COMBINED CLINICAL AND BASIC SCIENCE SEMINAR Selected and edited by Richard T. Silver, M.D. and Alexander G. Bearn, M.D. Department of Medicine, The New York Hospital-Cornell
Medical Center, New York, New York
Genetic Aspects of Diseases of Complement: An Explosion
Lecturers: MARGARET J. POLLEY, Ph.D ALEXANDER
G. BEARN, M.D.
New York, New York
From the Department of Medicine, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York 10021. Text of a lecture delivered on October 9, 1973. Requests for reprints should be addressed to Dr. Margaret J. Polley.
THE CLASSIC METHOD OF ACTIVATION COMPLEMENT
OF
Dr. Margaret J. Polley: Complement was first described [l] as a factor in serum which was necessary to supplement antibody in the induction of lysis of foreign red cells and bacteria-a complement to antibody, hence the name. It differed from antibody by two of its properties. First, it was heat labile: and second, it was not increased upon immunization. When first described, complement was considered to be a single component. Not long afterwards it was shown that it was not a single component, but that it comprised two components and these two components were named in order of their reactivity-Cl and C2 [2]. A little later, a third component was described and the sequence of reactivity of the three components was Cl, C2 and C3 [3]. Just a year later, a fourth component was described and called C4 since this was the order of its discovery. However, the sequence of reactivity of the four components was Cl, C4, C2 and C3 [4]. This was the situation for close to 30 years. It was believed that the complement system was composed of four factors, Cl, C4, C2 and C3. Late in the 1950’s it was found that what was being termed C3 at that time was not a single component. It was, in fact, composed of six components [ 5-71. Various terminologies were introduced by the people who identified these components. However, it was finally decided [8] to label these components numerically according to the sequence of reactivity, C3 being retained for the first of these six components to react. Thus the components in order of their reactivity became Cl, C4, C2, C3, C5, C6, C7, C8 and C9. Cl also was found not to be a single component. It was composed of three components, and to prevent further complication of terminology, lettered terminology was retained for these proteins, Clq, Clr and C 1s [9]. Thus the 11 components of the complement system in order of their reactivity became Clq, Clr, Cls, C4, C2, C3, C5, C6, C7, C8 and C9. Each of the components is a distinct serum protein and, except for Clq, each in its native form lacks biologic activity. Something has to trigger off the system and subsequent to such a stimulus, biologic activity is produced sequentially in each of the components. The stimulus clearly
which
activates
dependent
January 1975
the
system
on the interaction
The American
is not fully understood between
Journal
Clq
of Medicine
but is
and an antibody-
Volume
58
105
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
EA+ Clq,lr,ls-+EAClq,~,i;
EACl + C4
(EACI)
EAfi,4+
(1)
-EACl ,4,2
C2
C4i
(2)
C2i
--
EACl,4,2,3
EACi, 4,2 + C3
(3)
c3i
--
EAC1,4,2,3,+
C5,C6,C7+EACi,4,2,3j,6,7
I
(4)
C(5,6,7)i
--EAC1,4,2,3,5,6,7+ C8,9+EAC1,4,2,3,5,6,7,8,9 (5) Figure 1. The five phases of the complement reaction.
