Activity	Responsible Complement Protein
Host Defense Against Infections
Opsonization	Covalently bonded fragments of C3 and C4
Chemotaxis and leukocyte activation	C5a, C3a, and C4a; anaphylatoxin leukocyte receptors
Lysis of bacterial and mammalian cells	C5-C9 membrane attack complex
Interface Between Innate and Adaptive Immunity
Augmentation of antibody	C3b and C4b bound to immune complexes and to antigen
Responses	C3 receptors on B cells and antigen-presenting cells
Enhancement of immunologic memory	C3b and C4b bound to immune complexes and to antigen; C3 receptors on follicular dendritic cells
Disposal of Waste
Clearance of immune complexes from tissues	C1q; covalently bonded fragments of C3 and C4
Clearance of apoptotic cells

Adapted from Walport MJ: Complement, N Engl J Med 344:1058–1065, 2001.

Table 5-2

Initiators of Three Complement Activation Pathways

Pathway	Initiators
Classic	Immune complexes Apoptotic cells Certain viruses and gram-negative bacteria C-reactive protein bound to ligand
Alternate	Various bacteria, fungi, viruses, or tumor cells
Mannose-binding lectin	Microbes with terminal mannose groups

Adapted from Walport MJ: Complement, N Engl J Med 344:1058–1065, 2001.

1. Classic pathway

2. Alternative pathway

3. Mannose-binding lectin pathway

The three pathways (Fig. 5-1) converge at the point of cleavage of C3 to C3b, the central event of the common final pathway, which in turn leads to the activation of the lytic complement sequence, C5 through C9, and cell destruction (Fig. 5-2).

Figure 5-1 Early steps of complement activation.
The steps in the activation of the alternative, classical, and lectin pathways are shown. Note the sequence of events is similar in all three pathways, although they differ in their requirement for antibody and in the proteins used. (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders.)

Figure 5-2 Late steps of complement activation.
A, The late steps of complement activation start after the formation of the C5 convertase and are identical in the alternative and classical pathways. Products generated in the late steps induce inflammation (C5a) and cell lysis (the membrane attack complex [MAC]). B, The properties of the proteins of the late steps of complement activation are listed. (From Abbas AK, Lichtman AH: Basic immunology: functions and disorders of the immune system, updated edition, ed 3, Philadelphia, 2011, Saunders.)

Classic Pathway

The classic complement pathway is one of the major effector mechanisms of antibody-mediated immunity. The principal components of the classic pathway are C1 through C9. The sequence of component activation—C1, 4, 2, 3, 5, 6, 7, 8, and 9—does not follow the expected numeric order.

C3 is present in the plasma in the largest quantities; fixation of C3 is the major quantitative reaction of the complement cascade. Although the principal source of synthesis of complement in vivo is debatable, the majority of the plasma complement components are made in hepatic parenchymal cells, except for C1 (a calcium-dependent complex of the three glycoproteins C1q, C1r, and C1s), which is primarily synthesized in the epithelium of the gastrointestinal and urogenital tracts.

The classic pathway has three major stages:

1. Recognition

2. Amplification of proteolytic complement cascade

3. Membrane attack complex (MAC)

Recognition

The recognition unit of the complement system is the C1 complex—C1q, C1r, and C1s, an interlocking enzyme system. In the classic pathway, the first step is initiation of the pathway triggered by recognition by complement factor C1 of antigen-antibody complexes on the cell surface. When C1 complex interacts with aggregates of immunoglobulin G (IgG) with antigen on a cell’s surface, two C1-associated proteases, C1r and C1s, are activated. A single IgM molecule is potentially able to fix C1, but at least two IgG molecules are required for this purpose. The amount of C1 fixed is directly proportional to the concentration of IgM antibodies, although this is not true of IgG molecules. C1s is weakly proteolytic for free intact C2, but is highly active against C2 that has complexed with C4b molecules in the presence of magnesium (Mg²⁺) ions. This reaction will occur only if the C4bC2 complex forms close to the C1s.

The resultant C2a fragment joins with C4b to form the new C4bC2a enzyme, or classic pathway C3 convertase. The catalytic site of the C4bC2a complex is probably in the C2a peptide. A smaller C2b fragment from the C2 component is lost to the surrounding environment.

Amplification of Proteolytic Complement Cascade

Once C1s is activated, the proteolytic complement cascade is amplified on the cell membrane through sequential cleavage of complement factors and recruitment of new factors until a cell surface complex containing C5b, C6, C7, and C8 is formed.

The complement cascade reaches its full amplitude at the C3 stage, which represents the heart of the system. The C4bC2a complex, the classic pathway C3 convertase, activates C3 molecules by splitting the peptide, C3 anaphylatoxin, from the N-terminal end of the peptide of C3. This exposes a reactive binding site on the larger fragment, C3b. Consequently, clusters of C3b molecules are activated and bound near the C4bC2a complex. Each catalytic site can bind several hundred C3b molecules, even though the reaction is very efficient because C3 is present in high concentration. Only one C3b molecule combines with C4bC2a to form the final proteolytic complex of the complement cascade.

Membrane Attack Complex

The membrane attack complex (MAC) is a unique system that builds up a lipophilic complex in cell membranes from several plasma proteins. To initiate C5b fixation and the MAC, C3b splits C5a from the alpha chain of C5. No further proteinases are generated in the classic complement sequence. Other bound C3b molecules not involved in the C4b2a3b complex form an opsonic macromolecular coat on the erythrocyte or other target, which renders it susceptible to immune adherence by C3b receptors on phagocytic cells.

When fully assembled in the correct proportions, C7, C6, C5b, and C8 form the MAC (see Fig. 5-2, inset). The C5bC6 complex is hydrophilic but, with the addition of C7, it has additional detergent and phospholipid-binding properties as well. The presence of hydrophobic and hydrophilic groups within the same complex may account for its tendency to polymerize and form small protein micelles (a packet of chain molecules in parallel arrangement). It can attach to any lipid bilayer within its effective diffusion radius, which produces the phenomenon of reactive lysis on innocent so-called bystander cells. Once membrane bound, C5bC6C7 is relatively stable and can interact with C8 and C9.

The C5bC6C7C8 complex polymerizes C9 to form a tubule (pore), which spans the membrane of the cell being attacked, allowing ions to flow freely between the cellular interior and exterior. By complexing with C9, the osmotic cytolytic reaction is accelerated. This tubule is a hollow cylinder with one end inserted into the lipid bilayer and the other projecting from the membrane. A structure of this form can be assumed to disturb the lipid bilayer sufficiently to allow the free exchange of ions and water molecules across the membrane. Ions flow out, but large molecules stay in, causing water to flood into the cell. The consequence in a living cell is that the influx of sodium (Na⁺) ions and H₂O leads to disruption of osmotic balance, which produces cell lysis.

Alternative Pathway

The alternative pathway shows points of similarity with the classic sequence. Both pathways generate a C3 convertase that activates C3 to provide the pivotal event in the final common pathway of both systems. However, in contrast to the classic pathway, which is initiated by the formation of antigen-antibody reactions, the alternate complement pathway is predominantly a non–antibody-initiated pathway.

Microbial and mammalian cell surfaces can activate the alternative pathway in the absence of specific antigen-antibody complexes. Factors capable of activating the alternative pathway include inulin, zymosan (polysaccharide complex from surface of yeast cells), bacterial polysaccharides and endotoxins, and the aggregated IgG2, IgA, and IgE. In paroxysmal nocturnal hemoglobinuria (PNH), the patient’s erythrocytes act as an activator and result in excessive lysis of these erythrocytes. This nonspecific activation is a major physiologic advantage because host protection can be generated before the induction of a humoral immune response.

