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Eur J Clin Chem Clin Biochem 1997; 35(9):647-654
© 1997 by Walter de Gruyter · Berlin - New York
REVIEW
Biological Functions of Haptoglobin - New Pieces to an Old Puzzle
Wanda Dobryszycka
Department of Biochemistry, Faculty of Pharmacy, Wroclaw University of Medicine, Wroclaw, Poland
Summary: Haptoglobin, an "acute phase" protein, has different functions, which display genetic polymorphism.
The complex of haptoglobin with haemoglobin is metabolized in the heaptic reticuloendothelial system. Biosynthesis of haptoglobin occurs not only in the liver, but also in adipose tissue and in lung, providing antioxidant and
antimicrobial activity. Changes in the measured concentrations of haptoglobin in serum may help to assess the
disease status of patients with inflammations, infections, malignancy etc. (increases) as well as in haemolytic
conditions (decreases). Haptoglobin plays a role in stimulation of angiogenesis and has highly potent cholesterolcrystallization-promoting activity. Probably the most important biological function of haptoglobin consists in the
host defence responses to infection and inflammation, acting as a natural antagonist for receptor-ligand activation
of the immune system.
Introduction
Haptoglobin is a genetically determined a2-acidic glycoprotein with haemoglobin-binding capacity, present in
most body fluids of humans and other mammals. A positive acute-phase reactant, its plasma level is increased in
inflammation, infections of different aetiology, trauma,
tissue damage and malignant proliferation, but decreased in haemolytic conditions and in severe hepatocellular deficiency. Thus, changes in the measured
concentrations of haptoglobin have been used in the
diagnosis and evaluation of treatment response under
various pathological conditions (for reviews see I.e.
(1-5)).
Despite years of investigations and a variety of the
known biological activities, the physiological functions
of haptoglobin have remained enigmatic.
Haptoglobin Phenotypes and Functional Properties
Essentially, three major phenotypic forms of human haptoglobin, designated Hp 1-1, Hp 2-2, and Hp 2-1 are due
to two alleles HP 1 and HP 2. In its simplest form Hp
1-1 is a tetrachain structure composed of two a1 (light,
Μτ 9100, each 83 residues) and two β (heavy, Mr 40 000,
each 245 amino acids, containing carbohydrate) chains
linked by disulphide bridges.
Haptoglobin 2-2 and 2-1 are polymerized forms of
higher molecular mass, showing multiple bands in polyacrylamide gel electrophoresis. The polymorphism of
haptoglobin was found to be related to the α subunits
(the a2-chain comprising 142 residues is almost a duplicate of the c^-chain, although in a cross-over event some
amino acid residues have been deleted). Hp 2-1 forms
a linear polymeric series which may be represented as
(α1β)2(α2β)η, where η = 0,1,2 ... The formula of the
Hp 2-2 polymeric series is (a2 )m, where m = 3,4, 5 ...
Figure 1 shows the structure and subunit arrangements
in Hp 2-1 (4).
Ν
« κ 1
ι
15 Ά
1
S
68
93
127 1
?A8
I
179 J90
I
I
2«
24, c
β
.·.
r α2 ι · Ί
'i^i/*-
form SH
polymers
..HHrs·2Π 7?
105
-S-S~
83 Γ
α 7 chain
179190
219
s-S-
(α'· β)2· (α*. β)π where π = 0,1,2....
Fig. 1 Scheme of the structure and subunit arrangements in haptoglobin phenotype 2-1 (4).
648
A method called "crossed haemoglobin electrophoresis"
(6) has been designed for the study of haemoglobin
binding with some of the individual polymeric molecules of haptoglobin subtypes and variants.
The oligosaccharide moiety of haptoglobin consists of
N-acetylglucosamine, mannose, galactose, fucose and
sialic acid. There are 8 sites of glycosylation, forming
bi- and triantennary N-linked glycans, which are present
in equal proportions (7).
Additional allelic variation of the HP 1 allele is due to
the HP IF and HP IS alleles, where F indicates fast and
S indicates slow migration during electrophoresis (8).