antigen complex. Clq is thus the recognition unit of the system, and, subsequent to its interaction with an antibody-antigen complex, either in the fluid phase or on a membrane surface, each of the remaining components of the system becomes activated according to the described sequence (Figure 1). Subsequent to the interaction between Clq and the sensitized red cell (EA), enzymatic activity is produced in both Clr (CUP) and Cl.%. The next step in the reaction procedure is the interaction between EACi and C4 to form the intermediate complex EAm4 which has the-- ability to interact with C2 to form the complex EAC 1,4,2 and subsequent to the incorporation of C2 into this complex, a second enzymatic activity is produced (Ca). The next step in the reaction procedure is the interaction of-- this enzyme with C3 to form another complex, EAC1,4,2,3. The C4,2,3 enzyme, but not the C4,2 enzyme, has the specificity which enables it to interact with C5. The next step in the --reaction procedure is the interaction between EAC1,4,2,3 and
C5, C6 and C7 to form the intermediate complex EACl-7. And finally the terminal steps, the interaction of the EACl-7 complex with C8 and C9 which leads to the formation of the EAC1,4,2,3,5,6,7,8,9 complex and red cell lysis. Cell lysis is produced only subsequent to the action of all components of the system. (For a review of the reaction mechanism of complement, see [ lo] .) It is clear that with a system Control Mechanisms. such as this in which various enzymatic activities are produced sequentially, some control mechanisms must exist and there are, in fact, several. One of these is depicted in Figure 1 at each reaction step. Taking for example the reaction between Cl and C4, only part of the C4 (and that is less than 10 per cent of the C4 offered in the system) becomes incorporated into the active complex. Ninety per cent of the protein becomes biologically inactivated in the fluid phase of the reaction mixture. Similarly, at each step in the reaction procedure, only a certain percentage, and this percentage is usually less than 10 per cent of the component offered, becomes incorporated into the active complex. The remainder stays in the fluid phase as an inactive protein [lo]. The second control mechanism that exists is the presence in serum of specific inhibitors to the complement components. Several such inhibitors have already been described, and it is highly likely that most, if not all, of the components will be shown to have a corresponding specific inhibitor present in serum. THE ALTERNATE METHOD OF ACTIVATION OF COMPLEMENT A second method of activation of complement has been recently described [ 1 l] which, unlike the classic mechanism of activation described, is not dependent on antibody-antigen interaction. Figure 2 outlines both systems. As discussed earlier, activation of the Alternate
ClOSSiC
Endotoxin Yeast Cell Walls lnulin
Antigen-Antibody
I
c2 + c4
G
>C4,2
3 Proactivator
c3 1’1 c5 - 9
106
January 1975
The American Journal of Medicine
Volume 56
Figure 2. Two methods of the complement system.
of activation
GENETIC ASPECTS OF DISEASES OF COMPLEMENT
classic mechanism requires antibody-antigen interaction, C 1, C4 and C2, whereas the alternate mechanism does not require antibody-antigen interaction and bypasses the components Cl, C4 and C2. This system enters the previously described classic sequence at the C3 stage. The alternate mechanism is also composed of several factors [ 1 l- 131. However, not all of them have as yet been purified and isolated. The scheme for the alternate mechanism as indicated in Figure 2 is in circular form since activated C3 is an early as well as a late component of this system [ 131. Once C3 has been activated by any means that we are currently aware of, the activated C3 will itself activate the alternate mechanism until the active form of C3 has been destroyed. The means of destruction of activated C3 in a physiologic system is by a protein termed the C3b inactivator. This protein has the specificity to cleave activated C3 (C3b), thereby preventing the C3 from participating further in the reaction system [ 141. IN VITRO BIOLOGIC ACTIVITIES OF COMPLEMENT
Table I lists the various biologic activities of complement which are demonstrable in vitro. Cytolysis, which has been described, requires all 11 components. Immune adherence requires components up to the C3 stage of the reaction sequence [ 15,161. Opsonization, which generally requires components only up to C3, may be much enhanced under certain experimental conditions by the presence of C5 [ 171. Anaphylatoxin is a biologically active fragment cleaved from both C3 [ 161 and C5 [ 16,191, and chemotaxis is dependent on C3 [20], C5 [21] and the C5,6,7 complex [22]. Histamine release from platelets and mast cells is a function that requires components only up to C5 [23]. Virus neutralization under certain conditions has been shown to require only C4 [ 241. In studies performed with herpes simplex virus, it appeared to be the amount of protein on the surface of the virus rather than the specificity of which component was responsible for its neutralization. Kinin activity has been shown to be released from C4 and C2 subsequent to the action of Cl esterase (Ci) [ 251. Recently, by the study of rabbits which are genetically deficient in C6, it was shown that complement was also involved in the process of blood coagulation [26]. Just exactly where in the coagulation scheme complement is involved is not yet clear. However, it has been shown that the action of complement in blood clotting is associated with the platelet membrane [ 271. Finally, antibody to both C3 and C4 was shown to inhibit blood clot lysis [28].
TABLE I
Biologic Activities of Complement in Vitro
Cytolysis Immune adherence Opsonization Anaphylatoxin Leukocyte chemotaxis Histamine release from platelets and mast cells Virus neutralization Kinin Promotion of blood coagulation Blood clot lysis
WHAT IS THE IN VIVO SIGNIFICANCE COMPLEMENT?