A key feature of the alternative pathway is that the first three proteins of the classic activation pathway—C1, C4, and C2—do not participate in the cascade sequence. The C3a component is considered to be the counterpart of C2a in the classic pathway. C2 of the classic pathway structurally resembles factor B of the alternative pathway. The omission of C1, C4, and C2 is possible because activators of the alternative pathway catalyze the conversion of another series of normal serum proteins, which leads to the activation of C3. It was previously believed that properdin, a normal protein of human serum, was the first protein to function in the alternative pathway; thus, the pathway was originally named after this protein.

The uptake of factor B onto C3b occurs when C3b is bound to an activator surface. However, C3b in the fluid phase or attached to a nonactivator surface will preferentially bind to and therefore prevent C3b,B formation. C3b and factor B combine to form C3b,B, which is converted into an active C3 convertase, C3b,Bb. This results from the loss of a small fragment, Ba (glycine-rich α₂-globulin believed to be physiologically inert), through the action of the enzyme, factor D. The C3b,Bb complex is able to convert more C3 to C3b, which binds more factor B and the feedback cycle continues.

The major controlling event of the alternative pathway is factor H, which prevents the association between C3b and factor B. Factor H blocks the formation of C3b,Bb, the catalytically active C3 convertase of the feedback loop. Factor H (formerly β₁-H) competes with factor B for its combining site on C3b, eventually leading to C3 inactivation. Factors B and H apparently occupy a common site on C3b. The factor that is preferentially bound to C3b depends on the nature of the surface to which C3b is attached. Polysaccharides are called activator surfaces and favor the uptake of factor B on the chain of C3b, with the corresponding displacement of factor H. In this situation, binding of factor H is inhibited, and consequently factor B will replace H at the common binding site. When factor H is excluded, C3b is thought to be formed continuously in small amounts. Another controlling point in the amplification loop depends on the stability of the C3b,Bb convertase. Ordinarily, C3b,Bb decays because of the loss of Bb, with a half-life of approximately 5 minutes. However, if properdin (P) binds to C3b,Bb, forming C3b,BbP, the half-life is extended to 30 minutes.

The association of numerous C3b units, factor Bb, and properdin on the surface of an aggregate of protein or the surface of a microorganism has potent activity as a C5 convertase. With the cleavage of C5, the remainder of the complement cascade continues as in the classic pathway.

Mannose-Binding Lectin Pathway

Mannose-binding lectin is a member of a family of calcium-dependent lectins, the collectins (collagenous lectins), and is homologous in structure to C1q. Mannose-binding lectin, a pattern recognition molecule of the innate immune system, binds to arrays of terminal mannose groups on a variety of bacteria.

A deficiency of mannose-binding lectin is caused by one of three point mutations in its gene, each of which reduces levels of the lectin. After the discovery that the binding of mannose-binding lectin to mannose residues can initiate complement activation, the mannose-binding lectin–associated serine protease (MASP) enzymes were discovered. MASP activates complement by interacting with two serine proteases called MASP1 and MASP2. These components make up the mannose-binding lectin pathway.

BiologicAl Functions of Complement Proteins

The biological functions of the complement system fall into the following two general categories:

1. Cell lysis by the membrane attack complex (MAC)

2. Biological effects of proteolytic fragments of complement

The first category is the situation in which the MAC leads to osmotic lysis of a cell. The second category encompasses other effects of complement in immunity and inflammation that are mediated by the proteolytic fragments generated during complement activation. These fragments may remain bound to the same cell surfaces at which complement has been activated or may be released into the blood or extracellular fluid. In either situation, active fragments mediate their effects by binding to specific receptors expressed on various types of cells, including phagocytic leukocytes and the endothelium (Table 5-3).

Table 5-3

Selected Complement Components and Functions

Complement Component(s)	Function
C5-C9	Lysis of cells
C3B, IC3B	Opsonization in phagocytosis
C5A >C3A >>C4A	Anaphylatoxins/inflammation (vascular responses)
C5A	Polymorphonuclear leukocyte activation
Classic complement pathway, C3B, ?iC3b, C3dg	Immune complex removal B-lymphocyte activation

In contrast, the absence of an integral component of the classic, alternative, or terminal lytic pathways can lead to decreased complement activation and a lack of complement-mediated biological functions.

Alterations in Complement Levels

The complement system can cause significant tissue damage in response to abnormal stimuli. Biological effects of complement activation can occur as a reaction to persistent infection or an autoantibody response to self antigens. In these infectious or autoimmune conditions, the inflammatory or lytic effects of complement may contribute significantly to the pathology of the disease.

Complement activation is also associated with intravascular thrombosis, which leads to ischemic injury to tissues. Complement levels may be abnormal in certain disease states (e.g., rheumatoid arthritis, systemic lupus erythematosus [SLE]) and in some genetic disorders.

Elevated Complement Levels

The complement level can be elevated in many inflammatory conditions. Increased complement levels are often associated with inflammatory conditions, trauma, or acute illness such as myocardial infarction because separate complement components (e.g., C3) are acute-phase proteins. However, these elevations are common and nonspecific. Therefore, increased levels are of limited clinical significance.

Decreased Complement Levels

Low levels of complement suggest one of the following biological effects:

• Complement has been excessively activated recently.

• Complement is currently being consumed.

• A single complement component is absent because of a genetic defect.

Specific component deficiencies are associated with a variety of disorders (Table 5-4). Deficiencies of complement account for a small percentage of primary immunodeficiencies (<2%), but depression of complement levels frequently coexists with SLE and other disorders associated with an immunopathologic process (Box 5-1).

Table 5-4

Complement Deficiency in Human Beings

Deficiency	Associated Disease
C1q	SLE-like syndrome; decreased secondary to agammaglobulinemia
C1r	SLE-like syndrome; dermatomyositis, vasculitis, recurrent infections and chronic glomerulo-nephritis, necrotizing skin lesions, arthritis
C1s	SLE, SLE-like syndrome
C1 INH	Hereditary angioedema, lupus nephritis
C2	Recurrent pyogenic infections, SLE, SLE-like syndrome, discoid lupus, membranoproliferative glomerulonephritis, dermatomyositis, synovitis, purpura, Henoch-Schönlein purpura, hypertension, Hodgkin’s disease, chronic lymphocytic leukemia, dermatitis herpetiformis, polymyositis
C3	Recurrent pyogenic infections, SLE-like syndrome, arthralgias, skin rash
C3 inactivator	Recurrent pyogenic infections, urticaria
C4	SLE-like syndrome, SLE, dermatomyositis-like syndrome, vasculitis
C5	Neisseria infections, SLE
C5 dysfunction	Leiner’s disease, gram-negative skin and bowel infection
C6	Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome, vasculitis
C7	Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome, vasculitis
C8	Neisseria infections, xeroderma pigmentosa, SLE-like syndrome

Modified from Colten HR, Rosen FS: Complement deficiencies annual review of immunology, 10:809-834, 1992 and Nusinow SR, Zuraw BL, Curd JG: The hereditary and acquired deficiencies of complement, Med Clin North Am 69:487-504, 1985.

Deficiencies in any of the protein components of complement are usually caused by a genetic defect that leads to abnormal patterns of complement activation. If regulatory components are absent, excess activation may occur at the wrong time or at the wrong site. The potential consequences of increased activation are excess inflammation and cell lysis and consumption of complement components.

