Postscript

Definitions

Acute-phase response (or reaction) refers to a complex highly evolutionarily conserved innate multi-organ systemic defense reaction triggered by different stimuli (surgical or other trauma, burn injury, tissue ischemia/infarction, infection, stress, and inflammation) with the goal of restricting the area of damage, eliminating/isolating the damaging agent, reestablishing homeostasis and promoting healing.

Acute-phase protein generally defined as a blood protein primarily synthesized by hepatocytes as part of the acute phase response (APR) whose plasma concentration increases (positive acute-phase proteins) or decreases (negative acute-phase proteins) by at least 25 percent during inflammatory disorders. They are regarded as important components of the innate immune response to infection.

About the acute-phase response (APR)

A core part of the innate immune response and is observed across all animal species.
Evolved as an adaptive response to injury or infection but become non-adaptive in autoimmune or autoinflammatory diseases.
Includes changes in a broad array of metabolic, endocrine, and physiologic functions:
- Concentrations of many plasma proteins (acute-phase proteins).
- Neuroendocrine changes:
  - Fever (resetting of the thermostatic set point of the thermoregulatory center in the anterior hypothalamus).
  - Stimulation of corticotropin and cortisol production.
  - Increased adrenal secretion of catecholamines.
  - Stimulation of the production of arginine vasopressin.
- Hematological changes:
  - Leukocytosis
  - Anemia of inflammation
  - Thrombocytosis
- Alterations of vascular permeability
- Nutritional/metabolic changes:
  - Loss of muscle and negative nitrogen balance
  - Increased hepatic lipogenesis
  - Increased lipolysis in adipose tissue
  - Decreased gluconeogenesis
- Behavioral changes:
  - Anorexia
  - Somnolence
  - Lethargy

Acute phase proteins (APP)

Classified according to:
- Direction of changes in the blood:
  - Upregulated (positive) APPs:
    - Complement system:
      - C3
      - C4
      - C9
      - Factor B
      - C1 inhibitor
      - C4b-binding protein
      - Mannose-binding lectin
    - Coagulation and fibrinolytic system:
      - Fibrinogen
      - Plasminogen
      - Tissue plasminogen activator
      - Urokinase
      - Protein S
      - Vitronectin
      - Plasminogen-activator inhibitor 1
    - Antiproteases:
      - a1-Protease inhibitor
      - a1-Antichymotrypsin
      - Pancreatic secretory trypsin inhibitor
    - Iron homeostasis:
      - Ceruloplasmin
      - Haptoglobin
      - Hemopexin
      - Ferritin
      - Hepcidin
    - Others:
      - CRP
      - Serum amyloid A
      - α-2-Macroglobulin
      - α-l-antitrypsin
      - Fibronectin
  - Downregulated (negative) APPs:
    - Albumin
    - Transferrin
    - Pre-albumin (transthyretin)
    - Retinol-binding protein 4
    - Insulin-like growth factor I
- Kinetics of release:
  - First-phase APPs
  - Second-phase APPs

Acute-phase proteins and other systemic responses to inflammation. Gabay C, Kushner I. N Engl J Med. 1999 Feb 11;340(6):448-54.

- Extent of increase in blood levels:
  - Types:
    - Type I – 50%
    - Type II – two- to fivefold
    - Type III – > fivefold
      - c-reactive protein (CRP)
      - serum amyloid A (SAA)

Methods Enzymol. 1988;163:373-83

- - Minor vs. moderate vs. major:
    - Major (increase 10- to 100-fold)
      - often are observed to increase markedly within the first 48 h after the triggering event
      - often have a rapid decline due their very short half-life
      - Examples include:
        
        CRP
        
        Serum amyloid protein
    - Moderate (2- to 10-fold)
      - May increase more slowly and be more prolonged in duration
      - may be observed more often during chronic inflammatory processes
    - Minor (slight increase)
      - May increase more slowly and be more prolonged in duration
      - may be observed more often during chronic inflammatory processes
- Site of production:
  - Liver – majority are produced by liver hepatocytes
  - Extrahepatic:
    - Sites include:
      - Lymphatic tissue
      - Salivary gland
      - Periodontal tissues
      - Keratinocytes
    - While non-hepatocyte cells are capable of producing some APPs, they do not appear to be able to generate a system-wide acute phase response.
The acute-phase response may be associated with:
- Parallel changes in all acute phase proteins.
- Unique patterns of response in different patients with the same condition and between different conditions.
Typically, the magnitude of the acute-phase response is related to the severity of the inflammatory state or the extent of tissue injury, and the rapidity of change generally parallels magnitude of change.
While APPs play a role in the innate immune response, controlling and eliminating pathogens and initiating repair processes, they are used by the clinician to assess the severity and, in some cases, the prognosis of an ongoing inflammatory response or response to therapy.