OF
The most direct information has been obtained in answer to this question by the study of people who are deficient either in one of the components of complement or in one of the specific inhibitors to the components. One of the most Deficiency of the Inhibitor to Cl. widely studied deficiencies of the inhibitors is the genetically controlled deficiency of the inhibitor to Cl. The deficiency of this protein results in manifestation of the disease hereditary angioedema [29]. The clinical history of these patients shows recurrent attacks of acute, usually circumscribed and sometimes fatal, edema. During the asymptomatic period, C2 and C4 may be low and C3 is usually normal. However, during an attack, due to the absence of the inhibitor to Cl in the serum of these patients, Cl in its activated form can be demonstrated. Since activated Cl (Ci) is present, its two natural substrates, C2 and C4, are virtually absent. Kinin-like activity can be demonstrated in these serums and has been shown to be due to the action of Ci on C4 and C2 [30]. There are two types of people who lack the inhibitor to Cl. Type I in whom the Cl inhibitor protein cannot be demonstrated immunochemically or functionally, and type II in whom the Cl inhibitor protein can be detected immunochemically, but this protein is present as a functionally inactive analog of the normal protein
[311. Genetics of the Cl Inhibitor Deficiency. Dr. Bearn: If the Cl inhibitor protein is present but is biologically inactive, it suggests that a structural mutation affecting its binding site has taken place. Thus, although the Cl inhibitor protein is functionally inactive, the altered protein can be recognized since it is similar antigenically to the Cl inhibitor protein and will cross react with antibody to the normal protein. Such a mutational event is referred to as cross-reactive material positive (CRM+). When the protein is neither detectable immunochemically nor functionally, two possibilities can explain the genetic variation. The
January 1975
The American
Journal
of Medicine
Volume
58
107
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Hemolytic
matosus plement
Phagocytic
N0rlTlCll
[39,40]. The association between the comdeficiency and the disease process is not
clear.
C5
The Third Component (C3).
Deficient C5
Figure 3. Hypothetical diagrammatic rebresentation of a normal C5 molecule and a C5 molecule from a patient with C5 deficiency. first possibility the regulatory
is that the protein is absent because gene is defective and cannot turn on
the normal structural gene. Alternatively, it might mean that there is a structural mutation of the Cl inhibitor protein, as seen in type II people, where the mutational event is so disruptive that no immunochemically recognizable protein is synthesized. It is virtually impossible to distinguish between these two possibilities at the present time. Usually, classic inborn errors of metabolism are due to recessive traits rather than to dominant ones. In people deficient in the inhibitor to C 1, the situation appears to be different. One abnormal gene is sufficient for the person to manifest the disease, and the disease is present even in the heterozygous condition. Whether the disease is of type I or type II, whether immunochemically cross-reacting material is present or not, the disease is still inherited in an autosomal dominant fashion. Genetic studies of this disease have already told us two things: First, that a disease which appears clinically homogeneous may be heterogeneous genetically. And, second, that inborn errors of metabolism may, on occasion, be inherited in a dominant fashion.
Deficiency of the C3b Inactivator.
Dr. Polley: This is a deficiency of the inhibitor to the activated form of C3 (C3b). Only a single patient has so far been described with this defect. Due to the deficiency of this protein, the patient apparently has a continuous activation of the alternate mechanism of complement resulting in marked hypercatabolism of C3 and a much increased susceptibility to infection [32-341. GENETICALLY CONTROLLED DEFICIENCIES OF COMPLEMENT COMPONENTS IN MAN The Second Component (C2).
Whole complement hemolysis and C2 activity were found to be less than 5 per cent in these subjects [35,36]. Immune adherence and bactericidal activity also were much reduced. No C2 protein can be demonstrated immunochemically in these people [37,38]. In persons with this deficiency recognized in the past, there appeared to be no apparent undue susceptibility to infection. However, more recently, two patients with C2 deficiency have been described with lupus erythe-
108
January
1975
The American
Journal
of Medicine
Volume
In a patient deficient in C3, the bactericidal activity and chemotactic activity were low as was enhancement of phagocytosis. Only a single person with a genetic deficiency of C3 has been described and this subject has a history long susceptibility to infection [41].
of life
Fifth Component (C5).