Hypocomplementemia can result from the complexing of IgG or IgM antibodies capable of activating complement. Depressed values of complement are associated with diseases that give rise to circulating immune complexes. Because of the rapid normal turnover of the complement proteins—within 1 or 2 days of the cessation of complement activation by immune complexes—complement levels return to normal rapidly.

The following three types of complement deficiency can cause increased susceptibility to pyogenic infections:

1. Deficiency of the opsonic activities of complement

2. Any deficiency that compromises the lytic activity of complement

3. Deficient function of the mannose-binding lectin pathway

Increased susceptibility to pyogenic bacteria (e.g., Haemophilus influenzae, Streptococcus pneumoniae) occurs in patients with defects of antibody production, complement proteins of the classic pathway, or phagocyte function. The sole clinical association between inherited deficiency of MAC components and infection is with neisserial infection, particularly Neisseria meningitidis. Low levels of mannose-binding lectin in young children with recurrent infections suggest that the mannose-binding lectin pathway is important during the interval between the loss of passively acquired maternal antibody and the acquisition of a mature immunologic repertoire of antigen exposure.

Diagnostic Evaluation

During immune complex reactions, certain complement proteins become physically bound to the tissue in which the immunologic reaction is occurring. These proteins can be demonstrated in tissue by appropriate immunopathologic stains. The most frequent evaluation of complement is by serum or plasma assay (Table 5-5). Complement components (e.g., C3 and C4) can be assessed by nephelometry. These assays are useful for the diagnosis and monitoring of patients.

Table 5-5

Interpretation of Complement Activation by Individual Components

Complement Determination	Classic Pathway	Alternative Pathway	Improper Specimen^∗	Inflammation
C3	Decreased	Decreased	Normal	Increased
C4	Decreased	Normal	Decreased	Increased

^∗Results if specimen is improperly stored or too old.

Assessment of Complement

The procedures discussed next can be used in diagnostic immunology.

C1 Esterase Inhibitor (C1 Inhibitor)

C1 measures the activity and concentration of C1 inhibitor in serum. A deficiency of this protein is characteristic of hereditary angioedema (HAE; see later). Some patients demonstrate catalytically inactive protein.

C1r, C1s, C2, C3, C4, C5, C6, C7, C8

Homozygous deficiencies predispose a patient to autoimmune disease (especially SLE) and to arthritis, chronic glomerulonephritis, infections, and vasculitis.

C1q

The complement component C1q is evaluated in serum. Decreased levels can be demonstrated in patients with hypocomplementemic urticarial vasculitis, severe combined immunodeficiency (SCID), or X-linked hypogammaglobulinemia.

C1q Binding

This procedure measures the binding of immune complexes containing IgG1, IgG2, or IgG3 and IgM to the complement component C1q. High values of C1q binding are associated with the presence of circulating immune complexes of the type that interacts with the classic pathway of complement activation. This test can be useful as a prognostic tool at diagnosis and during remission of acute myelogenous leukemia.

C2

The most common complement deficiency is of C2. It is an autosomal recessive disorder; the C2 gene is on chromosome 6 in the major histocompatibility complex (MHC). The incidence is 1:28,000 to 1:40,000; the carrier state is 1.2% in the general population.

Half of patients with homozygous C2 deficiency have no symptoms; those with symptoms have infections with S. pneumoniae, N. meningitidis, and H. influenzae. Of symptomatic patients, 50% exhibit a lupus-like disorder with photosensitivity and rash.

C3

Also an acute-phase protein, elevated C3 levels can indicate an acute inflammatory disease. Although C3 lies at the junction of the two pathways, it is much more severely depressed when activation occurs via the alternative pathway. Extremely decreased levels are seen in patients with poststreptococcal glomerulonephritis and in those with inherited (C3) complement deficiency. This component is also decreased in cases of severe liver disease and in SLE patients with renal disease.

C3b Inhibitor (C3b Inactivator)

The C3b component of complement causes low complement C3 levels, the absence of C3PA in serum, and high C3b levels. A deficiency of C3b inhibitor is associated with an increased predisposition to infection.

C3PA (C3 Proactivator, Properdin Factor B)

The factor B component is consumed by activation of the alternative complement pathway. Assessment of C3PA indicates whether a decreased level of C3 results from the classic or alternative pathways of complement activation. Decreased levels of C3 and C4 demonstrate activation of the classic pathway. Decreased levels of C3 and C3PA with a normal level of C4 indicate complement activation via the alternative pathway (Table 5-5).

Activation of the classic pathway (and sometimes with accompanying alternative pathway activation) is associated with disorders such as immune complex diseases, various forms of vasculitis, and acute glomerulonephritis. Activation of the alternative pathway is associated with many disorders, including chronic hypocomplementemic glomerulonephritis, disseminated intravascular coagulation (DIC), septicemia, subacute bacterial endocarditis, PNH, and sickle cell anemia.

In SLE, both the classic and alternative pathways are activated.

C4

The C4 level often provides the most sensitive indicator of disease activity. C4 is also an acute-phase reactant. Elevated C4 levels can indicate an acute inflammatory reaction or a malignant condition. Measurement of C4 may demonstrate inflammation or infection long before it is clinically evident by standard assessment methods (e.g., total white blood count [WBC] and leukocyte differential, febrile response, or elevated erythrocyte sedimentation rate [ESR]).

C4 is destroyed only when the classic pathway is activated. A decreased C4 level with elevated anti–n-DNA and antinuclear antibody (ANA) titers confirm the diagnosis of SLE in a patient. In these cases of SLE, the periodic assessment of C4 can be useful for monitoring the progress of the disorder. Patients with extremely low C4 levels in the presence of normal levels of the C3 component may be demonstrating the effects of a genetic deficiency of C1 inhibitor or C4. Reduction of C3 and C4 components implies that activation of the classic pathway has been initiated.

C4 Allotypes

The antigenically distinct forms of C4A and C4B are located on chromosome 6 in the MHC. C4 allotypes in conjunction with specific human leukocyte antigens (HLAs) are markers for disease susceptibility.

C5

A genetic deficiency of the C5 component is associated with increased susceptibility to bacterial infection and is expressed as an autoimmune disorder (e.g., SLE). Patients with dysfunction of C5 (Leiner’s disease) are predisposed to infections of the skin and bowel, characterized by eczema. Their C5 level is normal, but the C5 component fails to promote phagocytosis.

C6

A decreased quantity of C6 predisposes an individual to significant neisserial (bacterial) infections.

C7

A decreased level of C7 is associated with Raynaud’s phenomenon, sclerodactyly, telangiectasia, and severe bacterial infections caused by Neisseria spp.

C8

A decreased quantity of C8 is associated with SLE. A C8 deficiency makes patients highly susceptible to Neisseria infections.

Select Complement Deficiencies

Properdin Deficiency

Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb). A deficiency leads to bacterial infections, often meningococcemia. This disorder is an X-linked recessive trait.

Hereditary Angioedema

This disorder is a deficiency in a complement protein. Infections are not usually a significant problem. HAE is autosomal dominant, unlike other complement deficiencies. Two types exist, type 1 (low antigen level and low functional protein) and type 2 (normal antigen level with low function).

Familial Mediterranean Fever

This defect in protease in peritoneal and synovial fluid is transmitted as an autosomal recessive trait on chromosome 16. Patients with this defect experience recurrent episodes of fever and inflammation in the joints and pleural and peritoneal fluids.