Homologous crossing over between HP 2 and either the
HP IF or the HP IS allele in HP 2/HP 1 heterozygotes
can change the usual type of HP 2 to three other forms:
HP 2SS, HP 2FF, and HP 2SF (9).
There is still no generally accepted satisfactory mechanism explaining the maintenance of genetic polymorphism in human populations. Interactions between
single- or multilocus genetic systems have been extensively examined in the search for evidence of selection
in humans.
Recently, several functional differences between haptoglobin phenotypes have been demonstrated, which appear to have important biological and clinical consequences (10). These include inter alia the reaction of
haptoglobin with haemoglobin (11), the antioxidant activity towards haemoglobin-stimulated lipid peroxidation (12), and the inhibitory effect on prostaglandin synthesis (13). In all these cases the strongest activity was
shown by haptoglobin type 1-1, in comparison with 2-1
and 2-2 types.
Haptoglobin Biosynthesis
Haptoglobin is known to be produced mainly in the
liver. Its expression is regulated at three different levels:
[a] developmental control for lack of expression in
foetal liver,
[b] tissue-specific control for selective expression in
hepatocytes, and
[c] modulation of expression during the acute phase
reaction (14).
Maximal expression of haptoglobin synthesis in the liver
requires cytokines (interleukins IL-1 and IL-6 type,
tumour necrosis factor TNF- , transforming growth
factor TGF-ß, leukaemia inhibitory factor LIF and others), and glucocorticoids, alone or in various combinations (15). Elevation of the haptoglobin gene transcription rate in acute phase liver relies essentially on an
increase in the binding affinity of hepatocyte regulatory
DNA-binding proteins. These include the family of
Dobryszycka: Biological functions of haptoglobin
nuclear factors and CCAAT-enhancer binding protein.
Isoform ß of the latter has been identified as a ligand to
the hormone response elements of haptoglobin (16).
Haptoglobin is expressed in specific cells of non-hepatic
origin, including adipocytes and lung cells. After inflammation had been induced in vivo, transcription of the
haptoglobin gene rose four-fold in lung (in mouse and
baboon), and six-fold in adipose tissue (in mouse), an
increase similar to that observed in the normal livers
(17, 18).
Unique exposure of the lung to the atmosphere and a
large number of potential injuries from chemical agents,
microorganisms, organic and inorganic dusts makes it
particularly vulnerable. Locally synthesized haptoglobin
provides a major source of antioxidant and/or antimicrobial activity in the mucous blanket as well as in the
alveolar fluid in the lung. This indicates a protective
role of haptoglobin against infection and in repairing
injured tissues.
Haptoglobin-Haemoglobin Complex
Haptoglobin combines specifically with haemoglobin,
the complex showing peroxidase activity in vitro. The
binding of haptoglobin to haemoglobin is one of the
strongest known non-covalent interactions in biology,
the association constant being greater than 10~15 mol/1
(4). The haptoglobin-haemoglobin complex in plasma is
rapidly cleared by the reticuloendothelial system in the
liver. Hence, iron from haemoglobin remains available
for further metabolic use. In haemolytic states (especially intravascular), haptoglobin levels are reduced. It
should be pointed out that such a decrease is considered
to be a secondary phenomenon, whereas a primary
decrease occurs during severe chronic hepatocellular deficiency (a failure in biosynthesis or in secretion by hepatocytes). Moreover, hypohaptoglobinaemia may result
from dissolution of haematomas and other foci of internal haemorrhage (19). Haptoglobin cannot be considered as a regular transport protein, but is regarded as a
suicidal one. On the other hand, haemoglobin binding is
apparently not an essential function, since the condition
of ahaptoglobinaemia is clinically silent. Moreover, under normal conditions there is more than enough circulating haptoglobin to bind the intravascular free haemoglobin that is formed daily. Some animals (e. g. cattle)
synthesize haptoglobin only in response to inflammation.
