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IMMUNOHAEMATOLOGY AND HUMAN BLOOD TRANSFUSION.

IMMUNOHEMATOLOGY;

Section I. THE IMMUNE RESPONSE AND THE INTERACTION OF ANTIGENS,

ANTIBODIES, AND COMPLEMENT IN IMMUNOHEMATOLOGY

1-1. BACKGROUND

Immunology, a field once dominated by bacteriologists, has become important to

scientists in many other areas. The field of immunohematology came into being when

Landsteiner discovered that the blood antigens (ABO) present on RBCs (RBCs) would

react with their respective antibodies present in plasma, and that this reaction had great

clinical significance. Since that time, many discoveries in this field have added to the

understanding of immune mechanisms operative in health and disease. It is important

that scientists working in areas associated with blood transfusion understand basic

immunology and try to be familiar with the recent advances in this field that might relate

directly to their work.

1-2. THE IMMUME RESPONSE

According to Roitt, the basic of immunology is memory, specificity, and the

recognition of "nonself". The original basis for this was the protection (immunity)

afforded by exposure to infectious illness. The first contact with an infectious organism

imprints some information (for example, memory) so that the body will recognize and

attack that organism when it encounters it in the future. The protection is usually

specific (for example, only against the original infecting organism). The body also has

to recognize that organism as being foreign (that is., "nonself"). The substance initially

responsible for an immune response is known as an antigen or more specifically an

immunogen.

ANTIGENS;

a. Antigens are substances that can induce a specific immunologic response orcan interact with specific antibody or immune cells "in vivo" or "in vitro". The immune response can be either humoral or cellular (paragraph 1-4). Blood group serology is mainly concerned with the humoral response that leads to the production of free antibody in the plasma. The antibodies, under appropriate conditions of reaction (temperature, pH, ionic strength, and so forth.), will react specifically with the antigen in some observable way (for example., agglutination, hemolysis).

b. An antigen contains structural chemical groups in a specific three-dimensional arrangement, known as antigenic determinants (epitopes), which are lacking or foreign to the immunized animal. Each antigen can contain many of these epitopes. The specific three-dimensional shape of these antigenic determinants, or chemical groupings, is what determines the specificity of its reaction with a particular antibody molecule.

c. An Important factor affecting the immunogenicity of an antigen is its molecular size. Immunogenic molecules are rarely less than 4,000 daltons. Much smaller molecules (for example, drugs such as penicillin) can be immunogenic if coupled to a

protein “carrier” of larger molecular weight. Such a molecule is termed a hapten and can be defined as a small molecule that, by itself, cannot stimulate antibody synthesis

but will combine with antibody once formed. Indeed, most of our basic understanding of

antigen specificity came from work by Landsteiner using haptens.

d. Blood group antigens are chemical groupings present on the RBC membrane.

We are only just beginning to learn the exact nature of these determinants. The ABH

antigens have been the most thoroughly studied and when present on RBCs are

predominantly glycolipids. A and B antigens are composed of the same fatty acids and

sugars, the difference in specificity being caused by the terminal sugar in the chain of

sugars joined to the fatty acid backbone. The specificity is a result not only of the

particular sugar but also the configuration of the end grouping it forms. As the sugars

responsible for A or B specificity (N-acetylgalactosamine and galactose, respectively)

are structurally identical except for the substitution of an hydroxyl group for an N-

acetylamino group at carbon atom number two, they serve as a good example of the

remarkable specificity of antigen-antibody reactions.

e. Proteins are direct gene products, whereas carbohydrates, such as the A and

B antigens, are indirect products of genes (for example, A or B genes). The direct

(protein) products of the A and B genes are enzymes that recognize and then transfer

specific sugars from their nucleotide carriers to specific acceptor molecules. Thus, the

A gene product is an N-acetyl-D-galactose-aminyltransferase enzyme and the B gene product in

a D-galactosyltransferase.

f. The biologic role of blood group antigens, if any, is at present unknown. The

ABH antigens are widely distributed throughout the body, being present on many types

of cells, organs, and body fluids. Some antigens such as Rh and Kell (K) appear to play

a part in cell membrane integrity. Rare individuals lacking Rh antigens (Rhnull) on their