Other Soluble Immune Response Mediators

Biological Response Modifiers

Biological response modifiers (BRMs) modulate an individual’s own immune response. There are four main sources of major BRMs secreted by mononuclear leukocytes:

1. B lymphocytes that secrete specific antibodies

2. T lymphocytes that secrete soluble mediators, such as interleukin-2 (IL-2) and other ILs, granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β)

3. Natural killer (NK) lymphocytes that secrete IFN-α

4. Monocytes and macrophages that secrete IFN-α, IL-1, and other ILs, TNF-α, and GM-CSF and monocyte colony-stimulating factor (M-CSF)

Biological response modifiers can be used therapeutically. The classes of immunotherapy are as follows:

• Active—use of microbial or chemical immunomodulators (adjuvants) in a specific or nonspecific form

• Adoptive—use of soluble mediators, such as ILs, to regulate components of the immune system

• Passive—transfer of preformed antibodies to tumorous recipients, such as monoclonal antibodies

• Restorative—application of soluble substances, such as interferons, for a wide range of diseases

Cytokines

Migratory inhibitory factor (MIF) was the first cytokine activity to be described. MIF performs a T cell–derived activity that immobilizes macrophage migration, which may cause retention and accumulation of phagocytes at sites of inflammation.

Research is ongoing and the list of individual cytokines steadily increases. Cytokines are synthesized and secreted by the cells associated with innate and adaptive immunity in response to microbial and other antigen exposures (Tables 5-6 and 5-7).

Table 5-6

Examples of Cytokines of Innate and Adaptive Immunity

Innate Immunity	Adaptive Immunity
Chemokines	IFN-γ
IFN type 1 (IFN-α, IFN-β)	IL-2
IL-1	IL-4
IL-6	IL-5
IL-10	IL-13
IL-12	Lymphotoxin (Lt)
IL-15	TGF-β
IL-18
TNF

IFN, Interferon; IL, interleukin; TGF, transforming growth factor; TNG, tumor necrosis factor.

Table 5-7

Comparative Features of Innate and Adaptive Immunity

	Type of Immunity
	Innate	Adaptive
Examples	TNF-α, IFN-β, IL-1, IL-12	IFN-γ, IL-2, IL-4, IL-5
Major cell source	Macrophages, NK cells	T lymphocytes
Major physiologic function	Mediators of innate immunity and inflammation (local and systemic)	Regulation of lymphocyte growth and differentiation Activation of effector cells (macrophages, eosinophils, mast cells)
Stimuli	LPS (endotoxin), bacterial peptidoglycans, viral RNA, T cell–derived cytokines (e.g., IFN-β)	Protein antigens
Quantity produced	Possibly high, detectable in serum	Usually low, usually undetectable in serum
Effects on body	Local and systemic	Usually local
Roles in disease	Systemic diseases	Local tissue injury
Inhibitors	Corticosteroids	Cyclosporine, FK-506

IFN, Interferon; IL, interleukin; LPS, lipopolysaccharides; TNF, tumor necrosis factor.

Adapted from Abbas AK, Lichtman AH, Pober JS: Cellular and molecular immunology, ed 4, Philadelphia, 2000, Saunders.

The generic term cytokines has become the preferred name for this class of mediators. Lymphokines is another term used to describe cytokines produced by activated lymphocytes. Cytokines produced by leukocytes that act on other leukocytes are also referred to by the imperfect but descriptive term interleukins (ILs). As cytokines are discovered and characterized, they are assigned a number using a standard nomenclature (e.g., IL-1).

Cytokines are polypeptide products of activated cells that control a variety of cellular responses and thereby regulate the immune response. Many cytokines are released in response to specific antigens; however, cytokines are nonspecific in that their chemical structure is not determined by the stimulating antigen. Most cytokines have multiple activities and act on numerous cell types. Hematopoietic and lymphoid cell compartments are regulated by a complex network of interacting cytokines. The colony-stimulating factors (CSFs) and ILs have been shown to play important roles in normal proliferation, differentiation, and activation of several hematopoietic and lymphoid lineages (Tables 5-8 and 5-9).