The deficiency of C5 in man [42] is interesting in that it is quite different from the other known complement deficiencies. In this deficiency state, certain of the biologic activities of the protein were found to be normal. The hemolytic activity of C5 was normal, as was the quantitation of C5 protein in the serum [ 171. However, serum from these people would not support chemotaxis or enhancement of phagocytosis of some organisms. Examples of such organisms were certain gram-negative bacteria, Staphylococcus aureus and yeast particles, opsonization of each of which was shown to be much enhanced in the presence of C5. Of interest was that the person with C5 deficiency demonstrated repeated local and systemic infections with those organisms, the phagocytosis enhanced by the presence
of which was shown to be of C5 [ 171. Genetics of C5 Deficiency. Dr. Bearn: This patient with C5 deficiency presented at the age of 3 months with failure to thrive, widespread refractory eczema and intermittent diarrhea [42]. Even though this is a very rare disease, more than one case has been described. Family studies have indicated that this complement deficiency is inherited as an autosomal dominant trait. This deficiency is quite unusual in that it appears that both the hemolytic activity of the protein and the serum protein concentration are normal. Figure 3 is a highly schematic drawing of a C5 protein molecule from a normal person and from a patient with C5 deficiency. As indicated, the hemolytic site is unaffected, but a mutational event has occurred at a different point, which has a pronounced affect on the phagocytic site. There is, as yet, no evidence that this is the correct explanation, but it is the kind of way in which mutational events may be expected to dissect out the functional characteristics of a protein molecule. In an analagous fashion, it would be possible for a mutational event to occur at the hemolytic site which would exclusively affect hemolysis and leave the phagocytic activity unchanged. During the course of this discussion we have tried to indicate the difference between a regulatory mutation and a structural mutation and the difficulty in eukaryotic organisms in distinguishing between the two. With a structural mutation, the protein is synthesized, but the mutational event affects the structural properties of
58
GENETIC ASPECTS OF DISEASES OF COMPLEMENT
the protein and often the amount of protein synthesized as well. On the other hand, with a regulatory mutation, no detectable protein is synthesized. In the absence of any detectable protein, it is clearly impossible to decide whether the mutation has affected the structure of the molecule. Colten [43] recently described an interesting experiment which was designed to distinguish between these two possibilities. Human HaLa cells in culture do not synthesize C4. Peritoneal cells derived from CIdeficient guinea pigs were mixed with the human HeLa cells and a guinea pig-human hybrid was produced. In the hybrid, C4 was present, and the C4 was unequivocally human. Thus, the structural gene for making C4 must have been present in the HeLa cells although it was not expressed. When these cells were hybridized with another cell line, the production of the complement component which was lying silent in the HeLa cells was turned on and thus the guinea pig-human hybrid produced human C4. THERAPEUJIC DEFICIENCIES
APPROACHES
TO COMPLEMENT
Dr. Polley: At this time, none of the C2-deficient subjects has needed treatment. A possible approach for the person deficient in C3 would be replacement of C3, that is to say, infusion with the highly purified protein. However, the value line of therapy is uncertain since the catabolic rate of C3 is very fast, approximately 50 hours. People with C5 deficiency have so far been treated successfully with fresh plasma. With the deficiency of the inhibitor to Cl, a different problem exists. Infusion of plasma to these people would supply Cl inhibitor, but it would at the same time also supply excess substrate (C2 and C4) for the activated enzyme C 1. This obviously might be dangerous. With the C3b inactivator deficiency, infusion of plasma might even be lethal since in this case one is infusing a large amount of C3 at the same time as the inactivator. Thus, with the deficiencies of the inhibitors, the preferential treatment would be infusion of the purified Cl inhibitor in those with hereditary angioedema and the purified C3b inactivator in those deficient in this protein. ANTIBODY PRODUCTION TO AN INFUSED PROTEIN BY A PERSON GENETICALLY DEFICIENT IN THAT PROTEIN It has been shown that the C4-deficient guinea pigs [44], CSdeficient mice [7,45] and CG-deficient rabbits [46] will produce specific antibody to the lacking protein following its infusion. As yet, it is not known whether CBdeficient people or CS-deficient people will produce antibody to the protein in which they are deficient. It is possible that the presence of some
II
TABLE
Correlation Between Clinical Disease and Pathways of Activation Involved in Complement Deficiency States Protein Remaining
Complement Component c2 c3 C4 c5 c5 C6
Species Man Man Guinea Man Mouse Rabbit
Clinically Affected
(% of normal)
? pig
+++ ++ ++ ++
Activation Pathways
0
Medical Center, New York, New York
Genetic Aspects of Diseases of Complement: An Explosion
Lecturers: MARGARET J. POLLEY, Ph.D ALEXANDER
G. BEARN, M.D.
New York, New York
From the Department of Medicine, The New York Hospital-Cornell Medical Center, 525 East 68th Street, New York, New York 10021. Text of a lecture delivered on October 9, 1973. Requests for reprints should be addressed to Dr. Margaret J. Polley.