RBCs often have an associated hemolytic anemia (“Rh-null syndrome”), whereas, in

contrast, rare individuals lacking A, B, and H antigens (Bombay phenotype) do not. It

has been suggested that this is because the ABH antigens are glycolipids projecting

above the cell membrane, whereas Rh appears to be lipoprotein, an integral part of the

RBC membrane. An association between a rare inherited defect of neutrophil

bactericidal function (chronic granulomatous disease) and the Kell blood group system

has recently been described. Another report suggests a possible relationship between

the Duffy blood group antigens and resistance to malaria. There are many other

associations of blood groups with disease, particularly malignancy; many of them are

purely statistical and their causes unknown.

An Important factor affecting the immunogenicity of an antigen is its molecular

size. Immunogenic molecules are rarely less than 4,000 daltons. Much smaller

molecules (for example, drugs such as penicillin) can be immunogenic if coupled to a

protein “carrier” of larger molecular weight. Such a molecule is termed a hapten and

can be defined as a small molecule that, by itself, cannot stimulate antibody synthesis

but will combine with antibody once formed. Indeed, most of our basic understanding of

antigen specificity came from work by Landsteiner using haptens.

d. Blood group antigens are chemical groupings present on the RBC membrane.

We are only just beginning to learn the exact nature of these determinants. The ABH

antigens have been the most thoroughly studied and when present on RBCs are

predominantly glycolipids. A and B antigens are composed of the same fatty acids and

sugars, the difference in specificity being caused by the terminal sugar in the chain of

sugars joined to the fatty acid backbone. The specificity is a result not only of the

particular sugar but also the configuration of the end grouping it forms. As the sugars

responsible for A or B specificity (N-acetylgalactosamine and galactose, respectively)

are structurally identical except for the substitution of an hydroxyl group for an N�

acetylamino group at carbon atom number two, they serve as a good example of the

remarkable specificity of antigen-antibody reactions.

e. Proteins are direct gene products, whereas carbohydrates, such as the A and

B antigens, are indirect products of genes (for example, A or B genes). The direct

(protein) products of the A and B genes are enzymes that recognize and then transfer

specific sugars from their nucleotide carriers to specific acceptor molecules. Thus, the

A gene product is an N-acetyl-D-galactose-aminyltransferase enzyme and the B gene product in

a D-galactosyltransferase.

f. The biologic role of blood group antigens, if any, is at present unknown. The

ABH antigens are widely distributed throughout the body, being present on many types

of cells, organs, and body fluids. Some antigens such as Rh and Kell (K) appear to play

a part in cell membrane integrity. Rare individuals lacking Rh antigens (Rhnull) on their

RBCs often have an associated hemolytic anemia (“Rh-null syndrome”), whereas, in

contrast, rare individuals lacking A, B, and H antigens (Bombay phenotype) do not. It

has been suggested that this is because the ABH antigens are glycolipids projecting

above the cell membrane, whereas Rh appears to be lipoprotein, an integral part of the

RBC membrane. An association between a rare inherited defect of neutrophil

bactericidal function (chronic granulomatous disease) and the Kell blood group system

has recently been described. Another report suggests a possible relationship between

the Duffy blood group antigens and resistance to malaria. There are many other

associations of blood groups with disease, particularly malignancy; many of them are

purely statistical and their causes unknown.

ANTIBODY SYNTHESIS

a. The Process of Antibody Synthesis. When an antigen enters the body, it

may evoke a humoral response, in which antibodies are synthesized by plasma cells

and released into the body fluids (for example, plasma), and/or a cellular response, in

which lymphocytes participate in cell-mediated immunity (for example, rejection of

transplanted tissue and delayed hypersensitivity). That two different responses were

present was originally shown by Chase and Landsteiner in the early 1940s when they

demonstrated that some kinds of immune reaction could be transferred from one animal

to another by the exchange of living cells, whereas others could be transferred by blood

serum. The cells required for the former experiment were lymphocytes. It was not until

the early 1960s that involvement of the lymphocyte was proven.