Formation of the haptoglobin-haemoglobin complex in
vivo can play a role in preventing the haemoglobindriven generation of hydroxyl radical and lipid peroxide
in areas of inflammation. On the other hand, haptoglobin
may bind analogous iron-binding proteins of microbial
origin and thus provide a novel microbicidal mechanism
Dobryszycka: Biological functions of haptoglobin
to augment the destruction of intracellular pathogens.
The haptoglobin-haemoglobin complex bound within
the phagolysosomes of phagocytic cells may generate
reactive oxygen species that are directly microbicidal
(20). The complex was found to display an inhibitory
effect on the endothelium-derived relaxing factor (nitric
oxide) (21).
Relevance of Haptoglobin in Clinical Medicine
Haptoglobin is present in most body fluids (serum,
urine, saliva, cerebrospinal fluid, amniotic fluid, ascites
etc.). Serum haptoglobin may be measured by determining the peroxidase activity of haptoglobin-haemoglobin
complex (the method of Jayle, who first described this
property (22)), electrophoresis (23), crossed affinoimmunoelectrophoresis with wheat germ agglutinin (24),
afrlnity-chromatography (25), immunodifrusion (26),
differential acid denaturation of haemoglobin and its
complex with haptoglobin (27), laser-immunonephelometry (28), ELISA system based on streptococcal haptoglobin receptors (29) etc. However, some of the methods are insufficiently precise for small amounts of haptoglobin, while others are based on arduous and expensive techniques which makes them of limited practical
value in clinical laboratories.
In our laboratory two immunosorbent ELISA systems
were developed, based either on the reaction with haemoglobin (Hb-Hp ELISA) (30) or with the lectin, concanavalin A (Con A-Hp-ELISA) (31). In both the systems,
antihaptoglobin antibodies (polyclonal or monoclonal)
are conjugated with horse-radish peroxidase. In the HbHp-ELISA, haemoglobin is bound to the β chain of haptoglobin; in the Con A-Hp-ELISA, the binding also occurs on the β chain, but the oligosaccharide moiety
(mannobiosyl core) is involved in the binding. These
methods enable the measurement of haptoglobin in concentrations higher than 5 μg/l. By means of another
ELISA test, it is possible to determine 10~10 mol/1 of
haptoglobin in biological fluids (32). Such highly sensitive methods are of special significance in measurements
of haptoglobin levels in biological fluids of low haptoglobin content, i. e. in cord sera and amniotic fluids (diagnosis of intrauterine foetal infections), effusions of
different aetiology (discrimination of transudative vs.
exudative ascites), in sera from haemolytic anaemias, in
the diagnosis of ahaptoglobinaemia, etc.
649
haptoglobin are significantly related to those of fibrinogen, absolute number of leukocytes, neutrophils, CD4+
Τ cells and the CD4+/CD8+ Τ cell ratio, and soluble
receptors of interleukins 6 and 2 (35). Ahaptoglobinaemia is common in the newborn but is present in 8090% of infants up to 3 months of age. This has been
attributed both to the haemolysis of foetal red cells and
to immaturity of the parenchymal cells and reticuloendothelial system of the liver, with respect to the biosynthesis and catabolism of haptoglobin, respectively.
Profound changes in serum haptoglobin level occur hi a
great variety of disease states. Haptoglobin should be
considered as a protein undergoing two pathophysiological phenomena: an increase in production during an inflammatory reaction and a decrease (as a primary phenomenon) in production during severe hepatocellular
deficiency (either a failure in biosynthesis or in the
secretion by hepatocytes), or a decrease (as a secondary
phenomenon) under haemolytic conditions (19, 36).
The haptoglobin serum level is increased up to 3—8 fold
in response to injury, i. e. surgery and burns, bacterial or
parasitic infections, ischaemic necrosis, connective tissue diseases, chemical irritants, malignancy (10, 33, 37).
