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Effect of sex hormones on the complement-related clinical disorder of hereditary angioedema.

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Hereditary angioedema i s a disease manifested
clinically by episodic attacks of swelling which tend to
last 3 days and which disappear without sequelae (1).
The angioedema is painless, nonpruritic, nonpitting,
brawny edema usually involving an extremity but
which can involve any portion of the trunk or head. At
times the angioedema attacks may involve the face and
pharynx. Swelling in the region of the face or pharynx
can lead to obstruction of the airway, and the incidence
of death in hereditary angioedema in all published series approaches 25%. Patients with this disease have, in
addition to peripheral swelling, attacks of severe abdominal pain. It has been shown that these attacks are
due to the development of edema in areas of the bowel
wall that is quite similar to the areas of edema in the extremities. Presumably the stretching of the bowel wall
leads to severe abdominal pain. It is interesting that the
attacks of abdominal pain may, but do not always, coincide with the attacks of peripheral swelling. The factors
which precipitate attacks of angioedema in this patient
group are not entirely clear. However, it is known that
many of the patients have a marked increase in attack
frequency at times of emotional stress, and in about half
of the patients trauma will precipitate an attack. One of
the most characteristic events which preceeds a lifethreatening attack of angioedema is dental manipulation and dental surgery. In this case the trauma begins
in the area of the mouth and the swelling may extend to
occlude the airway.
In the past the therapy of hereditary angioedema
From the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205.
Address reprint requests to Michael M. Frank, MD, NLH I,
LCl, Building 10, Room 1 IB12, Bethesda MD 20205.
Arthritis and Rheumatism, Vol. 22, No. 11 (November 1979)
was quite unsatisfactory. Unlike most of the angioedemas which are related to histamine release and mast cell
degranulation, these patients do not respond well to epinephrine, antihistamines, or glucocorticoids. Thus, even
now many textbooks of medicine say that there is no
therapy available for this disease.
There are a number of other interesting features
of this disorder. The disease follows a dominant inheritance pattern, and on the average 50% of the offspring
of a patient will also be afflicted with the disorder. However, not all patients with the biochemical defect characteristic of hereditary angioedema will manifest clinical symptoms. We do not know why some patients
manifest clinical symptoms and others with exactly the
same biochemical disorder do not.
The first major breakthrough in understanding
the pathophysiology of hereditary angioedema came in
the 1960s. Landerman and his colleagues noted that patients with this disease lacked a kallikrein inhibitor in
their Serum (2). Shortly thereafter, Donaldson found
that patients with the disorder lacked the inhibitor of
the first component of complement, C1 esterase (3). It
was soon shown that the inhibitor of Cl esterase also inhibited kallikrein, and this protein, the C1 esterase inhibitor, was found to be low or nonfunctional in patients with hereditary angioedema. Thereafter two
groups of patients were defined. All patients had low
functional levels of the C1 inhibitor. It was found that
about 85% of the patients had low levels of a protein
which appeared to be functionally normal. About 15%
of patients, however, had normal or elevated antigenetic
levels of a C1 inhibitor protein which appeared to be
Thus, there appeared to be two distinct types of
defects which could lead to this disorder: in one, the patient did not appear to have sufficient levels of the protein, and in the second, an abnormal protein was produced. The clinical symptoms and inheritance patterns
of the two sets of patients were identical. For these reasons, it was very difficult to see how a disease inherited
as a mendelian dominant disorder could lead to the development of these two types of protein abnormalities.
Most diseases characterized by protein deficiency are
inherited as a mendelian recessive disorder with heterozygous individuals having lowered levels of protein and
homozygous individuals having very low levels. In this
case the heterozygous individuals appeared to have the
disorder. Some rather complex schemes involving various suppressor genes, for example, were proposed to explain the inheritance.
The pathophysiologic basis of the attacks of hereditary angioedema has also been studied in the last
few years, and there is evidence that the disease is related to an abnormality in the complement system. The
complement cascade consists of a series of interacting
serum glycoproteinsthat are responsible for the production of many of the manifestations of inflammation and
under certain circumstances can be cytotoxic to target
cells. The early steps in the triggering of this pathway
involve the activation of a serum protein termed C1.
This protein acquires proteolytic activity on activation
which can be measured by showing the development of
ability to cleave a synthetic ester substrate. Thus, the activated C 1 is termed C 1 esterase. The function of C 1 esterase is to cleave the next two proteins in the complement cascade, C4 and C2, thereby continuing the
sequence of reactions. In serum there is normally present a protein termed the C1 esterase inhibitor which
functions to turn off the activated C1 once activation
has occurred. Thus this protein is a regulator of the
early steps of complement activation. The C1 esterase
inhibitor is deficient in hereditary angioedema.