b. Lymphocyte PopulatioNS. Stem cells from the bone marrow are thought to

differentiate to form two distinct lymphocyte populations. The cells that pass through

the thymus become known as T-lymphocytes (T-cells) and the others that are

independent of the thymus B-lymphocytes (B-cells). Although these lymphocytes look

similar by conventional light or electron microscopy, they do look very different by

scanning electron microscopy, and also they can be differentiated by a variety of

surface markers. Their functions are of course different, but there is mounting evidence

for the possibility of cooperation between the two systems.

c. T-Lymphocytes. Once leaving the thymus, where they are known as

thymocytes, the T-lymphocytes are immunocompetent, that is to say, capable of

participating in an immune response. This is the basis of cellular immunity.

T-lymphocytes constitute the greater part of the recirculating pool of small lymphocytes

and have a relatively long half-life. When they encounter an antigen (which may have

to be first processed by a macrophage), they transform to lymphoblasts (See

figure 1-1). These T-lymphoblasts, which have no demonstrable intracellular

immunoglobulin, have several functions:

(1) They divide further into primed antigen-sensitive cells, which provide

immunologic memory because of their long life span.

(2) They release a number of soluble factors (lymphokines) which mediate

delayed-type hypersensitivity.

(3) They are “killer” cells, which are cytotoxic for cells bearing the

histo-compatibility antigens of a graft or tumor cells.

(4) They may cooperate with the humoral system by triggering

B-lymphocytes.

B-Lymphocytes. The B-lymphocyte gets its name from the Bursa of

Fabricius, a lymphoid organ present in birds, which controls the production of

lymphocytes responsible for making humoral antibody. The equivalent organ in man has

yet to be found. Thymus-independent, or B-lymphocytes synthesize and excrete

specific antibodies (surface immunoglobulins) that serve as receptors for antigens.

When triggered by antigen, the B-lymphocytes change to plasma cells, which are

responsible for the excretion of free antibody into the body fluids (for example, humoral

antibody), see figure 1-1. There is much evidence to suggest that macrophages are

required to process antigens for appropriate presentation to lymphocytes before the

humoral response occurs. In addition, many antigens appear to require the cooperation

of both B- and T-lymphocytes. The mechanisms by which T- and B-lymphocytes

interact are complex and far from clear at present. As mentioned previously, it is

humoral antibodies that are dealt with routinely in blood transfusion science, but

possibly cellular reactions will increase in importance in the future.

e. Differentiation of T- and B-Lymphocytes. Approximately 25 percent of

human blood lymphocytes are B cells, 70 percent T cells, and 5 percent have neither T

nor B markers (they are called “null cells”). Immunoglobulins are readily demonstrable

on B, but not T-lymphocytes by immunofluorescence. T- but not B-lymphocytes will

form "spontaneous” rosettes with unsensitized sheep erythrocytes.

PRIMARY AND SECONDARY IMMUNE RESPONSES

a. Following a first exposure to a foreign antigen, specific antibodies can appear

after about five days, rise slowly to a modest level, remain for a variable period, then

gradually decline, eventually becoming undetectable, until further stimulation occurs.

The first antibodies produced in this primary response are usually lgM, but eventually

other immunoglobulins (for example, lgG) may appear. The type of antigen and the

route of administration will influence the pattern observed.

b. After the primary response, a second dose of the same antigen, given days or

even years later, will usually elicit an intense and accelerated secondary (memory)

response. The serum antibody usually begins to rise within two or three days, reaching

a peak in about 10 days. In this secondary response, lgM antibody is often transiently

produced, following a similar pattern to the primary response, but the predominant

antibody produced is lgG, which rises to a much greater concentration than the lgM, and

remains in the plasma much longer. The secondary response is sometimes called an

anamnestic response.