Haptoglobin measurement may help hi these cases to
assess the disease status of patients, and to monitor the
effects of therapy. This has been applied satisfactorily
in serial analyses of serum haptoglobin in patients with
ovarian carcinoma receiving chemotherapy, indicating
duration of remission and/or recurrence of malignancy
(38). Pretreatment haptoglobin levels were correlated
positively with initial size of the ovarian tumour obtained during laparotomy (39). In diabetic patients under
different metabolic control (retinopathy, nephropathy),
haptoglobin levels were parallel to the cut-off value
(10%) of glycated haemoglobin (40). Evolution of the
haptoglobin concentration in serum has been observed
during the early phase of acute myocardial infarction
(41). Even localized infections, resulting in increased
inflammation and tissue loss in the periodontium, elicit
systemic changes manifested by increases in haptoglobin levels (42).
Low levels of haptoglobin are found in intra- or extravascular haemolysis, ineffective erythropoiesis, severe
liver disease, genetic factors in seemingly normal individuals, and in a number of abnormal haemoglobin
diseases such as sickle cell anaemia, haemoglobin C disMean values of haptoglobin concentrations in normal ease and thalassaemias; also after adverse transfusion
human serum obtained from various laboratories differ reactions and autoimmune haemolysis as in acquired
significantly (0.5-1.5, 0.75-1.75, 0.7-1.3, 0.5-2.2 haemolytic anaemia. In an acute haemolytic crisis all of
g/1). In part these variations are explained by the differ- the haptoglobin is usually depleted because of the one
ences in levels of the haptoglobin types as well as by way transit of the complex with haemoglobin to the
the dependence on sex (5, 10, 33). Recently, the IFCC- liver, where it is catabolized. The cut-off level of haptostandardized reference range of haptoglobin in serum globin (0.2 g/1) has been used in the differential diagno(0.3-2.0 g/1) was reported (34). Mean values in plasma sis of a variety of primary and secondary haemolytic
650
processes vs. anaemias without haemolysis (43). In patients exhibiting haemolysis and concurrent acute phase
reaction, it should be taken into account that the decline
in serum haptoglobin concentration occurs significantly
faster than the serum haptoglobin increase in the acute
phase (44). Also, evaluation of intravascular haemolysis
by haptoglobin administration followed by measurement
has been used in surgery for prosthetic valve replacement (45).
Patients with unipolar major depression exhibited significantly higher haptoglobin plasma levels than healthy
subjects and patients with minor depression. Subjects
with the haptoglobin phenotype 2-2 had significantly
lower haptoglobin levels than the Hp 2-1 and Hp 1-1
carriers. The frequency of phenotypes of the Hp-1 gene
was significantly higher in patients with major depression (46, 47). Major depression is characterized by
hyperhaptoglobinaemia, which in turn is largely dependent on the haptoglobin phenotype, being significantly
related to activation of cell-mediated immunity. This
altered distribution of haptoglobin phenotypes and genes
suggests that genetic variation on chromosome 16 may
be associated with that illness. Moreover, depressed
patients showed significantly lower basal hypothalamic
pituitary-thyroid levels than patients with normal haptoglobin levels (48-50).
Abnormal changes in the fucosylation of haptoglobin
may reflect early stages in the development of liver disease, and they can be used as a marker for alcoholic
liver disease, but not for excessive alcohol consumption
or non-alcoholic liver disease (7, 51). Similar changes
in the fucose content in haptoglobin were found in
patients with rheumatoid arthritis (52).
A possible therapeutic application of human haptoglobin
consists of the selective removal of free haemoglobin
(exceeding 2.3—3.5 g/1) from the blood of patients suffering from severe traumatic shocks. This has been
applied in the control of acute bleeding from oesophageal varices caused by treatment with ethanolamine oleate sclerosant, thereby successfully protecting against
renal damage (53). Prophylactic haptoglobin infusion
has been used in peripheral blood stem cell transplantation in order to avoid the elevation of free haemoglobin
and haemoglobinuria (54).
The haptoglobin polymorphism has been used in paternity cases in forensic science for several years. Now,
thanks to modern laboratory techniques, haptoglobin
subtyping can also be carried out in dried blood stains
(55).