Causes

Large changes:
- Infection
- Trauma
- Surgery
- Burns
- Tissue infarction
- Inflammatory conditions
- Cancer
Moderate changes:
- Strenuous exercise
- Heatstroke
- Childbirth
Small changes:
- Psychological stress

Mechanisms

Functions of acute phase proteins:
- Acute phase proteins are thought to curtail pathogens and minimize tissue damage by several ways including inhibition of bacterial proteinases, modulation of iron homeostasis, increased activity of the complement system and elevation of pathogen recognition receptors. Examples include:
  - Removal of dead or dying cells:
    - C-reactive protein (CRP)
    - Serum amyloid P (SAP)
  - Detection and elimination of bacteria, fungi and toxins:
    - CRP
    - SAP
    - Pentraxin 3
    - Complement component 3
  - Regulation of tissue regeneration and blood clotting:
    - Fibrinogen
  - Regulation of iron homeostasis:
    - Hepcidin antimicrobial peptide
    - Transferrin
Mechanism of production of acute phase proteins:
- Inflammatory stimuli lead to local release of inflammatory cytokines.
- When inflammation becomes uncoupled from local checks and balances, the cytokine response becomes amplified and leads to increased circulating levels of corresponding cytokines (systemic inflammatory response).
- Circulating cytokines initiate the acute phase response, including:
  - Induction of fever
  - Changes in hormone secretion
  - Increased white blood cell and platelet production
  - Enhancement of expression and secretion of hepatic positive acute phase proteins and decreased expression and secretion of negative acute phase proteins
- Cytokines implicated in the acute phase response include:
  - Interleukin (IL)-6:
    - Widely viewed as the major inducer of acute phase protein production in hepatocytes.
    - However, APP production in IL-6–deficient mice is only partly impaired and varies depending on the stimulus, suggesting that other cytokines such as tumor necrosis factor (TNF)-α and IL-1β play a role.
  - IL-1β – required to stimulate the production of IL-6.
  - TNF-α
  - Interferon (IFN)-gamma
  - Transforming growth factor (TGF)-β
  - IL-8
- Cytokines are produced by a variety of cell types, but the most important sources are macrophages and monocytes at sites of inflammation.
- Combinations of cytokines have been found to have additive, inhibitory, or synergistic effects.
- The activation of acute phase gene expression is mediated by induction of combinations of transcription factors which recognize distinct target sequences in acute-phase-responsive promoter regions.
  - Interleukin (IL)-6:
    - Binds and activates the IL-6 receptor.
    - IL-6R activation leads to phosphorylation of signal transducer and activator of transcription 3 (STAT3).
    - STAT3 undergoes oligomerization of STAT3, nuclear import, binding to STAT response elements in DNA and regulation of transcription.
  - IL-1β and TNF-α both converge to regulate gene transcription by two main routes:
    - IL-1β- or TNF–α-dependent activation of the MAP kinase pathway results in activation of the CCAAT/enhancer binding protein beta (CEBPB) and activator protein 1 (AP-1).
    - These cytokines potently activate the nuclear factor κB (NF-κB) family of transcription factors (TFs). The nuclear import and activation of NF-κB is achieved by cytokine-dependent phosphorylation and subsequent degradation of the inhibitory protein IκB.

Venn diagram portraying cytokine-induced genes and their different response under the various treatments. In parentheses is the total number of genes induced under each condition (1.5-fold change, adjusted p-value ≤0.05, measured with CuffDiff). Prominent APR genes are highlighted. Nat Commun. 2017 Nov 29;8(1):1849.

Diagnosis

Complete blood count may show:
- Anemia (anemia of inflammation)
- Leukocytosis
- Thrombocytosis
Automated chemistry analyzers may show:
- Elevated serum:
  - C-reactive protein (CRP)
  - Ferritin
  - Haptoglobin
  - Fibrinogen
- Reduced serum:
  - Albumin
  - Transferrin
Assays not widely available for:
- - Serum amyloid A (SAA)
  - Plasma cytokines
Erythrocyte sedimentation rate:
- Indirect measurement of plasma concentration of fibrinogen plasma acute phase protein concentrations.
- Can be greatly influenced by the size, shape, and number of erythrocytes, as well as by other plasma constituents
  such as immunoglobulins.
Protein electrophoresis:
- Proteins resolved into:
  - Albumin
  - 4 fractions of globulins each composed of APPs and/or immunoglobulins:
    - α1 fraction:
      - α1-antitrypsin
      - α1-acid glycoprotein
    - α2 fraction:
      - α2-macroglobulin
      - Haptoglobin
    - β fraction:
      - Transferrin
      - SAA
      - Fibrinogen
      - CRP
    - γ fraction – mostly IgG
  - Protein electrophoresis does not enable quantitation of single proteins but rather of groups of proteins that are mediators of acute inflammatory process.
  - Protein electrophoresis electrophoretograms from commercial systems are thought to reveal changes
    in approximately 30 major serum proteins.

Schematic summary of the regulation of hepatic acute-phase protein synthesis by inflammatory mediators. Biol. Chem. 2021; 402(9): 1129–1145.