IMMUNIZATION TO BLOOD-GROUP ANTIGENS

a. Within a few months after birth, an infant makes anti-A and/or anti-B, if lacking

those antigens on its RBCs. Such antibodies are termed naturally occurring since they

have no apparent antigenic stimulus. Experiments in chicks have shown that these

antibodies probably develop as a result of exposure to bacterial antigens, closely

related chemically to blood group antigens (for example, Escherichia coli has an antigen

on its membrane closely resembling human B antigen). Naturally occurring antibodies

to antigens other than ABO are also often encountered, particularly in the I, Lewis, P,

and MN systems. These antibodies are usually lgM and react better at lower

temperatures.

b. Immune antibodies to blood group antigens usually develop as a result of

pregnancy, transfusion, or immunization (intential sensitization). Following

immunization, lgM antibodies are often seen first, followed by lgG antibodies, which

often predominate. These antibodies usually react better at 37ºC.

c. Antibodies other than anti-A or anti-B are usually called “irregular", "atypical",

or "unexpected” antibodies. The preferred term is unexpected.

d. There is extensive evidence in animals, such as mice, that the immune

response is genetically controlled (by the so-called Ir genes). It has been suggested

that this may apply in man also. Studies in man on the immune response to the Rh (D)

antigen indicate that approximately 30 percent of the Rho(D)-negative individuals

appear to be incapable of forming anti-Rho(D) even after repeated and/or large

transfusions of Rho(D)-positive blood. The antibody response in individuals who do

make antibody will depend on many factors, including the relative potency of the

antigen, the route of immunization, and the amount of blood given.

ANTIBODY STRUCTURE, FUNCTION, AND PROPERTIES

a. Plasma proteins with antibody activity are called immunoglobulins (lg).

During the last ten years, great advances have been made in defining their structure,

physiochemical properties, antigenic characteristics, serologic behavior, and biological

properties.

b. Each immunoglobulin molecule consists of basic units, each composed of

four polypeptide chains, two light chains (L) and two heavy chains (H), held together by

covalent disulfide bonds (S-S), and noncovalent interactions (see figure 1-2).

c. Five classes of immunoglobulins have been recognized on the basis of

antigenic differences in the heavy chain: lgG, lgA, lgM, lgD, and lgE (see figure 1-3). No

blood groups antibodies have yet been found to be lgD or lgE. There are two types of

light chains (kappa chain and lambda chain), which are common to, and found in, all

five immunoglobulin classes, but each individual immunoglobulin molecule has only one

type of light chain. Approximately 66 percent of the molecules of each class have

kappa light chains, and 33 percent have lambda light chains.

ANTIGEN-ANTIBODY REACTIONS IN BLOOD GROUP SEROLOGY

a. Antibodies may react with their specific antigens in a number of ways. The

following reactions have all been used to demonstrate “in vitro” antigen-antibody

reactions in blood transfusion science:

(1) Agglutination.

(2) Hemolysis.

(3) Inhibition.

(4) Absorption and elution.

(5) Precipitation.

(6) Complement-fixation.

(7) Radioimmunoassay.

(8) Fluorescence.

b. The first two methods are the most commonly used in blood group serology

and will be discussed in more detail. Inhibition and absorption/elution techniques,

although not used every day in the routine blood bank, are used regularly in the forensic

laboratory (for example, blood grouping of blood stains) and in reference laboratories.

Absorption techniques lead to a decrease in antibody activity following treatment of a

serum with RBCs having the appropriate antigens; elution refers to the technique used

to dissociate or remove antibody bound to sensitized RBCs. Precipitation, complement�

fixation, and radioimmunoassay have been utilized more in blood banks in the last few

years, particularly for the detection of hepatitis virus. Fluorescence has been used to

demonstrate blood group antigens (for example, ABH) in tissues.