THE CLASSIC METHOD OF ACTIVATION COMPLEMENT
OF
Dr. Margaret J. Polley: Complement was first described [l] as a factor in serum which was necessary to supplement antibody in the induction of lysis of foreign red cells and bacteria-a complement to antibody, hence the name. It differed from antibody by two of its properties. First, it was heat labile: and second, it was not increased upon immunization. When first described, complement was considered to be a single component. Not long afterwards it was shown that it was not a single component, but that it comprised two components and these two components were named in order of their reactivity-Cl and C2 [2]. A little later, a third component was described and the sequence of reactivity of the three components was Cl, C2 and C3 [3]. Just a year later, a fourth component was described and called C4 since this was the order of its discovery. However, the sequence of reactivity of the four components was Cl, C4, C2 and C3 [4]. This was the situation for close to 30 years. It was believed that the complement system was composed of four factors, Cl, C4, C2 and C3. Late in the 1950’s it was found that what was being termed C3 at that time was not a single component. It was, in fact, composed of six components [ 5-71. Various terminologies were introduced by the people who identified these components. However, it was finally decided [8] to label these components numerically according to the sequence of reactivity, C3 being retained for the first of these six components to react. Thus the components in order of their reactivity became Cl, C4, C2, C3, C5, C6, C7, C8 and C9. Cl also was found not to be a single component. It was composed of three components, and to prevent further complication of terminology, lettered terminology was retained for these proteins, Clq, Clr and C 1s [9]. Thus the 11 components of the complement system in order of their reactivity became Clq, Clr, Cls, C4, C2, C3, C5, C6, C7, C8 and C9. Each of the components is a distinct serum protein and, except for Clq, each in its native form lacks biologic activity. Something has to trigger off the system and subsequent to such a stimulus, biologic activity is produced sequentially in each of the components. The stimulus clearly
which
activates
dependent
January 1975
the
system
on the interaction
The American
is not fully understood between
Journal
Clq
of Medicine
but is
and an antibody-
Volume
58
105
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
EA+ Clq,lr,ls-+EAClq,~,i;
EACl + C4
(EACI)
EAfi,4+
(1)
-EACl ,4,2
C2
C4i
(2)
C2i
--
EACl,4,2,3
EACi, 4,2 + C3
(3)
c3i
--
EAC1,4,2,3,+
C5,C6,C7+EACi,4,2,3j,6,7
I
(4)
C(5,6,7)i
--EAC1,4,2,3,5,6,7+ C8,9+EAC1,4,2,3,5,6,7,8,9 (5) Figure 1. The five phases of the complement reaction.
