lipophylic compartments when not associated with proteins.4
Interestingly, in accordance with a pro-oxidant effect of heme, recent
data 5 provide evidence of a proinflammatory role in vivo.
These data show that intravenous heme administration results in
increased vasopermeability, adhesion molecule expression, and tissue
infiltration of leukocytes, which are the hallmarks of inflammation.
Usually, low amounts of extravascular hemolysis occur during
enucleation of erythroblasts and destruction of senescent erythrocytes, thus causing Hb release into plasma. However, under various
intravascular hemolysis-linked pathologic conditions, such as
hemorrhage, hemoglobinopathies, ischemia reperfusion, or malaria, large
amounts of free Hb are released.6 Once in the plasma, free
Hb rapidly dissociates in When the buffering capacity of plasma Hp is exceeded, Hb is quickly
oxidized to ferrihemoglobin, which, in turn, dissociates into globin
and ferriheme. Ferriheme then binds to albumin (Kd In the cells, the toxic effects of heme are counteracted by heme
oxygenase (HO), which breaks down the porphyrin ring into carbon
monoxide, iron, and biliverdin.9 Iron is rapidly
sequestered by ferritin, whereas biliverdin is converted by biliverdin
reductase to bilirubin. Hitherto, 3 isoforms of HO have been
identified, among which HO-1 is highly inducible by a great variety of
stimuli other than heme, including oxidative stress, heat shock, UV
radiation, ischemia reperfusion, heavy metals, cytokines, and nitric
oxide. HO-2 and HO-3 isoenzymes are constitutively expressed and
probably function in the normal heme capturing and
metabolism.10
We have previously reported the generation and analysis of Hp-null and
Hx-null mice.11,12 These studies have shown that both Hp
and Hx protect against the effects of intravascular hemolysis. This is
evident from the observation that both Hp-null and Hx-null mice, after
a strong hemolytic stress, suffer from renal injury due to oxidative
damage. Moreover, Hx-deficient mice present an altered Hp turnover
after hemolysis, since Hp persists in the circulation for several days
after the hemolytic stimulus.
In the present study, we show that after acute hemolysis, Hx is
up-regulated in Hp-null mice, similar to Hp up-regulation in Hx-null
mice. This suggested that the 2 proteins have a redundant function in
relation to each other. To test this hypothesis, we generated HpHx
double-knockout (dKO) mice and analyzed them under basal conditions and
after acute hemolysis, comparing their response to that of wild-type
and single-knockout mice.
HpHx dKO mice displayed no obvious alterations in Hb and heme
catabolism in a physiologic state but, after acute hemolysis, showed
enhanced splenomegaly and liver inflammation and fibrosis compared to
wild-type and single-null mice.
Generation of HpHx dKO mice
Phenylhydrazine treatment
Hb injection Blood was collected from anesthetized mice by retro-orbital bleeding. The red blood cells were purified by serum gradient, washed twice with PBS, and lysed by freezing/thawing. After centrifugation in a microcentrifuge for 20 minutes at 4°C for removal of red blood cell debris, Hb was quantified using a kit from Sigma (S527-A; Sigma), and the concentration was adjusted to 200 mg/mL with PBS. Age-matched adult males were injected into the tail vein with purified Hb at 0.5 mg/g body weight.Splenectomy Age-matched adult males of all genotypes were anesthetized with Avertin (2,2,2-tribromoethanol; Sigma-Aldrich, St Louis, MO). After surgical skin preparation, the spleen was exteriorized through a 1-cm left subcostal incision. The splenic artery and vein were double ligated, and the spleen was removed. The peritoneum and skin were closed in separate layers, using 4.0 absorbable suture. Mice were rested for 4 weeks before histological analysis.Histology and immunohistochemistry Tissues were dissected, fixed in 10% formalin for 24 hours, and embedded in paraffin. Microtome sections, 7 to 10 µm thick, were mounted onto TESPA (3-aminopropyl-thiethoxysilane; Sigma)-treated slides. Kidney sections were stained with hematoxylin and eosin, periodic acid Schiff (PAS), or Perls staining, according to standard procedures. The following parameters were chosen as indicative of morphological renal damage: brush border loss, red blood cell extravasation, and tubule dilatation. Liver sections were stained with hematoxylin and eosin or Masson Trichrome reaction. Leukocyte infiltration, hepatocyte degeneration, necrosis, and fibrosis were analyzed as parameters of liver injury.For immunohistochemistry, mice were perfused with PBS, organs