AGGLUTINATION

a. Background. It is convenient to consider antibody-mediated agglutination of

RBCs as involving two distinct stages. First, there is physical attachment of antibody to

the antigenic determinant on the RBC surface. This stage, representing the specific

immunochemical reaction, is referred to as sensitization. It may go on to involve the

binding or fixing of complement components. The second stage involves agglutination

of the sensitized cells. Agglutination results from collision of sensitized cells, allowing

cross-linking of cells to occur by the formation of antibody bridges. As the aim of blood

group serology is to obtain maximum sensitivity without loss of specificity, it is important

to understand and recognize the factors that influence the complex agglutination

phenomenon.

b. Factors Affecting the First Stage (Sensitization). Red blood cell

sensitization with antibody obeys the law of mass action. Thus, the reaction between

antigen on the RBC surface and antibody is reversible and the quantity of

cell-bound antibody at equilibrium will vary depending on the reaction conditions and the

equilibrium constant of the antibody. The reaction conditions should be designed to

maximize the quantity of cell-bound antibody at equilibrium in order to facilitate

detection of either blood group antigen or antibody. Some of these reaction conditions

are described below:

(1) Temperature. Most blood group antibodies show their greatest reactivity

over a restricted temperature range, some reacting optimally at 4ºC, others at 37ºC.

Antibodies reacting optimally at 37ºC have been described as “warm” antibodies, and

those reacting optimally at lower temperatures as “cold” antibodies. Agglutinins

(antibodies) having maximum reactivity at one temperature may have sufficient thermal

amplitude to be active at others. Antibody activity is usually tested at room temperature

and at 37ºC. Antibodies active at 37ºC are the most clinically significant, although “cold”

antibodies cannot be ignored if they have a wide thermal amplitude (for example, above

30ºC). Antibodies only reacting at lower temperatures may be of importance in patients

subjected to hypothermia.

(2) pH. The pH optima for antibody reactivity in most blood-group systems

have not been investigated. For anti-RhO(D), the optimum pH lies between 6.5 and 7.

Antibodies of other blood-group specificities may have different pH optima (for example,

some examples of anti-M react best at pH 5.5).

(3) Incubation time. Time is required for the antibody RBC reaction to reach

equilibrium. The amount of time required to reach this state will depend upon other

variables. The rate of antibody binding is greatest initially, so incubation times for

routine laboratory procedures may be relatively short (for example, 15 to 30 minutes).

(4) Ionic strength.

(a) The ionic strength of the reaction medium is one of the

physiochemical conditions that play an important role in the binding of antibody to RBC

antigens. Ionic strength is a measure of intensity of the electrical field resulting from

ions in solution. Electro-static forces (interaction of positive and negative charges) play

an important role in antibody reaction involving RBCs. Red blood cells carry a large

electronegative charge, which serves to keep them from spontaneously aggregating.

This enables them to function efficiently in oxygen transport by maintaining a maximum

surface area available for gas diffusion. When RBCs are suspended in an electrolyte

solution (0.85 percent NaCl), the cations (positive) are attracted to the negatively

charged RBCs, and the RBC becomes surrounded by a diffuse double layer (“ionic

cloud”), that travels with the RBC as if it were part of it. The outer edge of this layer is

called the surface of shear or the

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1-13 slipping plane. The effective charge (potential) of the RBC, called the zeta potential, is

determined at this plane and is responsible for the electrostatic repulsion between one

RBC and another.

(b) In the first stage of agglutination, reducing the ionic strength of the

medium decreases the electropositive clouds of cations surrounding the RBCs and

facilitates the interaction of electropositive lgG with the negatively charged RBC. This

absorption of antibody to the RBC reduces the electronegative charge of the RBC and

reduces the zeta potential, thereby accelerating the second stage. Experiments have

shown that the initial rate of association of

anti-RhO(D) with RhO(D)-positive RBCs is increased 1,000-fold by a reduction of ionic

strength from 0.17 to 0.03 (for example, instead of using 0.9 percent NaCI, 0.2 percent

NaCI in 7 percent glucose or 0.3M glycine is used as a RBC diluent).

(5) Antigen-antibody ratio. The rate at which antibody is bound to the cell,

and the quantity of antibody bound, depend on the concentration of cells and of

antibody. In general, an increase in sensitivity is obtained by increasing the amount of

antibody in relation to antigen. This is often achieved in the blood bank by using less

antigen in the form of weaker cell suspensions (for example, it is a more sensitive

technique to add one volume of two percent RBCs to two volumes of serum, than to add

one volume of ten percent RBCs to two volumes of serum). Some agglutination

reactions are weakened or even become negative in the presence of an excess of

antibody, the prozone reactions phenomenon. The optimal proportion of antigen to

commercial antiserum is usually determined by the manufacturer; the directions issued

with each antiserum should be followed.

c. The Second Stage (Agglutination).