The occurrence of polymorphism of the haptoglobin locus motivated many investigations directed at the determination of possible associations between haptoglobin
phenotype distribution and different disorders. Identifi-
Dobryszycka: Biological functions of haptoglobin
cation of persons at high risk for a disease on the basis
of haptoglobin phenotype could have been a rather stimulating goal of preventive medicine. Since a report on
the significant increase of haptoglobin type 1-1 among
leukaemia patients, compared with other cancer patients
as well as normal controls (56), numerous reports have
appeared (for comprehensive review see I.e. (10)).
Some examples follow: association of haptoglobin types
with serum lipids and apolipoproteins (increase of cholesterol and low density lipoprotein levels with an excess of HP 2 gene) (57), with sarcoidosis (excess of
HP 1 gene) (58), cirrhosis of the liver (excess of HP 1
gene) (59), chronic hepatitis C (excess of HP 1 gene)
(60), essential arterial hypertension (increase of Hp 2-2
phenotype) (61), haemoglobinopathies (increase in Hp
1-1 phenotype (62), immune response after hepatitis B
vaccination (decrease of immune response in subjects
with Hp 2-2), etc. (63).
Some of these investigations clearly show a significant
association, while others provide contradictory results.
For the time being, such inconclusive reports indicate
that the haptoglobin phenotype has little if any practical
use in clinical science.
Some Biological Activities
Recent data suggest that haptoglobin may have temporary but specific physiological activity, distinct from its
role in haemoglobin metabolism.
Haptoglobin is known to inhibit viral haemagglutination
and prostaglandin H synthase (64), to suppress lectinand polysaccharide-produced proliferation of lymphocytes and B-cell mitogenesis and to modulate macrophage function (65). Haptoglobin was found to agglutinate Group A of Streptococcus pyogenes, which possesses surface antigen T4. The reaction was phenotypedependent, i. e. haptoglobin 2-2 type showed higher activity than 2-1, while 1-1 type was inactive (29, 66).
A novel function of haptoglobin and the haptoglobinhaemoglobin complex is the induction of apoptosis of
hepatocarcinomatous Hep 3B cells (DNA fragmentation,
increase of transglutaminase expression). The antiproliferative cytotoxic effect of the complex is exerted via the
typical apoptotic pathway (67).
Stimulation of angiogenesis is a newly recognized biological function of haptoglobin. In experiments in vitro
and in vivo, the angiogenic effects of haptoglobin 2-2
was much more expressed than that of the 1-1 type,
while 2-1 type exhibited an intermediate activity. The
increased levels of haptoglobin found in chronic inflammatory conditions may play an important role in tissue
repair. In systemic vasculitis haptoglobin may also compensate for ischaemia by promoting development of collateral vessels (68).
651
Dobryszycka: Biological functions of haptoglobin
Biliary haptoglobin at its physiological concentration
has a highly potent cholesterol-crystallization-promoting
activity and thus becomes a candidate for major attention in understanding gallstone pathogenesis (69).
Trypanosomes are protozoan parasites of medical and
veterinary importance. Trypanosoma brucei rhodesiense
and Trypanosoma brucei gambiense infect humans,
causing African sleeping sickness. However, man is protected from T. brucei brucei, which can only infect animals, causing the disease Nagana in cattle. It was suggested that the protective factor may be "haptoglobinrelated protein" (70). However, recent data suggest that
the trypanolytic activity of normal human serum is due
to a second, less well-defined factor of high molecular
mass (71).
Haptoglobin may contribute by a humoral mechanism to
the bone resorption seen in chronic inflammatory lesions
such as rheumatoid arthritis, periodontitis and osteomyelitis (72). It has been shown that, beside paracrine stimulators of osteoclasts (e.g. IL-1, TNF, bradykinin,
thrombin), humoral factors induced by the inflammatory
process also participate in the resorption of bone. Haptoglobin stimulates prostaglandin E2 biosynthesis hi isolated osteoblasts, and it synergistically potentiates the
effect of bradykinin and thrombin. Thus it may contribute to the destruction of bone by inducing the formation
of prostanoids capable of stimulating bone resorption
(73).