Considerable information is now available about
the Cl inhibitor glycoprotein. It is known to be an acid
labile glycoprotein with a very high hexose content and
a molecular weight of about 100,OOO daltons. The site of
synthesis appears to be in the liver, although this fact is
based upon immunofluorescent analysis of tissue sections with anti-C 1 inhibitor antibody. Hepatocytes stain
with fluoresceinated anti-Cl inhibitor. The C1 inhibitor
protein functions as an inhibitor in many proteolytic
systems in serum and does not solely have the function
of inhibiting activated C1. Thus the inhibitor has inhibitory function in all of the Hageman factor dependent,
proteolytic enzyme pathways in serum including the fibrinolytic pathway, the clotting pathway, and the kinin
generating pathway. However, it is the only protein in
serum which efficiently inactivates activated C 1.
It is believed that attacks of hereditary angioedema are initiated by the activation of C1 in serum. It
has been shown in the test tube that plasmin can activate Cl, and one possible mechanism which has been
put forth is that small amounts of clotting lead to the
generation of small amounts of plasmin and it is this
plasmin that activates C1. In any case, once C 1 is activated in the serum of patients with this disorder, it is not
efficiently inactivated. The activated C1 cleaves C4 and
C2, its natural substrates. It is thought that cleaved C2
can interact with plasmin to generate a still smaller C2
cleavage fragment which has kinin-like activity and can
cause local edema formation. This kinin-like fragment
has been termed C-kinin.
There are ‘a series of observations which suggest
that endocrine factors play a role in this disease. However, it must be emphasized that this is a disorder which
affects males and females equally. It appears in early
life but tends to become much more severe at the time
of puberty in both males and females. In some women,
attacks of angioedema occur strikingly at the time of
menses, and some women have attacks of angioedema
only during their menstrual period, but this is not always the case. There is some variability about the effect
of pregnancy on attacks of hereditary angioedema;
however, in general, attacks tend to become less severe
in the last trimester of pregnancy and the trauma of delivery, to my knowledge, has never precipitated an attack. We made the observation that birth control pills,
particularly those high in estrogen content, make attacks of hereditary angioedema much more frequent,
and we have been able to treat some patients for this
disorder by simply discontinuing the use of birth control
pills. The most striking observation in terms of endocrine factors and hereditary angioedema, first reported
by Spalding in 1960, was that the drug methyltestosterone can ameliorate symptoms of this disorder
(4). Subsequent work showed that impeded androgens,
that is, androgens with reduced masculinizing effect including fluoxymesterone and oxymetholone, tended to
cause amelioration of symptoms (5). Because this disorder was much less severe at the time of delivery and
because of some other endocrine considerations mentioned, we decided to explore further the use of hormones in the treatment of this disease.
The first hormone we studied was the pro-
gestational agent provera. We treated patients with proVera in a double-blind study for a period of some
months. All our patients tended to feel better while taking provera, however, no statistically significant difference in attack frequency or severity was noted. We then
turned to other endocrine factors. We wanted a drug
that was derived from an anabolic or androgen-like steroid-like methyltestosterone but that could be given to
women without the difficulty of masculinizing side effects. Since increasing estrogen levels made the disorder
more severe, a drug that decreased estrogen levels was
also considered beneficial. Drs.Gelfand, Alling, and I,
in consultation with our endocrinologic colleague, Dr.
Richard Sherins, decided to study the effect of an
androgenic agent, danazol, which had not previously
been used in treatment of an immunologic disorder. In
our study patients were given danazol or a placebo in an
extensive double-blind trial (6). A total of 93 courses of
drug or placebo were given. Of 47 courses of placebo
therapy, 44 were marked by attacks of hereditary angioedema, and 3 courses were not attended by attacks. Of
46 courses of danazol therapy, 45 were attack-free and 1
was attended by an attack of hereditary angioedema.
Thus, the drug was strikingly effective in reducing attacks of this disorder.