dissected, fixed in 10% formalin, and embedded in paraffin. Microtome
sections, 7 to 10 µm thick, were analyzed with the following antibodies: rat monoclonal anti-mouse F4/80 antigen (MCA497R; Serotec,
Oxford, United Kingdom), rabbit polyclonal anti-mouse hemoglobin
(catalog no. 55447; ICN, Irvine, CA), and rabbit polyclonal anti-human
HO-1 (SPA-896; StressGen, BC, Canada), which cross-react also with the
mouse proteins. Briefly, the sections were deparaffinised, rehydrated,
and treated as follows: 10 minutes with 0.1% triton in Tris
[tris(hydroxymethyl)aminomethane]-buffered saline (TBS), 10-20 minutes with 3% hydrogen peroxide solution, 5 minutes with TBS,
saturated with blocking buffer (3% milk, 10% normal swine serum in
TBS) for 20 minutes, followed by antibody incubation overnight at
4°C. The following secondary antibodies were used: biotynilated swine
anti-rabbit IgG and biotynilated rabbit anti-rat IgG (Dako A/S,
Denmark). Immunoreactivity was detected with the StreptABComplex/HRP
system (Dako) and developed with DAB (methanol 3,3' diamino-benzidine;
Boehringer Mannheim, Mannheim, Germany). The slides were then
counter-colored rapidly with hematoxylin and mounted with DPX (BDH
Laboratory Supplies, Leicester, United Kingdom). Liver cells
counts were made on a microscope at × 20 magnification using an image
analyzer (Image Pro Plus 4.0). Positive cells were counted on 4 randomly chosen fields within an area of 7 × 10 Western blot To determine Hp and Hx levels, 1 µL plasma, collected from the tail vein, was separated on 6% (Hp) or 10% (Hx) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotted onto nitrocellulose membranes (Amersham, Buckinghamshire, United Kingdom), and probed overnight with a goat antiserum against human Hp (Sigma H 5015; Sigma) or with a goat antiserum against human Hx (AES-217; Harlan Sera-Lab, Crawley Down, Sussex, United Kingdom) that also cross-react with mouse Hp or Hx, respectively. Filters were then incubated with horseradish peroxidase-conjugated rabbit anti-goat IgG (Southern Biotechnology Associates, Birmingham, AL) and developed with an enhanced chemiluminescence (ECL) detection system (Amersham). Quantitation of the band intensity was performed by densitometry using a Biorad system (Biorad, Munchen, Germany).For HO-1 detection, protein extracts were made by homogenization in 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 5 mM NaCl, and 50 mM EDTA (ethylenediaminetetraacetic acid) with protease inhibitors (aprotinin, leupeptin, pepstatin; Sigma). Protein concentration was determined with the Biorad protein assay system (Biorad), and 50 µg protein extracts were separated on a 12% SDS-PAGE, blotted onto nitrocellulose membranes (Amersham), and probed with a rabbit polyclonal antibody to HO-1 (SPA-896; StressGen). Filters were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Southern Biotechnology Associates) and developed with an ECL detection system (Amersham). RNA slot blot Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden, Germany) and analyzed as described by Sambrook et al.13 Filters were sequentially probed with -32P dCTP random primer labeled probes to mouse serum
amyloid A (SAA3) and P (SAP) components, rat 1-acid
glycoprotein (-AGP), and mouse  -actin. Quantitation of the slot
intensity was performed by densitometry, using a Biorad system (Biorad).
Blood and urine analyses Blood was collected from anesthetized mice by retro-orbital sampling, and blood cell counts were determined using an automatic cell counter. Renal damage was monitored by following blood urea nitrogen (BUN) concentrations in serum with a diagnostic kit (Sigma). Urine Hb content was measured with the Bayer Hemastix system (Bayer, Milano, Italy).Statistical analysis All data were expressed as mean ± SD or mean ± SEM and analyzed with the Student t test. Differences were considered significant when P < .05.
Analysis of basal and induced levels of Hp and Hx in single-knockout mice Wild-type and Hp and Hx single-null mice were subjected to acute hemolysis by intraperitoneal injection of a single dose of hyd of
0.15 mg/g body weight. Both basal and hyd-induced plasma levels of
Hp and Hx were analyzed by Western blotting.