Notes about selected acute phase proteins

C-reactive protein (CRP):
- Pentameric molecule consisting of 5 identical subunits of 23 kDa each.
- First identified and described in 1930 via its ability to bind to the C-fragment of Streptococcus pneumoniae.
- Synthesized mainly in the liver in response to interleukin-6 (and interleukin-1).
- Functions include:
  - Activation of the classical complement pathway via C1q binding.
  - Binding to human immunoglobulin Fc𝛾 receptors and opsonization of biological particles for macrophages.
- In healthy individuals, serum CRP levels are low (typically <1 mg/dl).
- In acute phase response, CRP plasma concentrations increase within a few hours, and can reach levels up to 1000-fold compared to normal.
- This effect is mediated by:
  - Cytokines:
    - IL-6, enhanced by IL-1β
  - Downstream signals, including:
    - STAT3
    - C/EBPs
    - NF-kB
Hepcidin:
- Master regulatory of iron homeostasis.
- Biologically active hepcidin is a peptide of 25 amino acids.
- Generated primarily in hepatocytes.
- Mature hepcidin is secreted to plasma and circulates bound to α2-macroglobulin.
- Hepcidin exerts its biological action by binding to ferroportin and promoting its phosphorylation, internalization and lysosomal degradation.
- Inflammatory regulation of hepcidin gene expression involves transcriptional control through a signal transducer and activator of transcription (STAT) site on the hepcidin promoter:
  - Ligand binding to the IL-6 receptor in turn leads to activation of Janus kinases that phosphorylate STAT3.
  - IL-6 promotes the phosphorylation of STAT3 (signal transducer and activator of transcription 3), which translocates to the nucleus and activates hepcidin transcription upon binding to a proximal promoter element, effectively promoting hypoferremia through down-regulation of ferroportin iron export activity from duodenal enterocytes and RES macrophages.
Ferritin:
- Ubiquitous and highly conserved iron-binding protein synthesized in liver hepatocytes.
- In vertebrates, the cytosolic form consists of 2 subunits, termed H and L.
- Twenty-four ferritin subunits assemble to form the apoferritin shell.
- Each apoferritin molecule of 450 000 d can sequester up to approximately 4500 iron atoms.
- normal range for blood ferritin is:
  - For men, 24 to 336 micrograms per liter.
  - For women, 11 to 307 micrograms per liter.
- Main function is to serve as the depot site for iron storage (iron sequestration) inside cells.
- Small quantities of ferritin are also present in human serum, serum ferritin:
  - Is iron-poor
  - Resembles ferritin L immunologically
  - May contain a novel “G” (glycosylated) subunit
- Ferritin gene expression and serum ferritin levels are elevated in conditions of iron overload and inflammation.
- Inflammatory cytokines such as TNF-α and interleukin 1 (IL-1), regulate ferritin translation through an “acute phase box” in the 5′ region of its transcript, which includes multiple NF-kappa-B binding sites.
Fibrinogen:
- 340‑kDa soluble glycoprotein synthesized in liver hepatocytes.
- Involved in blood clotting, homeostasis, inflammation and tissue repair.
- Consists of three fibrinogen chains are encoded by three genes:
  - FGA for Aα
  - FGB for Bβ
  - FGG for γ
- The tree chains undergo a degree of co-regulation to generate mRNA at balanced levels and sustain fibrinogen secretion from the liver.
- In healthy adults, fibrinogen plasma levels are ~150‑400 mg/dl.
- Gene expression and serum fibrinogen levels are elevated in conditions of inflammation (levels can increase by up to 20‑fold).
- IL-6 is the major inducer of fibrinogen gene expression:
  - When hepatocytes are stimulated with IL-6, transcription of all three genes increase simultaneously.
  - Following removal of IL-6, they coordinately return to normal level.
- The IL-6 response is mediated by STAT3 binding sites (IL-6RE, CTGGGAA motif).
- Glucocorticoids synergize with IL-6 to induce fibrinogen genes expression.
- One of the key leukocyte-derived APR cytokines, IL-6 (previously known as hepatocyte stimulatory factor, HSF) stimulates a coordinated increase in fibrinogen mRNA and protein expression, with the majority of regulation felt at the transcriptional level

Regulation of fibrinogen gene expression. A stylized fibrinogen promoter region is shown, representing any of the three fibrinogen genes. Shown is the promoter in the acute-phase inflammatory response. IL-6 signaling leads to STAT3 activation (phosphorylation) and interaction with IL-6REs, increasing fibrinogen mRNA production. Thrombosis and Haemostasis 108.3/2012.

Albumin:
- The most abundant protein in plasma:
  - Concentration approximately 35–50 g/L.
  - Represents 60–65% of total proteins.
- Synthesized exclusively by hepatocytes
- Constantly released by the liver at rate of about 14 grams per day in healthy adult subjects.
- Half-life of approximately 19 days.
- Expressed as single-copy gene, which encodes:
  - Single polypeptide chain of 585 amino acids
  - Molecular mass of 66.5 kDa
- Expression is regulated mainly at the transcriptional level; during the acute-phase reaction there is a down regulation of albumin transcription
Transferrin:
- 76-kDa glycoprotein that is mainly produced in the liver.
- The major iron-transport protein in the human body.
- Has a half-life of approximately 8 days in the serum.
- One transferrin molecule can carry two ferric ions (Fe3+).
- Concentration of human serum transferrin is approximately 200–300 mg/dL
- Expression decreases during inflammation.

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