Table 5-8

Origin and Immunoregulatory Activity of Cytokines

Cytokines	Origin	Prominent Biological Activities
Interleukins (ILs)
IL-1 superfamily	Both IL-1α and IL-1β are produced by monocytes-macrophages and dendritic cells.	Original members: IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1RA). IL-1α and IL-1β are proinflammatory cytokines involved in immune defense against infection. IL-1RA is a molecule that competes for receptor binding with IL-1α and IL-1β, blocking their role in immune activation. Principal function of IL-1—mediator of host inflammatory response to infections and other inflammatory stimuli. These cytokines increase the expression of adhesion factors on endothelial cells to enable transmigration of leukocytes to sites of infection and reset the hypothalamic thermoregulatory center, leading to increased body temperature (fever), which helps the immune system fight infection. IL-1 also important in regulation of hematopoiesis.
IL-2 (formerly “T-cell growth factor”)	Helper T cells	Has high capacity to induce activation of almost all clones of cytotoxic cells. Increases cytotoxic functions of T killer and NK cells; promotes production of perforins and IFN-γ by these cells. Activates monocytes-macrophages to synthesize and secrete TNF-α, IL-1β, IL-6, IL-8, G-CSF, and GM-CSF.
IL-3 (formerly “multicolony colony-stimulating factor”)	Activated T cells	Promotes expansion of early blood cells (hematopoiesis) that differentiate into all known mature cell types. Supports growth and differentiation of T cells from bone marrow through immune response.
IL-4	T cells, mast cells	Induces differentiation of naive helper T cells (Th0 cells) to Th2 cells. On activation by IL-4, Th2 cells subsequently produce additional IL-4. Cell that initially produces IL-4 and induces Th0 differentiation has not been identified. Early activation of resting B cells—upregulates MHC class II production (induces HLA-DR molecules on B cells, macrophages) and governs B cell isotype switching to IgG1 and IgE. Key regulator in humoral and adaptive immunity.
IL-5	Helper T cells type 2 (Th2) and mast cells	Principal function—activate eosinophils and serve as link between T cell activation and eosinophilic inflammation. Stimulates growth and differentiation of eosinophils and activates mature eosinophils (IL-5 expressed on eosinophils). Growth and differentiation–inducing factor for activated T and B cells; induces class-specific B cell differentiation (IgA production).
IL-6	Macrophages, T cells, osteoblasts	Functions in innate immunity and adaptive immunity; in the latter, stimulates growth of B cells that have differentiated into antibody producers. IL-1, TNF, and IL-6 appear to be major factors that induce the acute-phase response.
IL-7	Stromal cells of red bone marrow and thymus	Stimulates proliferation of lymphoid progenitors; important for proliferation during certain stages of B cell maturation and in T cell and NK survival, development, and homeostasis. IL-7 has recently been shown to have therapeutic potential and safety in several clinical trials designed to demonstrate T cell restoration in immunodeficient patients.
IL-8	Macrophages and certain types of epithelial cells (e.g., endothelium)	Potent stimulator of neutrophils in chemotaxis. Activates “respiratory burst” and release of specific and azurophilic granular contents.
IL-9	T cells (specifically by CD4+ helper cells)	Promotes proliferation of T cells, thymocytes, and mast cells. Supports proliferation of some T cell lines and of bone marrow–derived mast cell progenitors; supports growth of erythroid blast-forming units.
IL-10	Monocytes, Th2 cells, B cells	Inhibits activated macrophages; displays potent abilities to suppress antigen-presenting capacity of APCs. Released by cytotoxic T (Tc) cells to inhibit the actions of NK cells during immune response to viral infection. IL-10 is stimulatory toward certain T cells, mast cells, and B cells. It can downregulate the synthesis of other ILs.
IL-11	Bone marrow stroma	Acts in a manner similar to IL-6 on hematopoietic progenitor cells. IL-11 has been shown to synergize with IL-3 to stimulate production of megakaryocyte and myeloid progenitors and to increase number of Ig-secreting B lymphocytes in vivo and in vitro.
IL-12 (NK stimulatory factor)	B cells, macrophages	Although it shares functional properties of enhancing cytotoxic function of NK cells and activated T cells with IL-2, IL-12 appears to act through a distinct mechanism independent of IL-2. Biological actions of IL-12 include stimulating production of IFN-γ by NK and T cells, stimulating differentiation of naive T cells into Th1 cells, and enhancing cytolytic functions of activated NK cells and CD8+ Tc cells. Growth factor for activated NK-LAK cells.
IL-13	T cells	Possesses many biological effects similar to IL-4 but appears to have less effect on T or B cells than IL-4. Major action of IL-13 on macrophages is to inhibit their activation and to antagonize IFN-γ. Important mediator of allergic inflammation and disease. Functions of IL-13 overlap considerably with those of IL-4, especially changes induced on hematopoietic cells, but these effects are probably less important given the more potent role of IL-4. IL-13 acts more prominently as a molecular bridge linking allergic inflammatory cells to nonimmune cells, altering physiologic function. It is associated primarily with induction of airway disease and also has antiinflammatory properties.
IL-14 (high-molecular-weight B cell growth factor [HMW-BCGF])	T cells and malignant B cells	Acts as B-cell growth factor (BCGF) in proliferation of normal and cancerous B cells. Hyperproduction of IL-14 enables progression of B-cell non-Hodgkin’s lymphoma (NHL-B); conversely, its antibodies slow down growth of NHL-B.
IL-15	T cells	Biologically similar to IL-2; acts as synergist, particularly in LAK cell induction process; increases antitumoral activities of T-killer and NK cells and can be chemoattractant for T lymphocytes; endogenous IL-15 is key condition for IFN-γ synthesis. IL-15 produced in response to viral infection and other signals that trigger innate immunity; homologous to IL-2. Function of IL-15 is to promote proliferation of NK cells. Maintenance of memory cells does not appear to require persistence of the original antigen; instead, survival signals for memory lymphocytes are provided by cytokines such as IL-15.
IL-16	Monocytes, CD8+ lymphocytes, B lymphocytes	Acts as a T-cell chemoattractant; increases mobility of CD8+ and CD4+ T cells and, with IL-2, promotes their activation. IL-16 is found in B lymphocytes. Recruits and activates many other cells expressing CD4 molecule, including monocytes, eosinophils, and dendritic cells.
IL-17	CD4+ lymphocytes	Induces granulopoiesis through G-CSF; can reinforce antibody-dependent tumor cell destruction; participates in regulation of many cytokines (IL-1, IL-4, IL-6, IL-10, IL-12, IFN-γ). Histamine and serotonin increase production of IL-17. IL-17 mimics many proinflammatory actions of TNF-α and TNF-β.
IL-18	Macrophages	Acts as synergist with IL-12 in some effects, especially induction of IFN-γ production and inhibition of angiogenesis; high IFN-γ production under integrated effect of IL-18 and IL-12 suppresses tumor growth. IL-18 stimulates production of IFN-γ by NK cells and T cells, synergistic with IL-12.
IL-19	Monocytes	Lipopolysaccharides (LPS) and GM-CSF stimulate synthesis of IL-19, which is then upregulated in monocytes. Biological function similar to that of IL-10; regulates functions of macrophages and suppresses activities of Th1 and Th2.
IL-20	Activated keratinocytes, monocytes	Biological activities similar to those of IL-10 and can stimulate tumor growth. Regulates proliferation and differentiation of keratocytes during inflammation, particularly inflammation associated with the skin. Causes expansion of multipotential hematopoietic progenitor cells.
IL-21	Various lymphocytes	Regulates hematopoiesis and immune response and influences development of lymphocytes; similar to IL-2 and IL-15 in antitumor defense system; promotes high production of T lymphocytes, fast growth and maturation of NK cells, and fast growth of B lymphocytes. Has potent regulatory effects on immune cells, interacting with cell surface IL-21 receptor, expressed in bone marrow cells and various lymphocytes.
IL-22	Activated T cells	Similar to IL-10, but does not prohibit production of proinflammatory cytokines through monocytes in response to LPS; somewhat similar to IFN-α, -β, and -γ.
IL-23	—	Newly discovered cytokine that shares some in vivo functions with IL-12. IL-23 is important part of inflammatory response against infection; as proinflammatory cytokine, it enhances T cell priming and stimulates production of proinflammatory molecules (IL-1, IL-6, TNF-α, NOS-2, chemokines), resulting in inflammation.
IL-24	Activated monocytes-macrophages, Th2 cells	Appears to participate in cell survival and proliferation by inducing rapid activation of particular transcription factors called STAT-1 and STAT-3; predominantly released by and acts on nonhematopoietic (skin, lung, reproductive) tissues. Performs important roles in wound healing and cancer; cell death occurs in cancer cells and cell lines after exposure to IL-24.
IL-25	Th2 cells, mast cells	Biologically characterized as a member of IL-17 cytokine family. Supports proliferation of cells in lymphoid lineage. Induces production of other cytokines (IL-4, IL-5, IL-13) in multiple tissues, which stimulate the expansion of eosinophils. Important molecule in controlling immunity of the gut; implicated in chronic inflammation associated with gastrointestinal tract; identified in chromosomal region associated with autoimmune diseases such as inflammatory bowel disease (IBD), although no direct evidence suggests that IL-25 plays a role in IBD.
IL-26	Expressed in certain herpesvirus-transformed T cells, but not in primary stimulated T cells	Induces rapid phosphorylation of transcription factors STAT-1 and STAT-3, which enhance IL-10 and IL-8 secretion and expression of CD54 molecule on surface of epithelial cells.
IL-27	—	Has important function in regulating activity of B and T lymphocytes; belongs to the IL-12 family.
IL-28	—	Plays role in immune defense against viruses.
IL-29	—	Plays important role in host defenses against microbes; its gene is highly upregulated in cells infected with virus.
IL-30 (also IL27p28)	New name of p28, a subunit of IL27	Interleukin 30 (IL-30), a member of the long-chain four-helix bundle cytokine family, and EBI3 form the IL-27 heterdimer, which is expressed by APCs. IL-27 triggers expansion of antigen-specific naive CD4-positive T cells and promotes polarization toward a Th1 phenotype with expression of IFN-γ. IL-27 acts in synergy with IL-12 and binds to WSX1.
IL-31	Produced preferentially by Th2 cells. Receptor subunits expressed in activated monocytes and unstimulated epithelial cells.	Believed to play role in skin inflammation.
IL-32	Monocytes-macrophages	Can induce cells of immune system (e.g., monocytes-macrophages) to secrete TNF-α in addition to chemokines such as IL-8. Induces expression of TNF-α and IL-8 in THP-1 monocytic cells. Expression of IL-32 is induced in human peripheral lymphocyte cells after mitogen stimulation, in human epithelial cells by IFN-γ, and in NK cells after exposure to IL-12–IL-18 combination. Involved in activation induced cell death. Expression of IL-32 is upregulated in T killer and NK-cells after cell activation, and IL-32β is predominant isoform in activated T cells. IL-32 is expressed specifically in T cells undergoing cell death; enforced expression of IL-32 induces apoptosis; downregulation rescues the cells from apoptosis.
IL-33	Helper T cells	Induces type 2 cytokine production from Th cells. Mediates biological effects by interacting with orphan IL-1 receptor, activating intracellular molecules in certain signaling pathways that drive production of type 2 cytokines (e.g., IL-4, IL-5, IL-13) from polarized Th2 cells. Constitutive expression of IL-33 is found in smooth muscle cells and bronchial epithelial cells. Expression in primary lung or dermal fibroblasts and keratinocytes is inducible by treatment with TNF-α and IL-1β; these two cytokines only induce low-level expression in dendritic cells and macrophages.
Interferons (IFNs)
IFN-α	Leukocytes	Antiviral, increased MHC class I expression.
IFN-β	Fibroblasts, epithelial cells	Antiviral, increased MHC class I expression.
IFN-γ	T cells, NK cells	Major macrophage activator; induces MHC class II molecules on many cells and can synergize with TNF; augments NK cell activity; antagonist to IL-4.