The basal level of Hp was significantly higher in Hx-null than in
wild-type mice. Moreover, the
In Hp-null mice, the basal Hx level was comparable to that of wild-type
mice, but at 1 and 3 days after The overexpression of Hp and Hx, after hemolytic stress, in Hx- and Hp-deficient mice, respectively, suggested that in single-knockout strains, reciprocal compensatory effects could be taking place in the resolution of the acute phase. Generation of HpHx dKO mice HpHx dKO mice were generated by breeding single Hp and Hx knockout mice. The HpHx dKO mice were viable and fertile. Furthermore, histological analysis of liver, kidney, spleen, heart, brain, and bone marrow showed no evident abnormalities and/or tissue lesions due to oxidative damage, abnormal iron deposition, or inflammation (not shown), thus indicating that, under physiologic conditions, the simultaneous lack of Hp and Hx did not affect the clearance of free plasma Hb and heme.Induction of acute hemolysis HpHx dKO mice were then analyzed after hemolytic stress. We used the well-established model ofhyd treatment to induce acute hemolysis.14,15 Mice of all genotypes were injected
intraperitoneally with a single dose of hyd (0.15 mg/g body weight)
that is nonlethal. Hemolysis was evident on day 1 by hemoglobinemia,
hemoglobinuria, and marked depression in hematocrit level. On day 1, Hb
urine content was within 450 to 600 µg/L both in wild-type and Hp and Hx single- and double-knockout mice. Hematocrit fell to similar values
in all genotypes (Hp+/Hx+/: 25.6% ± 2.76%;
Hp /Hx+/: 24.6% ± 0.85%;
Hp+/Hx/: 21.03% ± 4.58%;
Hp/Hx/: 21.67% ± 2.35%, n = 3).
Recovery of blood parameters was almost complete 7 days after hyd
injection, as hematocrit returned to normal levels in all mice
(Hp+/Hx+/: 43.8% ± 5.52%;
Hp/Hx+/: 42.6% ± 5.3%;
Hp+/Hx/: 42.9% ± 4.92%;
Hp/Hx/: 44.3% ± 4.56%, n = 3),
thus indicating that the double mutation in Hp and Hx genes did not
affect either the severity of hemolysis or the recovery of normal
hematologic values.
Reduced Hb deposits in the kidney of HpHx dKO mice after acute hemolysis The kidney is considered to be the most sensitive organ to heme overload.3 Hb and heme overload were evaluated on perfused kidney sections by immunohistochemistry with an anti-Hb antibody and on renal extracts by Western blotting with an anti-HO-1 antibody. Renal histopathological changes were evaluated by PAS staining.On day 1 after
PAS staining did not reveal any obvious morphological abnormalities in mice of all genotypes, thus showing that Hb overload did not cause histopathological changes. Kidney function was assayed by determining BUN serum level in all
genotypes. BUN was equal in wild-type, single-, and double-null mice at
0, 3, and 7 days after Reduced susceptibility of HpHx dKO mice to a lethal hemolytic stress A lethal hemolytic stress was obtained by increasing the injected dose ofhyd to 0.2 mg/g body weight. Hemolysis was evident on day 1 by hemoglobinemia, hemoglobinuria, and marked depression in hematocrit
level to about 20% to 25% in all genotypes. Susceptibility to this
dose of hyd was significantly different in HpHx dKO mice compared
with the other genotypes: whereas 80% of wild-type and single-knockout
mice died within a week after injection, death occurred in only 50% of
HpHx dKO mice (Figure 3A). Mortality
occurred mostly in the first 3 days of hyd treatment.
Immunohistochemical and histological analyses showed Hb accumulation
near the apical membrane of proximal tubules of the kidney of dying
mice and evident signs of renal damage as tubular dilatation, brush
border loss, and flattened epithelium (Figure 3B). These data suggest
that renal dysfunction is the cause of death in these mice, in
agreement with previously reported data and clinical
observations.3,11,12
Interestingly, histological alterations were strongly attenuated or
absent in surviving mice, mostly in HpHx dKOs (Figure 3B), thus
indicating that the simultaneous lack of Hp and Hx protected double-null mice from acute renal failure by reducing Hb deposits on
the tubular cells of the kidney. Backing up these data is the observation that, in surviving mice, one day after Pronounced splenomegaly in HpHx dKO mice after acute hemolysis Because the spleen and the liver are the main sites for free plasma Hb and heme clearance after hemolysis, a detailed analysis of these organs was carried out.After acute hemolysis, mice of all genotypes developed splenomegaly.
