Physiology, Acute Phase Reactants

Cellular Level

Macrophages sense bacterial products through Toll-like receptors (TLR) and regulate the inflammatory response. TLR4 senses the gram-negative bacterial lipopolysaccharide (LPS). Downstream signaling leads to the activation of the transcription factor, nuclear factor kappa beta (NF-kB), and increased production of IL-6. IL-6 activates liver production of other acute-phase proteins. Tissue injury and trauma can also activate the acute-phase reaction (APR). The purpose of acute-phase proteins released during APR is to participate in blood coagulation, defense against infection, transport metabolites, nutrients and hormones, and maintenance of homeostasis. IL-6, IL-1beta, TNF-alpha, glucocorticoids, and growth factors are the primary mediators of acute-phase proteins (APP) gene expression.[2][3]

APPs are divided into positive and negative APPs. The concentrations of positive APPs increase during inflammation, and the concentration of negative APPs decrease. Positive APPs include C-reactive protein, haptoglobin, angiotensinogen, alpha1-acid glycoprotein, serum amyloid A, lipopolysaccharide-binding protein (LBP), ferritin, alpha1-antitrypsin, hepcidin, fibrinogen, serum amyloid A, vitronectin, procalcitonin, among others. Negative APPs include albumin, transthyretin (prealbumin), antithrombin, and transferrin.

Erythrocyte sedimentation rate (ESR): ESR measures the distance that red blood cells in the anticoagulated blood fall in a vertical tube after one hour. Fibrinogen reduces the charge on the red blood cell surface, which causes them to aggregate rapidly. As a result, ESR increases. ESR is used to indirectly measure the amount of fibrinogen by determining the rate at which erythrocytes settle inside a vertical tube in one hour. The ESR level starts to rise within 24 to 48 hours of inflammation. An increase in ESR can be indicative of inflammation.[4]

C-reactive protein: CRP is a marker of bacterial inflammation and has a higher sensitivity than ESR, and is a direct measure of the inflammatory response. It was first discovered by Tillet and Francis in 1930 when they showed it reacted to the C-polysaccharide of Streptococcus pneumoniae in patients with pneumococcal pneumonia. It belongs to a highly conserved family of proteins referred to as pentraxins, which are typified by five protomers around a central pore, and its half-life does not change between health and disease, making the production rate the sole determinant of plasma concentrations.[2][3] The normal range for CRP is between 2 to 10 mg/L. The CRP levels start to rise after 4 to 6 hours and peaks by 36 to 50 hours. Levels of CRP can increase 100- to 1000-fold during acute inflammation. The main functions of CRP are to help promote phagocytosis and the innate immune response against foreign infectious pathogens.[4]

Procalcitonin: PCT is a 14.5 kDa peptide. IL-6, IL-1, and TNF-alpha stimulate its secretion. However, it appears that secretion is not activated by IFN-gamma (produced mainly in response to viral infections), making it a sensitive marker of bacterial infections.[5] 

Function

Procalcitonin: Under normal conditions, procalcitonin is produced by the parafollicular (C cells) of the thyroid gland. It is then converted to calcitonin and released from C cells. However, during inflammation, LPS, microbial toxins, or inflammatory mediators can activate the procalcitonin gene in other tissues, including the liver, kidney, adipocytes, pancreas, colon, and brain. Unlike the procalcitonin produced by the thyroid gland, procalcitonin produced in response to inflammation is directly released into blood circulation. Procalcitonin is a sensitive marker for following the progression of infections, especially for pneumonia and sepsis. Levels of procalcitonin can be used to guide antibiotic therapy.

C-reactive protein: CRP binds to several pathogens acting as an opsonin. It can also bind to degenerating cells and cell remnants. CRP also activates complement by the classical C1q pathway. CRP is used as a clinical measurement of ongoing inflammation.[3]

Serum amyloid A: The role of SAA is to function as an inhibitor of many biological processes, including fever, platelet activation, mobilization of monocytes, and chemotactic effect on various immune cells. In tissues, SAA attracts and modulates inflammatory cells and inhibits respiratory burst. As an APP, SAA influences HDL cholesterol transport. SAA can bind to the LPS comparable to LPS binding protein (LBP). The prolonged elevation of SAA can lead to secondary amyloidosis.[6]

Hepcidin: Hepcidin inhibits iron absorption in the intestinal mucosal cells by binding to the ferroportin and inhibits iron transport by binding to ferroportin in macrophages. Increased hepcidin during inflammation causes anemia of chronic disease. [2][3]

Haptoglobin: Intravascular hemolysis releases free hemoglobin in the circulation. Free hemoglobin is an oxidizing agent and can cause tissue damage. Bacteria can utilize heme for the iron requirement. Haptoglobin is a scavenger protein that has antioxidant, antimicrobial, and anti-inflammatory properties. It is an antioxidant because it removes free hemoglobin from the blood, and it is antimicrobial because it reduces iron availability to the pathogens. Its anti-inflammatory properties are due to its binding to CD11b/CD18 integrins on neutrophils.[7] Because haptoglobin binds to hemoglobin, levels of haptoglobin decrease during intravascular hemolysis. As a positive APP, its levels increase during inflammation. Therefore, haptoglobin levels can appear within normal limits in patients with intravascular hemolysis and inflammation.