antigen complex. Clq is thus the recognition unit of the system, and, subsequent to its interaction with an antibody-antigen complex, either in the fluid phase or on a membrane surface, each of the remaining components of the system becomes activated according to the described sequence (Figure 1). Subsequent to the interaction between Clq and the sensitized red cell (EA), enzymatic activity is produced in both Clr (CUP) and Cl.%. The next step in the reaction procedure is the interaction between EACi and C4 to form the intermediate complex EAm4 which has the-- ability to interact with C2 to form the complex EAC 1,4,2 and subsequent to the incorporation of C2 into this complex, a second enzymatic activity is produced (Ca). The next step in the reaction procedure is the interaction of-- this enzyme with C3 to form another complex, EAC1,4,2,3. The C4,2,3 enzyme, but not the C4,2 enzyme, has the specificity which enables it to interact with C5. The next step in the --reaction procedure is the interaction between EAC1,4,2,3 and
C5, C6 and C7 to form the intermediate complex EACl-7. And finally the terminal steps, the interaction of the EACl-7 complex with C8 and C9 which leads to the formation of the EAC1,4,2,3,5,6,7,8,9 complex and red cell lysis. Cell lysis is produced only subsequent to the action of all components of the system. (For a review of the reaction mechanism of complement, see [ lo] .) It is clear that with a system Control Mechanisms. such as this in which various enzymatic activities are produced sequentially, some control mechanisms must exist and there are, in fact, several. One of these is depicted in Figure 1 at each reaction step. Taking for example the reaction between Cl and C4, only part of the C4 (and that is less than 10 per cent of the C4 offered in the system) becomes incorporated into the active complex. Ninety per cent of the protein becomes biologically inactivated in the fluid phase of the reaction mixture. Similarly, at each step in the reaction procedure, only a certain percentage, and this percentage is usually less than 10 per cent of the component offered, becomes incorporated into the active complex. The remainder stays in the fluid phase as an inactive protein [lo]. The second control mechanism that exists is the presence in serum of specific inhibitors to the complement components. Several such inhibitors have already been described, and it is highly likely that most, if not all, of the components will be shown to have a corresponding specific inhibitor present in serum. THE ALTERNATE METHOD OF ACTIVATION OF COMPLEMENT A second method of activation of complement has been recently described [ 1 l] which, unlike the classic mechanism of activation described, is not dependent on antibody-antigen interaction. Figure 2 outlines both systems. As discussed earlier, activation of the Alternate
ClOSSiC
Endotoxin Yeast Cell Walls lnulin
Antigen-Antibody
I
c2 + c4
G
>C4,2
3 Proactivator
c3 1’1 c5 - 9
106
January 1975
The American Journal of Medicine
Volume 56
Figure 2. Two methods of the complement system.
of activation
GENETIC ASPECTS OF DISEASES OF COMPLEMENT
classic mechanism requires antibody-antigen interaction, C 1, C4 and C2, whereas the alternate mechanism does not require antibody-antigen interaction and bypasses the components Cl, C4 and C2. This system enters the previously described classic sequence at the C3 stage. The alternate mechanism is also composed of several factors [ 1 l- 131. However, not all of them have as yet been purified and isolated. The scheme for the alternate mechanism as indicated in Figure 2 is in circular form since activated C3 is an early as well as a late component of this system [ 131. Once C3 has been activated by any means that we are currently aware of, the activated C3 will itself activate the alternate mechanism until the active form of C3 has been destroyed. The means of destruction of activated C3 in a physiologic system is by a protein termed the C3b inactivator. This protein has the specificity to cleave activated C3 (C3b), thereby preventing the C3 from participating further in the reaction system [ 141. IN VITRO BIOLOGIC ACTIVITIES OF COMPLEMENT
Table I lists the various biologic activities of complement which are demonstrable in vitro. Cytolysis, which has been described, requires all 11 components. Immune adherence requires components up to the C3 stage of the reaction sequence [ 15,161. Opsonization, which generally requires components only up to C3, may be much enhanced under certain experimental conditions by the presence of C5 [ 171. Anaphylatoxin is a biologically active fragment cleaved from both C3 [ 161 and C5 [ 16,191, and chemotaxis is dependent on C3 [20], C5 [21] and the C5,6,7 complex [22]. Histamine release from platelets and mast cells is a function that requires components only up to C5 [23]. Virus neutralization under certain conditions has been shown to require only C4 [ 241. In studies performed with herpes simplex virus, it appeared to be the amount of protein on the surface of the virus rather than the specificity of which component was responsible for its neutralization. Kinin activity has been shown to be released from C4 and C2 subsequent to the action of Cl esterase (Ci) [ 251. Recently, by the study of rabbits which are genetically deficient in C6, it was shown that complement was also involved in the process of blood coagulation [26]. Just exactly where in the coagulation scheme complement is involved is not yet clear. However, it has been shown that the action of complement in blood clotting is associated with the platelet membrane [ 271. Finally, antibody to both C3 and C4 was shown to inhibit blood clot lysis [28].
TABLE I
Biologic Activities of Complement in Vitro
Cytolysis Immune adherence Opsonization Anaphylatoxin Leukocyte chemotaxis Histamine release from platelets and mast cells Virus neutralization Kinin Promotion of blood coagulation Blood clot lysis
WHAT IS THE IN VIVO SIGNIFICANCE COMPLEMENT?