Spleen-body weight ratio was measured under basal conditions and after
acute hemolysis (Figure 4A). On day 0, spleens of HpHx dKO mice were significantly larger than spleens of
wild-type mice (spleen-body weight ratio was 0.56% ± 0.02% vs
0.33% ± 0.03%, P < .001). Furthermore, after acute
hemolysis, enlargement of the spleen was much more significant in HpHx
dKO mice. The highest spleen-body weight ratio was reached on day 3 (1.47% ± 0.1% in HpHx dKO mice vs 0.98% ± 0.03% in wild-type
mice, P < .001). On the other hand, spleens of Hp-null
and Hx-null mice, under basal conditions, were similar to those of
wild-type mice (spleen-body weight ratio was 0.37% ± 0.01% and
0.38% ± 0.01%, respectively), whereas after acute hemolysis, they
were significantly lower than those of HpHx dKO mice: the highest
spleen-body weight ratio on day 3 was 1.22% ± 0.06% in Hp-null mice
and 1.08% ± 0.06% in Hx-null mice.
Histological analysis of HpHx dKO spleens, under basal conditions, did
not show any abnormality in cellularity and tissue organization in
spite of their larger size. However, the further enlargement in size
after The reticuloendothelial system was analyzed with an antibody against the macrophage lineage specific antigen, F4/80, and an anti-HO-1 antibody that labeled heme-activated macrophages. In all genotypes, most of HO-1-expressing macrophages were seen in the marginal zone around the white pulp. Histologically, no differences were observed, in either the number or the distribution of activated macrophages among HpHx dKO, wild-type, and single-knockout mice (Figure 4B). Marked liver inflammation in HpHx dKO mice after acute hemolysis Histological examination of the HpHx dKO liver revealed no abnormality under basal conditions. Moreover, in untreated animals of all genotypes, only few scattered HO-1-positive cells were detectable.On day 1 after injection of a nonlethal dose of
From day 1 to day 3 after The state of liver inflammation was analyzed 7 days after
Therefore, the simultaneous lack of Hp and Hx affected the reticuloendothelial system after acute hemolysis, resulting in a state of severe hepatic injury. The phenotype of single-null mice clearly demonstrated that overexpression of the remaining protein maintained a significantly lower number of heme-activated Kupffer cells than in HpHx double-null mice, thus causing only a limited inflammatory reaction. Pronounced liver inflammation in splenectomized HpHx dKO mice Marked liver inflammation and fibrosis seen in HpHx dKO mice 7 days afterhyd treatment indicated that, when spleen buffer capacity was exceeded, the simultaneous lack of Hp and Hx caused hepatic injury. To further confirm the anti-inflammatory action of Hp
and Hx, we used a different experimental model in which mice of all
genotypes were splenectomized and analyzed at increasing times after
surgical operation without pharmacological treatment. One and 2 months
after splenectomy, histological sections of the liver of Hp and Hx
single-null mice showed weak signs of inflammation, determined by
leukocyte infiltrates, compared with wild-type mice (not shown). In
contrast, the liver of HpHx dKO mice presented evident signs of
inflammation with cellular vacuolization, leukocyte infiltration, and
necrotic areas (Figure 7Ai-ii). Moreover,
2 months after splenectomy, the livers of most (5 of 7) HpHx dKO mice
showed marked fibrosis (Figure 7Aiii-iv). The state of systemic inflammation was also evaluated by determining the expression of other
acute phase proteins. The expression of serum amyloid A (SAA3) and P
(SAP) components and of 1-acid glycoprotein (-AGP) remained at the basal level in wild-type and single-null mice 2 months
after splenectomy, but increased several-fold in HpHx dKO mice (Figure
7B).
Thus, when the splenic filtering activity was missing, the simultaneous lack of Hp and Hx caused severe liver inflammation and fibrosis.
Hp and Hx, being scavengers of Hb and heme, respectively, from
circulation are considered plasma-protective proteins against intravascular hemolysis.3 In this study, we analyzed their positive effects by evaluating the response of Hp and Hx single- and
double-knockout mice to a nonlethal hemolytic stress. We showed that
the simultaneous lack of Hp and Hx caused significant alterations in
the spleen, liver, and kidney, whereas the single deficiency in either
Hp or Hx gene generated a phenotype quite similar to that of wild-type
mice. This is probably due to the overexpression of the remaining
protein in single-null mice, that is Hx in Hp Following acute hemolysis, the spleen of HpHx dKO mice became much more
enlarged than that of wild-type and single-null mice. Splenomegaly was
clearly evident 5 hours after The liver of HpHx dKO mice presented important alterations, after a
nonlethal hemolytic stress, compared with the other genotypes. Particularly, even if immunohistochemical data did not show differences in Hb accumulation after acute hemolysis, the liver of HpHx dKO mice
presented a higher number of HO-1-expressing Kupffer cells compared
with wild-type and single-null mice. Moreover, 7 days after Kupffer cells, together with sinusoidal cells, are thought to play
prominent roles in maintaining liver homeostasis.17 Our data demonstrate that, after acute hemolysis, HO-1 expression in
Kupffer cells, by degrading excess heme, provided the most important
protective system for the liver. Higher plasma levels of unbound Hb and
heme could account for the increased number of HO-1-expressing cells
in HpHx dKO mice, as well as for the state of liver inflammation.