Ferritin: The role of ferritin is to sequesters iron to inhibit microbial iron scavenging. During malignancy and infection, ferritin concentrations are elevated to reduce free iron available to tumor cells or pathogens, respectively. It is upregulated by proinflammatory cytokines. Some organisms like pseudomonas cause ferritin levels to drop because they have virulence factor siderophores (pyoverdine and pyocyanin) that chelate and import iron.

Fibrinogen: The role of fibrinogen as a coagulation factor is to promote endothelial repair. Fibrinogen also has a C3 complement function. Fibrinogen correlates with ESR.

Alpha-1 antitrypsin (AAT): AAT is a serine protease inhibitor (serpin) that breaks down neutrophil elastase. It protects the cells against neutrophil elastase activity. AAT deficiency can cause hepatitis, liver cirrhosis, and panacinar emphysema.

Others: Ceruloplasmin (Cp) contains copper, and it has histaminase-and ferroxidase-activity. Cp also scavenges Fe2+ and free radicals. Alpha2-macroglobulin (a2MG) binds to the proteolytic enzymes. Alpha1-glycoprotein (a1AGP) influences T-cell function and binds to the steroids such as progesterone. Alpha1-antichymotrypsins inhibit leukocytes and lysosomal proteolytic enzymes.

Transferrin: Transferrin is a negative APP. Macrophages internalize transferrin to sequester iron and inhibit microbial iron scavenging.

Albumin: Albumin is a negative APP, and its production is decreased to conserve amino acids for positive APPs.[1]

Related Testing

The best accepted clinical measures of acute inflammation are CRP and ESR. CRP has the advantage of being more sensitive and easily measured on automated platforms by nephelometry and turbidimetry in the majority of clinical laboratories and is a direct readout of the APR. ESR is an indirect measure of APR proteins, mainly fibrinogen. Both can provide results within hours. However, the quantification of some APPs such as alpha-1antitrypsin (AAT), alpha-1-acid glycoprotein, alpha-2 macroglobulin is not as well validated and standardized as CRP. Fibrinogen rises much later than CRP, and its concentration only increased around two folds.[2] 

Assessing intravascular hemolysis with haptoglobin can be inaccurate without tests such as bilirubin, lactate dehydrogenase, and hemoglobin. Haptoglobin levels increase during inflammation and decrease during hemolysis. In patients with hemolysis and inflammation, levels can appear normal.

Procalcitonin is a sensitive marker for sepsis and can be used to guide treatment. Procalcitonin has replaced CRP as a diagnostic parameter in sepsis because PCT has higher sensitivity than CRP in sepsis. CRP is no longer used as a diagnostic parameter in sepsis, but it can be useful in the follow-up.

Ferritin levels are increased in inflammatory conditions, and levels of transferrin, a negative APP that transports iron, decrease. Ferritin sequesters iron. Prolonged inflammation or malignancy can lead to anemia of chronic disease. Ferritin, transferrin, and serum iron are part of the iron panel laboratory studies. Iron deficiency anemia and anemia of chronic disease can both present as microcytic anemia. They can be distinguished by assessing ferritin and transferrin levels. Anemia of chronic disease patients will have elevated ferritin and low transferrin. Iron deficiency anemia patients will have lower ferritin and elevated transferrin. Serum iron will be low in both patients for different reasons. In iron deficiency, low serum iron could be due to malnutrition, increased demand, or hemorrhage. In anemia of chronic disease, low serum iron is from impaired distribution of iron mainly due to hepcidin-mediated reduced absorption from enterocytes and macrophages.[8]

Pathophysiology

There are some disease states that are causally related to APPs, and some are associated with them. The central role of fibrin during hemostasis and thrombosis is well known. However, fibrinogen also increases the risk of bleeding and thrombosis in many pathologies, including inflammation, infection, neurologic disease, and cancer. CRP can activate complement. In cardiac infarction, CRP has a key role in some forms of tissue alteration. Elevated levels of CRP are associated with the risk of atherosclerosis. [9]

The prolonged elevation of serum amyloid A (SAA) can eventually lead to secondary amyloidosis. Amyloidosis is caused by amyloid fibrils (misfolded SAA) depositing extracellularly in various organs, including the heart, liver, tongue, spleen, hematologic, and spleen. Patients can develop symptoms of restrictive cardiomyopathy or arrhythmia, macroglossia, hepatomegaly, splenomegaly, cough, and dyspnea. There is a casual relationship between SAA and amyloid fibrils. However, the cause of misfolded SAA is not fully understood. Sustained high SAA levels, amyloid enhancing factor, apolipoprotein-E4, impaired SAA-degrading proteases, and many other factors have been implicated. Some of the diseased states with a prolonged elevation of SAA include chronic infection, rheumatoid arthritis, familial Mediterranean fever (FMF), inflammatory bowel disease, and malignancy.[10]