Haptoglobin as a Ligand of Immune Cells
Exogenous haptoglobin is concentrated within granulocytes and monocytes in considerable quantities. It is not
synthesized de novo and is exocytosed following neutrophil activation. Neutrophils possess two specific binding
sites for haptoglobin with an affinity similar to that for
binding of the lectin, concanavalin A. Native haptoglobin blocks the human neutrophil response to a variety of
agonists with defined plasma membrane receptors. Of
the various functional parameters assessed, neutrophil
respiratory burst activity (superoxide production) and
the rise in intracellular calcium were inhibited by haptoglobin. This suggests that haptoglobin may act as a natural antagonist for receptor-ligand activation of the immune system (74).
Normal haptoglobin binds to granulocytes and monocytes. Among lymphocytes, haptoglobin predominantly
binds to cells that express CD lib/CD 18 receptor (NK
cells) and to a minor extent CD4+ and CD8+ cells (75).
The MAC-1 or CD lib/CD 18 molecule belongs to the
integrin family. These integrins play a major role in numerous biological processes such as inflammation and
immune function. Patients deficient for such receptors
suffer from persistent neutrophilia, pyogenic infections
and wound healing.
Modulation of granulocyte activity by haptoglobin is especially interesting when considering the existence of a
feedback loop among monocytes and hepatocytes in the
control of inflammatory response. Therefore, one of the
likely roles of haptoglobin seems to be in the host defence responses to infection and inflammation.
CD22 is a cell-surface receptor of resting mature B
cells that recognizes sialic acid (Sia) in the natural
structure, Sia(a2-6)Gal(ßl-4)GlcNAc. It probably directs potential interactions between mature B cells during inflammatory states. High affinity CD22 ligands
are subunits of immunoglobulin M and haptoglobin.
Such a mechanism may be biologically relevant (76).
The membrane carbohydrate antigen, sialyl Lewisx
[Sia(a2-3)Gal(ßl-4)Fuc(al-3)GlcNAc], is involved
in cellular adhesive interactions, in leukocyte trafficking, inflammation, thrombosis and probably in the metastasis of some tumour cells. Expression of this antigen in haptoglobin structure may modify disease processes or reflect some pathological changes (77).
Oncofoetal Haptoglobins
Analysis of the genomic clones for haptoglobin has resulted in the identification of a "haptoglobin-related protein" (Hpr) gene, which contains a number of retroviruslike sequences and is adjacent to the haptoglobin gene.
Hpr synthesis is not restricted to the liver: alternative
sites of synthesis were found to be the placenta or decidua, and breast cancer cells (5). Recently it was shown
that the Hpr gene is expressed in the human hepatoma
G2 and leukaemia molt-4 cell lines (78).
A macromolecular form of haptoglobin from ascitic
fluid of ovarian cancer patients (a haptoglobin gene
modified by the effect of malignant growth?) has been
shown to possess potent immunosuppressive properties
(79, 80). It might serve as a negative feedback regulator
of immune response produced by activated macrophages. Its production would be responsible for dysfunction of the immune system in cancer patients. Oncofoetal haptoglobins display similar functions in pregnancy
and neoplasia, aiding development of the placenta and
foetus while facilitating tumour invasion and metastasis
(modulating the host response to a malignancy so as to
enable it to survive and expand clonally). Specific
changes in the degree of branching of the carbohydrate
moiety hi "neoplastic" haptoglobin have been used in
measurements of concanavalin -bound haptoglobin in
the differentiation of malignant from non-malignant
ovarian tumours (81).
Despite over 50 years of research and the discovery of
multiple aspects of haptoglobin structure and function,
652
gaps and inconsistencies in our knowledge of this protein are readily apparent. Biological activities described
in the present article may give an impression of embarras de richesse. Haptoglobin seems to function in association with the immune system, i. e. to protect the host
Dobryszycka: Biological functions of haptoglobin
against all the dangers of an acute phase reaction. This
appears to be the most important function of this protein.
Our knowledge of this complex area continues to
evolve, but we are still arranging many pieces of the
puzzle.
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Received January 2I/March 10/April 4, 1997
Corresponding author: Prof. Dr. Wanda Dobryszycka, Department
of Biochemistry, Szewska 38/39, PL-50-139 Wroclaw, Poland