Blood group antibodies were characterized empirically before the immunoglobulin(1) Once RBCs are sensitized, they may or may not directly agglutinate.

classes were recognized. Those antibodies that could produce agglutination in a saline

medium were called “complete” antibodies or ‘bivalent” antibodies, and those that did

not were called "incomplete" antibodies or “univalent” antibodies. Current evidence

indicates that all antibodies are at least bivalent; that is, each molecule has at least two

antigen-combining sites. The term incomplete antibody is used to denote an antibody

that reacts with, but fails to cause visible agglutination of a saline suspension of RBCs

possessing the corresponding antigenic determinant; such antibodies tend to be of

class lgG.

(2) The failure of “incomplete" antibodies to produce agglutination in a saline

environment may be a result, in part, of location, number, and mobility of antigenic

determinants on the RBC surface, of the size and configuration of the antibody

molecule, and of the electrostatic forces involved.

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1-14 (3) It has been suggested that the zeta potential, mentioned previously, is

the most important factor In explaining why most lgG antibodies do not directly

agglutinate RBCs, the span of the lgG molecules not being sufficient to bridge adjacent

RBCs under the conditions created by the electrostatic forces keeping the RBCs apart.

The same workers suggested that lgG antibodies agglutinated RBCs in the presence of

albumin because albumin raises the dielectric constant (charge dissipating power) of

the suspending medium, thus lowering the zeta potential, allowing RBCs to come close

enough together for agglutination to occur. They also suggested that proteolytic

enzymes (for example, papain, ficin, bromelin, and trypsin) produce the same final

effect by cleaving sialic acid from the RBC membrane, thus reducing the zeta potential.

It should be noted that lgG antibodies (for example, lgG anti-A and -B) do sometimes

directly agglutinate saline-suspended RBCs; this may be a result of the large number of

antigenic sites present, the orientation of these antigens above the surface of the RBC

membrane, and/or the clustering of these antigens during the antigen-antibody

interaction. Recently some workers have argued that zeta potential may not be the

most important factor involved in these reactions.

HEMOLYSIS

Some blood group antibodies can activate the complement cascade (see

paragraph 1-11), leading to Iysis of RBCs possessing the appropriate antigens.

Antibodies showing this characteristic are termed hemolysins and usually will

agglutinate or sensitize RBCs in the absence of complement. Examples of blood-group

antibodies that can sometimes act as hemolysins are anti-A, -B, -A, B, -I, -i, -Lea , -Leb , -

Lex , -Jka , -Jkb , -PP1Pk (TJa ), -Vel. Some of these antibodies and others may sensitize

the RBCs with complement, but not hemolyze them. This complement sensitization can

be detected by the antiglobulin test.

THE CLASSIC COMPLEMENT PATHWAY

a. Activation of the classic pathway can be initiated by a number of substances,

the best known of these, and probably the most important, being the immunoglobulin

molecule. Only one molecule of lgM on the cell membrane is necessary to activate the

complement system (two subunits of the lgM molecule combining with adjacent

antigens on the membrane). In contrast, it is thought that lgG needs to form a "doublet”;

that is to say, two separate lgG molecules have to combine with adjacent antigens on

the cell membrane as close together as 250 to 400 A, before they are able to activate

C1. Only certain subclasses of lgG are able to activate complement through this

pathway. IgG3 is the most efficient, followed by IgG1; IgG2 is the least efficient. IgG4

does not activate the complement; neither does IgA.