Alpha-1 antitrypsin (AAT) is released from the liver and acts as a serine protease inhibitor (serpin) that protects the cells from neutrophil elastase activity. AAT deficiency is caused by a mutation in the SERPINA1 gene. The mutation is more common in European descendants. The production of AAT in individuals with the mutation is dependent on the allele type. There are three alleles for the AAT gene: M, S, and Z with autosomal codominant inheritance. The normal allele for the SERPINA1 gene is M, and AAT production in homozygous (PiMM) individuals is normal. The S mutation causes a moderate decrease in AAT production, and the Z mutation causes a significant decrease. Therefore, the severity of the disease is dependent on the genotypic expression. Individuals with two normal alleles, PiMM (protease inhibitor MM), have 100% expression of normal protein and have normal levels of AAT. Individuals with PiMS have 80% of normal serum levels of AAT. Individuals with PiSS, PiMZ, and PiSZ have 40-60% serum levels of AAT. Severe AAT deficiency is in individuals homozygous of the Z allele (PiZZ). They produce 10% of the normal serum AAT.

AAT gene mutation induces AAT protein conformation change in the structure. It affects the liver and the lungs. In the liver, AAT accumulates because of impaired secretion. As a result, there is hepatocyte destruction leading to hepatitis and liver cirrhosis. In the lungs, the absence of AAT causes uninhibited neutrophil elastase activity. It leads to the destruction of pulmonary architecture and panacinar emphysema.[1]

Clinical Significance

APPs are non-specific markers of inflammation, and the tests used should be interpreted in conjunction with history, physical examination, and other laboratory tests and imaging. Their levels will be elevated during both acute and chronic inflammation. However, the highest levels are attained in acute inflammation during an acute infection or after trauma resulting in CRP of 50 to 100 mg/L and ESR exceeding 50 mm/hour.

The best recent evidence relates to procalcitonin (PCT). PCT levels can be used to guide treatment in patients with pneumonia. PCT levels greater than 0.25 mcg/L correlate with bacterial infections of the lower respiratory tract. After 2 or 3 days of treatment, lower PCT levels can facilitate the decision to discontinue pneumonia antibiotic treatment. PCT levels greater than 0.5 ng/mL can confirm sepsis. PCT should not be used for the diagnosis of pneumonia or for deciding if the antibiotics are necessary to treat pneumonia. It should only be as a guide antibiotic treatment.[11]

The normal ESR value for men is 0 to 15 mm/hour and for women 0 to 20 mm/hour. Factors that increase ESR include infection, inflammation, malignancy, pregnancy, autoimmune diseases (SLE, RA, GCA, polymyalgia rheumatic, thyroiditis), multiple myeloma, Waldenstrom macroglobulinemia, anemia, macrocytosis, and old age. Factors that decrease ESR include hypogammaglobulinemia, hypofibrinogenemia, microcytosis, spherocytosis, sickle cell disease, polycythemia, and extreme leukocytosis (e.g., chronic lymphocytic leukemia).

Some of the APRs, like CRP, are unique because they can be used in cardiovascular risk assessment for patients. It has also been shown in patients with acute coronary syndromes that elevated CRP levels assayed by the high sensitivity (hsCRP) assay are indicative of poor cardiovascular prognosis. Poor prognosis includes increased mortality, post-myocardial infarction, and unstable angina, among others. In patients without ASCVD, a  hsCRP between 3 to 20 mg/L, on two occasions at least six weeks apart, confers an increased risk for ASCVD provided a nidus for inflammation has been excluded.[12]

CRP is a highly sensitive marker for detecting inflammation. It is not specific to any disease or organ and has a half-life of 24 hours. In patients with systemic lupus erythematosus (SLE), CRP is often within normal limits, and ESR is generally elevated. In SLE patients with elevated high-sensitivity CRP (hsCRP), an infection should be ruled out because elevated hsCRP is a predictor for active infection with high specificity in patients with SLE.[13]

The cell-mediated response to endothelial injury causes giant-cell arteritis (GCA). It involves monocytes differentiating into giant cells and macrophages within the vessel walls producing IL-6. As mentioned above, IL-6 is an AP protein that mediates the systemic response to inflammation like fever, weight loss and causes the liver to produce other acute-phase proteins. It is also responsible for elevated ESR in patients with GCA. Tocilizumab is an IL-6 receptor inhibitor used in patients with GCA, and it can reduce relapses and lower glucocorticoid requirements to maintain disease remission.[14]