IFN, Interferon; TNF, tumor necrosis factor; G-CSF, granulocyte colony-stimulating factor; GM, granulocyte-macrophage; MHC, major histocompatibility complex; NK, natural killer; APCs, antigen-presenting cells.

From Abbas AK, Lichtman AH: Basic Immunology ed 3 Update, Saunders, 2011, pp. 258-259 and http://www.gene.ucl.ac.uk/nomeclature/genefamily/il.php, 2007.

Table 5-9

Immunoregulatory Activity of Other Cytokines

Factor	Target Cells	Prominent Biological Activities
Tumor Necrosis Factor (TNF)
TNF-α (cachectin)	Macrophages, NK cells	Local inflammation, endothelial activation
TNF-β (lymphotoxin)	T cells, B cells	Killing, endothelial activation
Tumor necrosis family	T cells, mast cells
CD40 ligand		B-cell activation, class switching
TNF Family
CD27 ligand	T cells	Stimulates T-cell proliferation
CD30 ligand	T cells	Stimulates T- and B-cell proliferation
Chemokines
Membrane cofactor protein (MCP-1)	Macrophages, others	Chemotactic for monocytes

Adapted from Claman HN: The biology of the immune response, JAMA 268:2790–2796, 1992; Janeway C, Travers P: Immunobiology, ed 3, New York, 1997, Garland; and Abbas AK, Lichtman AH, Pober JS: Cellular and molecular immunology, ed 4, Philadelphia, 2000, Saunders.

Cytokines have a variety of roles in host defense. In innate immunity, cytokines mediate early inflammatory reactions to microbial organisms and stimulate adaptive immune responses. In contrast, in adaptive immunity, cytokines stimulate proliferation and differentiation of antigen-stimulated lymphocytes and activate specialized effector cells (e.g., macrophages).

Cytokines are very potent, even in minute concentrations. Their action is usually limited to affecting cells in the local area of their production, but they can also have systemic effects. As a group, cytokines differ in molecular structure but share the following actions:

• Secrete cytokines in rapid bursts, synthesized in response to cellular activation.

• Bind to specific membrane receptors on target cells.

• Regulate receptor expression in T and B cells, which drives positive amplification or negative feedback.

• Act on different cell types.

• Excite the same functional effects with multiple cytokines (redundancy).

• Act close to the site of synthesis on the same cell or on a nearby cell.

• Influence the synthesis and actions of other cytokines.

Cytokines act on other cells by bonding to cytokine receptors on the surface of cells. Individual cytokines have characteristic functions and differ in how they transduce signals as a result of binding. All cytokine receptors consist of one or more transmembrane proteins whose extracellular portions are responsible for cytokine binding and whose cytoplasmic portions are responsible for initiating the intracellular signaling pathways. These six pathways are as follows:

1. Janus kinase (JAK/STAT) pathway

2. Tumor necrosis factor (TNF) receptor signaling by TRAFs (tumor necrosis receptor–associated factors)

3. TNF receptor signaling by death domains

4. Toll receptor signaling

5. Receptor-associated tyrosine kinases

6. G-protein signaling

Interleukins

Many different individual and superfamilies of ILs have been identified. A characteristic of ILs is that secreted peptides and proteins mediate local interactions between leukocytes but do not bind antigen. ILs include molecules that are made by and act on lymphocytes.

Interleukins have widely overlapping functions. These molecules modulate inflammation and immunity by regulating growth, mobility, and differentiation of lymphoid cells. Each of the ILs has been shown to be a distinct molecule by gene cloning and sequencing. In addition, each IL functions through a separate receptor system.

Interferons

The interferons are a group of cytokines discovered in virally infected cultured cells. This interference with viral replication in the cells by another virus led to the term interferon.

The IFNs are one of the body’s natural defensive responses to foreign components (e.g., microbes, tumors, antigens). IFNs are among the most broadly active physiologic regulators, enhancing the expression of specific genes, inhibiting cell proliferation, and augmenting immune effector cells. IFNs have been demonstrated to act as antiviral agents, immunomodulators, and antineoplastic agents.

Type I IFNs mediate the early innate immune response to viral infections. They consist of two distinct groups of proteins, IFN-α and IFN-β that are structurally different but that bind to the same cell surface receptor and induce similar biologic responses.

IFN-γ is the principal macrophage-activating cytokine and serves a critical function in innate immunity and in specific cell-mediated immunity. It stimulates expression of MHC class I and class II molecules and costimulates antigen-presenting cells (APCs), promotes the differentiation of naive CD4+ T cells to the helper T cell type 1 (Th1) subset and inhibits the proliferation of Th2 cells. In addition, IFN-γ acts on B cells to promote switching to certain IgG subclasses, activates neutrophils, and stimulates the cytolytic activity of natural killer (NK) cells. It is also antagonistic to IL-4. IFN-γ is of most immunologic interest because of its diverse effects on the immune response. Its ability to augment the activity of many cytokines has resulted in clinical trials in a number of different diseases.

Tumor Necrosis Factor

Tumor necrosis factor is the principal mediator of the acute inflammatory response to gram-negative bacteria and other infectious microbes. TNF is responsible for many of the systemic complications of severe infections. The TNF receptor family stimulates gene transcription or induces apoptosis in a variety of cells. The gene-encoding TNF-α is located in the HLA region between the HLA-DR and HLA-B loci.

TNF-α and TNF-β share similar activities. The principal physiologic functions of TNF are as follows: (1) to stimulate the recruitment of neutrophils and monocytes to sites of infection; and (2) to activate these cells to eradicate microbes.

In low concentrations, TNF acts on leukocytes and endothelium to induce acute inflammation. At moderate concentrations, TNF mediates the systemic effects of inflammation. In severe infections, TNF is produced in large amounts and causes clinical and pathologic abnormalities (e.g., septic shock). When TNFs gain access to the circulation during infection, they mediate a series of reactions that induce shock and can result in death. The syndrome known as septic shock is a complication of severe gram-negative bacterial sepsis.

Hematopoietic Stimulators

Stem Cell Factor (c-kit Ligand)

Stem cell factor is a cytokine that interacts with a tyrosine kinase membrane receptor, the protein product of the cellular oncogene c-kit. The cytokine that interacts with this receptor is called c-kit ligand, or stem cell factor, because it acts on immature stem cells.

Stem cell factor is needed to make bone marrow stem cells responsive to other CSFs but it does not cause colony formation itself. Stem cell factor may also play a role in sustaining the viability and proliferative capacity of immature T cells in the thymus and mast cells in mucosal tissues.