b. Appropriate interaction of antibody with antigen leads to sequential activation

of the complement system, often ending in cytolysis. This involves a series of

protein-protein interactions resulting in the generation of a series of cellular

intermediates bearing successively bound complement components. An

antibody-sensitized erythrocyte is designated EA, and successive complement

components are designated by numbers, for example, EACI, EAC1, 4. The protein

components of complement circulate in the plasma in an inactive state, and once

activated are designated by a bar over the component number, for example, C1. The

activation process usually is achieved by cleavage of the next complement molecule

into fragments, which are designated by lower case letters, for example, C3a, C3b. The

activated products usually have enzymatic properties; thus the whole pathway is an

enzymatic cascade similar to the coagulation cascade (see figure 1-4). The system is

held in check by the instability of the complexes formed and the naturally occurring

inhibitors and inactivators present in normal plasma (for example, C3b INA).

c. The pathway consists of three operationally defined functional units, the

recognition unit (C1), the activation unit (C4, C2, C3), and the membrane attack unit

(C5, C6, C7, C8, C9).

(1) Recognition unit. C1 is a complex of three proteins held together by

calcium. C1q is a collagen-like protein with binding sites for lgG and gM; C1r is the

activating enzyme of the critical catalytic site of the C1 complex, C1s is a proenzyme,

activated by C1r. When C1 collides with an antigen-antibody complex (EA), it is bound

to the Fc fragment of the immunoglobulin molecule through the C1q subunit. This

activates C1r and subsequently C1s by cleavage of a single polypeptide chain.

(2) Activation unit. This unit is assembled in two stages. Activated C1(C1s)

acts on native C4 by cleaving the molecule into C4a and C4b. The major fragment C4b

attaches to the cell membrane. A shower of fragments is produced by a single CIs

enzyme, so that many C4b molecules may cluster around the EAC1 site on the cell.

C1s also cleaves native C2 into two fragments; the major C2 fragment, C2a, combines

with C4b on the cell membrane to form an active complex C4b2a (C3 convertase), that

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1-16 has enzymatic activity directed against C3. Magnesium ions are necessary for the

formation of the C4b2a complex. C3 is cleaved by the C4b2a complex into two

molecules, C3a and C3b. The smaller C3a (MW 10,000) does not bind to the cell

membrane, but is released into the fluid phase as a mediator of inflammation

(anaphylatoxin I). The C3b molecule (MW 175,000) binds to the cell membrane and

can also bind to its own activation enzyme. As the C4b2a complex is an enzyme, it can

react more than once, and produce a shower of C3b fragments each time. Only the

C3b fragments that become bound adjacent to the C4b2a enzyme, however, are

believed to participate in the next reaction, in which C5 is cleaved.

(3) Membrane attack unit. Some of the C3b molecules combine with C4b2a

to form C4b, 2a, and 3b, which will cleave the C5 molecule into C5a (anaphylatoxin II)

and C5b. This is the last enzymatic reaction in the pathway. C5b appears to bind C6

and C7 by absorption. The resulting trimolecular complex attaches to the cell

membrane and binds C8 and C9. Fully assembled, the membrane attack complex

consists of one molecule of C5b, C6, C7, C8, and up to six molecules of C9. It has a

molecular weight of about one million. The end result of the pathway is lysis of the cell

(see figure 1-4).

d. Electron microscopy shows that lesions start appearing in the cell membrane

after C8 is absorbed, although the cell does not Iyse until C9 is complexed. It is not

understood how these lesions are made. In most instances, the lesions are not large

enough to allow the hemoglobin molecule to escape directly through the lesion, so it is

thought that cell lysis is caused by an osmotic effect. When cells are attacked by

complement, they swell until the cell membrane is ruptured. The cause of the swelling

is salt and water entering the cell. Mayer has postulated a theory he calls his

“doughnut” hypothesis: a stable hole is produced by the assembly of a rigid,

doughnut-shaped structure in the lipid bilayer of the cell membrane. The hole forms a

channel connecting the inside of the cell with the extracellular fluid. The outside of the

doughnut could be composed of nonpolar polypeptides, that is, protein chains that were

hydrophobic; the interior would need polar peptides so that it could be hydrophilic. He

suggests that C5b, C6, C7, C8, and C9 may be the proteins that form the doughnut or

funnel shape, penetrating the lipid bilayer of the membrane.