However, several other factors could contribute to hepatic injury. The
state of activation of Kupffer cells has been associated with liver
injury, as these cells produce proinflammatory cytokines such as tumor
necrosis factor Therefore, in HpHx dKO mice, unbound Hb and heme, overexpression of HO-1, and the state of activation of Kupffer cells might contribute toward the high level of liver inflammation and fibrosis. The proinflammatory effects of Hb and heme were further evidenced by the analysis of splenectomized HpHx dKO mice. Indeed, when the liver took on the role of scavenging free Hb and heme from circulation, it became prone to developing inflammation and fibrosis. It could be possible that Hb and heme, not bound by Hp and Hx, respectively, account for this situation. The kidney of HpHx dKO mice, one day after The difference in Hb accumulation between wild-type and HpHx dKO mice
could be explained by a defect in Hb reabsorption and/or degradation by
tubular cells in HpHx dKO mice. According to this view, the Hp/Hx
system would affect the ability of tubular cells to take up and/or
degrade filtered Hb. On the other hand, there is an interesting
correlation between enhanced splenomegaly in HpHx dKO mice and the
reduction of Hb deposits in the kidney. These data suggested that Hb
accumulation in the spleen could protect the kidney from Hb overload
and could explain the reduced mortality rate of HpHx dKO mice compared
with the other genotypes. Indeed, when we used a lethal hemolytic
stress at the LD50 for HpHx dKO mice, 80% of wild-type and
single KOs died in the first 3 days from acute renal failure due to Hb
accumulation in proximal tubules of the kidney. Previously reported
data on Hp- and Hx-null mice had shown a higher mortality in
Hp-deficient than in wild-type mice and renal damage due to oxidative
stress in both Hp The protective effects of Hp and Hx on the liver could be explained by different mechanisms. One intriguing possibility is that these proteins modulate the inflammatory response by triggering specific intracellular signals. Consistent with this view is the recently reported identification of the CD163 antigen as the specific receptor for the Hb-Hp complex.7 CD163 is a member of the scavenger receptor cysteine-rich superfamily, restricted to the monocyte-macrophage lineage and is thought to have an important role in the down-regulation of inflammatory processes.21,22 On the other hand, although a specific receptor for the heme-Hx complex has not yet been identified, several in vitro studies have shown that, following exposure to heme-Hx complexes, important events involved in cell survival and response to stress take place, such as activation of the N-terminal c-jun kinase and nuclear translocation of NFkB.23 Therefore, characterization of these receptors and analysis of receptor-null mice will be useful in understanding the mechanisms of response to hemolytic stress. We conclude that Hp and Hx protect the spleen from excessive enlargement and the liver from inflammation and fibrosis in those pathological conditions that are characterized by hemolysis. Moreover, either of the 2 proteins alone can control spleen and liver homeostasis. Interestingly, in humans, a variant of the Hp gene has been reported, and the Hp2 allele has been associated with an increased risk for inflammatory complications.24,25 Our data support these observations, indicating that plasma levels of Hp and Hx can influence the inflammatory status in patients suffering from hemolytic disorders.
We thank S. K. Moestrup, M. Cirillo, M. Mostert, and V. Poli for critical reading of the manuscript; C. Camaschella for helpful discussion; and A. Gariboldi and A. Grillo for performing hematologic analyses.
Submitted April 29, 2002; accepted July 4, 2002.
Prepublished online as Blood First Edition Paper, July 25, 2002; DOI 10.1182/blood-2002-04-1270.
Supported by Contributo Consiglio Nazionale delle Ricerche-Ministero dell'Istruzione, dell'Università e della Ricerca (CNR-MURST) Legge 95/95 (L.S.), and by P. F. Biotecnologie CNR and MURST ex-60% (F.A.).
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: Emanuela Tolosano, Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5bis, 10126, Turin, Italy; e-mail: emanuela.tolosano{at}unito.it.
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Top
Abstract
Introduction
1 pM) and heme (Kd < 1 pM), respectively. They are expressed mainly in the liver and belong
to the family of acute phase proteins, the synthesis of which may be
induced by several cytokines following inflammatory
processes.
wild type
(Hp+/Hx+/), Hp-null
(Hp