Colony-Stimulating Factors

A variety of CSFs, such as granulocyte-CSF (G-CSF) and GM-CSF, are also made by T cells. These pathways provide a link between the lymphoid and hematopoietic systems. For example, G-CSF and GM-CSF regulate the production of granulocytes and monocytes, thus enabling the T cell system to promote the inflammatory response.

The biological activity of CSF is measured by its ability to stimulate hematopoietic progenitor cells to form colonies in semisolid medium. These proteins are necessary for the survival, proliferation, and differentiation of precursor cells of the immune system.

CSFs are potentially important in the treatment of human disease. GM-CSF has been used in a number of clinical trials to increase circulating leukocytes in patients with AIDS, other immunocompromised patients (e.g., those recovering from chemotherapy), and bone marrow transplant recipients.

Transforming Growth Factors

As with the IFNs, transforming growth factors (TGFs) were identified as products of virally transformed cells. These factors were found to induce phenotypic transformation in non-neoplastic cells and subsequently were termed transforming growth factors. TGF-β is a group of five cytokines released by many cell types, including macrophages and platelets. TGF-β is known to be a potent inhibitor of IL-1–induced T cell proliferation.

The principal action of TGF-β in the immune system is to inhibit the proliferation and activation of lymphocytes and other leukocytes. It inhibits the proliferation and differentiation of T cells and the activation of macrophages.

Chemokines

Chemokines are a large family of structurally homologous cytokines that stimulate transendothelial leukocyte movement from the blood to the tissue site of infection and regulate the migration of polymorphonuclear leukocytes (PMNs) and mononuclear leukocytes within tissues (see Chapter 3). The largest family consists of CC chemokines that attract mononuclear cells to sites of chronic inflammation, such as monocyte chemoattractant protein 1 (MCP-1). A second family of chemokines consists of CXC chemokines, of which IL-8 (CXCL8) is the prototype. CXCL8 attracts PMNs to sites of acute inflammation, activates monocytes, and may direct the recruitment of these cells to vascular lesions. The third family, CX3, forms a cell adhesion receptor capable of arresting cells under physiologic flow conditions. TNF-α–converting enzyme can cleave CX3CL1 from the cell membrane.

Other functions of various chemokines include the following:

• Increasing the affinity of leukocyte integrins for their ligands on endothelium (e.g., intercellular adhesion molecule-1 [ICAM-1], ICAM-2, vascular cell adhesion molecule-1 [VCAM-1])

• Regulating the traffic of lymphocytes and other leukocytes through peripheral lymphoid tissues

• Maintaining normal migration of immune cells into lymphoid organs or other specialized cells to particular sites

Assessment of Cytokines

Defects in cytokine production can lead to autoimmunity (Table 5-10). Traditional methods for assessment of cytokines include the following:

Table 5-10

Defects in Cytokine Production that Can Lead to Autoimmunity

Cytokine or Protein	Defect	Disorder
IL-1 receptor antagonist	Underexpression	Arthritis
IL-2 IL-7 IL-10	Overexpression	IBD
IL-2 receptor	Overexpression	IBD
IL-10 receptor	Overexpression	IBD
IL-3	Overexpression	Demyelinating syndrome
TNF-α	OverexpressionUnderexpression	IBD, arthritis, vasculitis SLE
IFN-γ	Overexpression in skin	SLE
TGF-β	Underexpression	Systemic wasting syndrome, IBD
TGF-β receptor in T cells	Underexpression	SLE

Adapted from Davidson A, Diamond B: Autoimmune diseases, N Engl J Med 345:340–350, 2001.

IL, interleukin; IBD, inflammatory bowel disease; TNF, tumor necrosis factor; SLE, systemic lupus erythematosus; IFN, interferon; TGF, transforming growth factor.

• Bioassays

• Enzyme-linked immunosorbent assay (ELISA)

• Intracellular staining

• Ribonuclease protection assay

• Polymerase chain reaction (PCR)

Newer methods of measurement include the following:

• Multiplexed assay using the FlowMetrix; quantifies multiple cytokines simultaneously

• Intracellular staining using flow cytometry

• Cord blood mononuclear cells stimulated by allergens (celELISA).

• Real-time PCR for lymph nodes or spleen

• Enzyme-linked immunosorbent spot (ELISPOT) assays

• Enhanced immunoassays for cytokines

• Biotrak assay—high-sensitivity ELISA

Acute-Phase Proteins

The acute-phase response is an innate body defense. This response is a nonspecific indicator of an inflammatory process.

Overview

A group of glycoproteins associated with the acute-phase response are collectively called acute-phase proteins or acute-phase reactants. The various acute-phase proteins rise at different rates and in varying levels in response to tissue injury (e.g., inflammation, infection, malignant neoplasia, various diseases or disorders, trauma, surgical procedures, drug response). The increased synthesis of these proteins takes place shortly after a trauma and is initiated and sustained by proinflammatory cytokines.

The main biological sign of inflammation is an increase in the ESR. In addition to the ESR, measurement of the plasma concentration of acute-phase reactants is usually a good indicator of local inflammatory activity and tissue damage. More than 20 acute-phase proteins have a definable role in inflammation (Box 5-2). These reactants constitute most of the serum glycoproteins (Table 5-11).

Box 5-2 Major Applications of Acute-Phase Protein Measurements

• Monitoring the progress of diagnosed disease activity

• Assessing response to therapy in inflammatory diseases (e.g., rheumatoid arthritis, juvenile chronic arthritis, ankylosing spondylitis, Reiter’s syndrome, psoriatic arthropathy, vasculitis, rheumatic fever)

• Detection of complications of a known disease (e.g., immune complex deposition, postsurgical infection)

Table 5-11

Examples of Clinically Useful Acute-Phase Proteins

Protein	Normal Concentration (g/L)	Concentration in Acute Inflammation (g/L)	Response Time (hr)
C-reactive protein	0.0008-0.004	0.4	6-10
α₁-Antichymotrypsin	0.3-0.6	3.0	10
α₁-Antitrypsin	2.0-4.0	7.0	24
Orosomucoid	0.5-1.4	3.0	24
Haptoglobin	1.0-3.0	6.0	24
Fibrinogen	2.0-4.5	10.0	24
C3	0.55-1.2	3.0	48-72
C4	0.2-0.5	1.0	48-72
Ceruloplasmin	0.15-0.6	2.0	48-72

Acute-phase reactants include C-reactive protein (CRP), inflammatory mediators (e.g., complement components C3 and C4), fibrinogen, transport proteins such as haptoglobin, inhibitors (e.g., α₁-antitrypsin), and α₁-acid glycoprotein. Profiles of inflammatory changes yield detailed information but rarely provide major evidence for diagnosis or treatment.

Produced by the liver under the control of IL-6, CRP is a parameter of inflammatory activity. Serum concentrations can increase 1000-fold with an acute inflammatory reaction. Persistent increases in CRP can also occur in chronic inflammatory disorders (e.g., autoimmune disease, malignancy).

CRP is prominent among the acute-phase proteins because its changes show great sensitivity. Changes in CRP are independent of those of ESR and parallel the inflammatory process. CRP is a direct and quantitative measure of the acute-phase reaction and, as a result of its fast kinetics, provides adequate information about the actual clinical situation (see later). In contrast, ESR is an indirect measure of the acute-phase reaction. It reacts much slower to changes of inflammatory activity and is influenced by other factors. ESR can be falsely normal in conditions such as polyglobulinemia, cryoglobulinemia, and hemoglobinopathy. ESR may also be spuriously high in the absence of inflammation in patients with anemia or hypergammaglobulinemia.