Our first surprise occurred, however, when we
analyzed sera of patients receiving drug or placebo for
the levels of C4 and C1 inhibitor. The majority of patients with hereditary angioedema have low levels of the
C1 inhibitor. These patients have activated Cl in their
sera and this activated C1 cleaves C2 and C4, therefore
most patients have low levels of C2 and C4 in their sera
as well. We had previous experience with a nonendocrinologically active drug in treatment of this disease,
epsilon amino caproic acid, a plasmin inhibitor. A
double-blind study demonstrated the effectiveness of
this drug in controlling the disease, although there were
a number of difficulties in its use clinically. It was believed that the mechanism of action of this drug was to
prevent activation of plasmin and thereby prevent activation of C1 by plasmin. However, patients receiving
the drug did not have any alteration in their levels of C2
or C4. In striking contrast, we found that patients receiving danazol therapy had a return to normal of their
C2 and C4 levels in many instances. Even more striking
was the finding that many patients treated for hereditary angioedema with danazol had a return of serum
levels of C1 esterase inhibitor to normal (Figure 1).
Thus, this disorder, showing a dominant mendelian inheritance pattern and characterized by low levels of a
x 60 days
&A rnglday
800 mglday
2 1
' ' ' '
1 '
Figure 1. Response of a patient with hereditary angioedema to danam1 therapy. W-levels of CI esterase inhibitor performed on 3 occasions prior to therapy. The patient was then treated with 600 mg danaml/day for 60 days. 0 = level of the inhibitor protein on the various
days sampled. Dotted line represents the lower limit of normal. (Reprinted from the New England Journal of Medicine 295:1448-1448,
serum protein, could be completely corrected biochemically in many patients by treatment with an anabolic
steroid, danazol. The time course of correction was examined, and it was found that C l inhibitor levels
tended to rise fairly rapidly. In some patients levels began to rise within a day or two, and in most the effect
was maximal within a week. At about the same time Dr.
W. Rosse and his group were finding similar but more
modest changes in blood levels using other synthetic
androgens (7). At this time we still do not know the
mechanism of action of these drugs. The drug is probably not producing its effect by altering the level of
other hormones. Rather it appears likely that the mechanism of danazol action involves direct induction of C1
inhibitor protein synthesis by the hepatocyte, although
this point has not yet been satisfactorilyproved.
Study of the effect of drug therapy in patients
with hereditary angioedema can give us far greater insight into the genetic basis of this disorder (8). Perhaps
the most interesting information has come from a study
of a series of patients with abnormal C1 esterase inhibitor protein. As previously mentioned, these patients
synthesize a functionless protein, although C 1 esterase
inhibitor can be found in the serum in normal or elevated levels. We found the treatment of patients with
the abnormal protein was also attended by a relief of
symptoms. Moreover, C4 levels in these patients also
tended to return to normal. It was therefore of great interest to analyze the serum of these patients and deter-
mine the effect of the drug. First, we found that on danazol therapy patients with the abnormal protein variant
of hereditary angioedema developed an increased level
of C1 esterase inhibitory activity in their serum. It was,
of course, possible that the material which inhibited C1
esterase in the serum of these patients was not the protein Cl esterase inhibitor, but one of the other inhibitory proteins in sera. We therefore prepared an anti41
esterase inhibitor protein immunoabsorbent column.
Passage of a patient’s serum over such a column removed C1 esterase inhibitor and removed all of the inhibitory material from the serum. Thus, the material in
the sera of patients with the abnormal protein that inhibited Cl esterase was indeed the Cl esterase inhibitor.
Further analysis of the sera of these patients was
possible because of an interesting biochemical variant
present in some of them. As originally noted by Rosen
(9) and independently by Laurell (lo), some of the patients with the abnormal variant protein have a Cl inhibitor which is unusually heavy in molecular weight
and is bound in serum to albumin. Thus they have an
antigenetically normal, functionless protein which is
bound to serum albumin. O n crossed immunoelectrophoresis this protein can be easily identified because of its unusual electrophoretic mobility and its albumin association. We treated patients with the
albumin-bound protein with danazol. After the appearance of normal levels of C1 esterase inhibitor activity,
we took aliquots of the patients’ sera and passed them
over an anti-albumin immunoabsorbent column. We
found that the anti-albumin immunoabsorbent removed
a great deal of the C1 esterase inhibitor activity, presumably that bound to albumin. However, the level of
functional C 1 esterase inhibitor activity was unchanged
after removing the albumin-bound C1 esterase inhibitor. These two experiments proved not only that C1 esterase inhibitor was responsible for the inhibitory activity in serum, but also the albumin-bound protein was
not the protein responsible for the inhibitory activity.
The results suggested that the patients were synthesizing
normal Cl esterase inhibitor protein. It was possible to
take an aliquot of the serum and pass it over a gel filtration column and thereby remove the heavy albuminbound protein. We were able to show that on danazol
therapy a peak of normal C1 esterase inhibitor protein
appeared and increased in concentration over time. This
peak corresponded with the normal C1 esterase inhibitor functional activity.