OF
The most direct information has been obtained in answer to this question by the study of people who are deficient either in one of the components of complement or in one of the specific inhibitors to the components. One of the most Deficiency of the Inhibitor to Cl. widely studied deficiencies of the inhibitors is the genetically controlled deficiency of the inhibitor to Cl. The deficiency of this protein results in manifestation of the disease hereditary angioedema [29]. The clinical history of these patients shows recurrent attacks of acute, usually circumscribed and sometimes fatal, edema. During the asymptomatic period, C2 and C4 may be low and C3 is usually normal. However, during an attack, due to the absence of the inhibitor to Cl in the serum of these patients, Cl in its activated form can be demonstrated. Since activated Cl (Ci) is present, its two natural substrates, C2 and C4, are virtually absent. Kinin-like activity can be demonstrated in these serums and has been shown to be due to the action of Ci on C4 and C2 [30]. There are two types of people who lack the inhibitor to Cl. Type I in whom the Cl inhibitor protein cannot be demonstrated immunochemically or functionally, and type II in whom the Cl inhibitor protein can be detected immunochemically, but this protein is present as a functionally inactive analog of the normal protein
[311. Genetics of the Cl Inhibitor Deficiency. Dr. Bearn: If the Cl inhibitor protein is present but is biologically inactive, it suggests that a structural mutation affecting its binding site has taken place. Thus, although the Cl inhibitor protein is functionally inactive, the altered protein can be recognized since it is similar antigenically to the Cl inhibitor protein and will cross react with antibody to the normal protein. Such a mutational event is referred to as cross-reactive material positive (CRM+). When the protein is neither detectable immunochemically nor functionally, two possibilities can explain the genetic variation. The
January 1975
The American
Journal
of Medicine
Volume
58
107
COMBINED CLINICAL AND BASIC SCIENCE SEMINAR
Hemolytic
matosus plement
Phagocytic
N0rlTlCll
[39,40]. The association between the comdeficiency and the disease process is not
clear.
C5
The Third Component (C3).
Deficient C5
Figure 3. Hypothetical diagrammatic rebresentation of a normal C5 molecule and a C5 molecule from a patient with C5 deficiency. first possibility the regulatory
is that the protein is absent because gene is defective and cannot turn on
the normal structural gene. Alternatively, it might mean that there is a structural mutation of the Cl inhibitor protein, as seen in type II people, where the mutational event is so disruptive that no immunochemically recognizable protein is synthesized. It is virtually impossible to distinguish between these two possibilities at the present time. Usually, classic inborn errors of metabolism are due to recessive traits rather than to dominant ones. In people deficient in the inhibitor to C 1, the situation appears to be different. One abnormal gene is sufficient for the person to manifest the disease, and the disease is present even in the heterozygous condition. Whether the disease is of type I or type II, whether immunochemically cross-reacting material is present or not, the disease is still inherited in an autosomal dominant fashion. Genetic studies of this disease have already told us two things: First, that a disease which appears clinically homogeneous may be heterogeneous genetically. And, second, that inborn errors of metabolism may, on occasion, be inherited in a dominant fashion.
Deficiency of the C3b Inactivator.
Dr. Polley: This is a deficiency of the inhibitor to the activated form of C3 (C3b). Only a single patient has so far been described with this defect. Due to the deficiency of this protein, the patient apparently has a continuous activation of the alternate mechanism of complement resulting in marked hypercatabolism of C3 and a much increased susceptibility to infection [32-341. GENETICALLY CONTROLLED DEFICIENCIES OF COMPLEMENT COMPONENTS IN MAN The Second Component (C2).
Whole complement hemolysis and C2 activity were found to be less than 5 per cent in these subjects [35,36]. Immune adherence and bactericidal activity also were much reduced. No C2 protein can be demonstrated immunochemically in these people [37,38]. In persons with this deficiency recognized in the past, there appeared to be no apparent undue susceptibility to infection. However, more recently, two patients with C2 deficiency have been described with lupus erythe-
108
January
1975
The American
Journal
of Medicine
Volume
In a patient deficient in C3, the bactericidal activity and chemotactic activity were low as was enhancement of phagocytosis. Only a single person with a genetic deficiency of C3 has been described and this subject has a history long susceptibility to infection [41].
of life
Fifth Component (C5).