Synthesis and Catabolism

All the acute-phase proteins are synthesized rapidly in response to tissue injury. The elevation is twofold to fivefold in certain disease states. In addition, strenuous exercise triggers an inflammatory response similar to that in sepsis. Indices of the inflammatory response, especially to exercise, include leukocytosis, release of inflammatory mediators and acute-phase reactants, tissue damage, priming of various white blood cell lines, production of free radicals, activation of complement, coagulation, and fibrinolytic cascades.

Acute-phase proteins have different kinetics and various degrees of increase. Some, the negative acute-phase proteins, actually decrease, possibly resulting from a loss of protein from the vascular space. In addition, acute-phase proteins can be modified by causes other than inflammation (e.g., low fibrinogen level in DIC, very low haptoglobin level in hemolysis, elevated α₁-acid glycoprotein [orosomucoid] in renal insufficiency, elevated transferrin level in iron deficiency). In addition, liver insufficiency or leakage through the kidney or gut lesions can lower these reactants.

The rate of change and peak concentration of separate acute-phase reactants vary with the component and the clinical situation. In acute inflammation, CRP and α₁-antichymotrypsin levels become elevated within the first 12 hours. The levels of complement components, C3 and C4, and ceruloplasmin do not rise for several days.

Acute-phase proteins do not always change in parallel. This mismatch in acute-phase protein levels is most often the result of increased catabolism and elimination from the circulation of certain proteins. Differences may also be caused by discrepancies in rates of synthesis. Most acute-phase proteins have half-lives of 2 to 4 days, but CRP has a half-life of 5 to 7 hours. Thus, the CRP level falls much more rapidly than that of the other acute-phase proteins when the patient recovers.

C-Reactive Protein

Traditionally, CRP has been used clinically for monitoring infection, autoimmune disorders and, more recently, healing after a myocardial infarction (MI). Levels of CRP parallel the course of the inflammatory response and return to lower undetectable levels as the inflammation subsides. CRP demonstrates a large incremental change, with as much as a 100-fold increase in concentration in acute inflammation, and is the fastest responding and most sensitive indicator of acute inflammation. CRP increases faster than ESR in responding to inflammation, whereas the leukocyte count may remain within normal limits despite infection. An elevated CRP level can signal infection many hours before it can be confirmed by culture results; therefore, treatment can be prompt. Because of these characteristics, CRP is the method of choice for screening for inflammatory and malignant organic diseases and monitoring therapy in inflammatory diseases.

Elevations of the CRP level occur in about 70 disease states, including septicemia and meningitis in neonates, infections in immunosuppressed patients, burns complicated by infection, serious postoperative infections, MI, malignant tumors, and rheumatic disease. Measurement of CRP may add to the diagnostic procedure in select cases (e.g., differentiation between bacterial and a viral infection). An extremely elevated CRP level suggests a possible bacterial infection (see later, procedure description). In general, CRP is advocated as an indicator of bacterial infection in at-risk patients in whom the clinical assessment of infection is difficult to make, but a lack of specificity rules out CRP as a definitive diagnostic tool.

Levels of CRP rise after tissue injury or surgery. In uncomplicated cases, the CRP level peaks about 2 days after surgery and gradually returns to normal levels within 7 to 10 days. If the CRP level is persistently elevated or returns to an increased level, it may indicate underlying sepsis preceding clinical signs and symptoms and should alert the clinician to postoperative complications.

In clinical practice, CRP is particularly useful when serial measurements are performed. The course of the CRP level may be useful for monitoring the effect of treatment and for early detection of postoperative complications or intercurrent infections. In rheumatoid arthritis (RA), the CRP level reflects short-term and long-term disease activity. Monitoring of CRP levels allows for early prediction of response to a particular drug, often months before clinical and radiologic confirmation are possible. In disorders such as RA, CRP can be used to assess the effect of antiinflammatory drugs (e.g., aspirin) and the nature of their action. Aspirin-like drugs do not suppress acute-phase proteins in inflammation, allowing optimal therapy in the shortest time and minimizing ongoing inflammation and joint damage. Assessment of CRP is also valuable in monitoring therapy and disease activity in other arthritides. Rheumatic fever and Crohn’s disease can also be monitored by CRP. In addition, CRP level assessment has been found to enhance the value of traditional enzyme measurements in MI.

In a number of chronic inflammatory diseases, however, CRP is an unreliable indicator. CRP values may be normal when other acute-phase proteins are altered in disorders such as SLE, dermatomyositis, and ulcerative colitis. SLE shows little or no CRP response, despite apparently active inflammation.

Both CRP and low-density lipoprotein (LDL) cholesterol levels are known to be elevated in persons at risk for cardiovascular disease. CRP level may be a stronger predictor of cardiovascular events than LDL cholesterol, an established benchmark of cardiovascular risk.

Other Acute-Phase Reactants

α₁-Antitrypsin is an acute-phase protein that increases in acute inflammatory reactions. Generalized vasculitis, such as in immune complex disease, may result in inappropriately low levels of α₁-antitrypsin, probably resulting from increased elimination of complexes with leukocyte lysosomal enzymes.

Defects in the complement components C3a and C5a and the opsonin C3b result in serious infections. In addition, immune complex disease and gram-negative bacteremia result in low levels of complement components, particularly C3 and C4, because the components are consumed during complement activation. Acute inflammation leads to normal or slightly elevated levels. If both disorders are present, complement consumption may be masked, making it deceptive to use complement measurement as the only index of immune complex deposition in disease. The detection of complement breakdown products is more useful than the measurement of total complement component concentrations. It is more desirable to measure C3 breakdown products than total C3 in conditions such as peritonitis or pancreatitis.

Lymphomas may result in a marked increase in C1 esterase inhibitor, with little other change. Ceruloplasmin, often measured as serum copper, is used to monitor Hodgkin’s disease; increases are considered specific indicators of relapse. Although not definitely established, ceruloplasmin monitoring may provide similar information in non-Hodgkin’s lymphoma.

Assessment Methods

Inflammation almost always follows acute tissue damage. Diagnostic categories of acute inflammation can include bacterial causes and nonbacterial causes such as trauma, chronic inflammation, and viral disease. Many laboratory tests have been advocated for the early diagnosis of acute inflammation: total WBC count (including the absolute count and percentage of band and segmented neutrophils, as determined by a 100-cell differential count on a peripheral blood smear), acute-phase proteins, and the ESR.

The ESR (“sed rate”) is a nonspecific indicator of disease, with increased sedimentation of erythrocytes seen in acute and chronic inflammation and malignancies. Although nonspecific, the ESR is one of the most frequently performed laboratory tests.

In addition to these hematologic tests, several tests are of direct value in immunologic testing. These procedures include a simple phagocytic cell function test and the determination of CRP.

CASE STUDY

Signs and Symptoms

A 39-year-old woman was admitted for a cholecystectomy. She had a history of chronic cholecystitis; recent x-ray studies revealed stones in the gallbladder and a large stone in the biliary duct (Fig. 5-3). During surgery, a large stone was removed from the duct, and a cholangiogram showed no further obstructions of the hepatic or common bile ducts.

Figure 5-3 Radiographs of gallbladder (contrast dye).
A, Normal gallbladder *(arrow).* B, Gallbladder filled with stones *(arrow)*.

The patient became febrile 1 day after surgery. A 48-hour postoperative complete blood count (CBC) and CRP were ordered (Fig. 5-4). On the seventh postoperative day, she had abdominal pain and began vomiting. A CBC, ESR, CRP, and blood culture were ordered at that time. Immediately after drawing the blood work, the patient was started on a broad-spectrum antibiotic and discharged on hospital day 15.