When patients with the nonalbumin-bound variant protein were treated with danazol, they had an in-
crease in the antigenic level of C1 esterase inhibitor and
inhibitory activity appeared in their sera. In these patients it was impossible to separate the antigenetically
normal but inactive protein from the normal protein
which had the same molecular weight and electrophoretic mobility. However, careful quantitative
analysis suggested that the danazol therapy was leading
to the formation of normal, functionally active inhibitor
protein whose presence was simply masked by the abnormal, functionally inactive protein. Thus, both of
these variants responded in the same way to danazol
In all, these studies suggest that patients with hereditary angioedema have one normal and one abnormal gene. This would explain the dominant inheritance
pattern in which heterozygous individuals have the disorder. In the common genetic variant the abnormal
gene codes for no protein. Patients make C1 esterase inhibitor because of the presence of the one normal gene.
When C1 esterase acts to destroy the functional activity
of C1, it binds stoichiometrically to the C1 protein.
Present evidence suggests that as many as 4 molecules
of Cl esterase inhibitor bind to 1 activated C1. Thus,
when the C 1 esterase inhibitor functions, it binds the C1
and this complex can be cleared rapidly from the circulation. Such a hypothesis would explain why patients
with the usual form of hereditary angioedema have only
20 or 30940 of the normal protein rather than the 50%
that might be expected. There is high utilization of the
protein following its interaction with Cl esterase. Presumably 20% of the normal protein concentration in
serum is not sufficient to prevent attacks of angioedema.
The patients with the abnormal variant also have 2
genes, one coding for the normal protein and one coding for abnormal variant protein. The normal protein is
synthesized and used in a way similar to that in the
usual form of the disease. The abnormal protein is also
synthesized, but its serum concentration is not lowered
because it does not bind efficiently to activated C1.
Thus, it tends to accumulate in the circulation. When
the serum of such a patient is studied, the high levels of
abnormal protein are striking. It has not been previously appreciated that many of these patients have
lower levels of a normal functional variant.
In all, these studies demonstrate that a hormonal
probe can provide much information about an “immunologic’’ disease. These studies have been very rewarding because not only have they given us further insight
into hereditary angioedema, but they have provided relatively nontoxic therapy for this complex disorder. It is
hoped that further studies of the endocrinologic aspects
of this disease will provide much more information on
the role of hormones in the function of the complement
1. Frank MM, Gelfand JA, Atkinson J P Hereditary angioedema: the clinical syndrome and its management. Ann
Intern Med 84580-93, 1976
2. Landerman NS, Webster ME, Becker EL, Ratcliff H E
Hereditary angioneurotic edema. 11. Deficiency of inhibitor for serum globulin permeability factor and/or plasma
kallikrein. J Allergy 33:330-341, 1962
3. Donaldson VH, Evans RR: A biochemical abnormality in
hereditary angioneurotic edema. Am J Med 35:37-44, 163
4. Spaulding WB: Methyltestosterone therapy for hereditary
episodic edema (hereditary angioneurotic edema). Ann
Intern Med 53:73945, 1960
5 . Davis PJ, Davis FB, Charache P Long-term therapy of
hereditary angioedema (HAE): preventive management
with fluoxymesterone and oxymetholone in severely affected males and females. Johns Hopkins Med J 135:39198, 1974
Gelfand JA, Sherins RJ, Alling DW, Frank MM: Treatment of hereditary angioedema with danazol reverse of
clinical and biochemical abnormalities. N Engl J Med
295:1444-1448, 1976
Rosse WF, Logue GL, Silberman HR, Frank MM: The
eaect of synthetic androgens in hereditary angioneurotic
edema: alterations in C1 inhibitor and C4 levels. Trans
Assoc Ann Phys 89:122-32, 1976
Gadek JE, Hosea SW,Gelfand JA, Frank MM: Response
of variant hereditary angioedema phenotypes to danazol
therapy: genetic implications. J Clin Invest, 64:28&286,
Rosen FS, Alper CA, Pensky J, Klemperer MR, Donaldson VH: Genetically determined heterogeneity of the C1
esterase inhibitor in patients with hereditary angioneurotic edema. J Clin Invest 202143-2149, 1971
Laurell AB, Martensson 1U:Cl inactivator protein complexed with albumin in plasma from a patient with angioneurotic edema. Eur J Immunol 1:46, 197I
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sex, effect, complement, clinical, disorder, hereditary, related, hormone, angioedema
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