The deficiency of C5 in man [42] is interesting in that it is quite different from the other known complement deficiencies. In this deficiency state, certain of the biologic activities of the protein were found to be normal. The hemolytic activity of C5 was normal, as was the quantitation of C5 protein in the serum [ 171. However, serum from these people would not support chemotaxis or enhancement of phagocytosis of some organisms. Examples of such organisms were certain gram-negative bacteria, Staphylococcus aureus and yeast particles, opsonization of each of which was shown to be much enhanced in the presence of C5. Of interest was that the person with C5 deficiency demonstrated repeated local and systemic infections with those organisms, the phagocytosis enhanced by the presence
of which was shown to be of C5 [ 171. Genetics of C5 Deficiency. Dr. Bearn: This patient with C5 deficiency presented at the age of 3 months with failure to thrive, widespread refractory eczema and intermittent diarrhea [42]. Even though this is a very rare disease, more than one case has been described. Family studies have indicated that this complement deficiency is inherited as an autosomal dominant trait. This deficiency is quite unusual in that it appears that both the hemolytic activity of the protein and the serum protein concentration are normal. Figure 3 is a highly schematic drawing of a C5 protein molecule from a normal person and from a patient with C5 deficiency. As indicated, the hemolytic site is unaffected, but a mutational event has occurred at a different point, which has a pronounced affect on the phagocytic site. There is, as yet, no evidence that this is the correct explanation, but it is the kind of way in which mutational events may be expected to dissect out the functional characteristics of a protein molecule. In an analagous fashion, it would be possible for a mutational event to occur at the hemolytic site which would exclusively affect hemolysis and leave the phagocytic activity unchanged. During the course of this discussion we have tried to indicate the difference between a regulatory mutation and a structural mutation and the difficulty in eukaryotic organisms in distinguishing between the two. With a structural mutation, the protein is synthesized, but the mutational event affects the structural properties of
58
GENETIC ASPECTS OF DISEASES OF COMPLEMENT
the protein and often the amount of protein synthesized as well. On the other hand, with a regulatory mutation, no detectable protein is synthesized. In the absence of any detectable protein, it is clearly impossible to decide whether the mutation has affected the structure of the molecule. Colten [43] recently described an interesting experiment which was designed to distinguish between these two possibilities. Human HaLa cells in culture do not synthesize C4. Peritoneal cells derived from CIdeficient guinea pigs were mixed with the human HeLa cells and a guinea pig-human hybrid was produced. In the hybrid, C4 was present, and the C4 was unequivocally human. Thus, the structural gene for making C4 must have been present in the HeLa cells although it was not expressed. When these cells were hybridized with another cell line, the production of the complement component which was lying silent in the HeLa cells was turned on and thus the guinea pig-human hybrid produced human C4. THERAPEUJIC DEFICIENCIES
APPROACHES
TO COMPLEMENT
Dr. Polley: At this time, none of the C2-deficient subjects has needed treatment. A possible approach for the person deficient in C3 would be replacement of C3, that is to say, infusion with the highly purified protein. However, the value line of therapy is uncertain since the catabolic rate of C3 is very fast, approximately 50 hours. People with C5 deficiency have so far been treated successfully with fresh plasma. With the deficiency of the inhibitor to Cl, a different problem exists. Infusion of plasma to these people would supply Cl inhibitor, but it would at the same time also supply excess substrate (C2 and C4) for the activated enzyme C 1. This obviously might be dangerous. With the C3b inactivator deficiency, infusion of plasma might even be lethal since in this case one is infusing a large amount of C3 at the same time as the inactivator. Thus, with the deficiencies of the inhibitors, the preferential treatment would be infusion of the purified Cl inhibitor in those with hereditary angioedema and the purified C3b inactivator in those deficient in this protein. ANTIBODY PRODUCTION TO AN INFUSED PROTEIN BY A PERSON GENETICALLY DEFICIENT IN THAT PROTEIN It has been shown that the C4-deficient guinea pigs [44], CSdeficient mice [7,45] and CG-deficient rabbits [46] will produce specific antibody to the lacking protein following its infusion. As yet, it is not known whether CBdeficient people or CS-deficient people will produce antibody to the protein in which they are deficient. It is possible that the presence of some
II
TABLE
Correlation Between Clinical Disease and Pathways of Activation Involved in Complement Deficiency States Protein Remaining
Complement Component c2 c3 C4 c5 c5 C6
Species Man Man Guinea Man Mouse Rabbit
Clinically Affected
(% of normal)
? pig
+++ ++ ++ ++
Activation Pathways
0
