Cytokines and Inflammatory Response in the Fetus and Neonate

Molecular Mechanisms

TNF is a pleiotropic cytokine that is a key regulator of the inflammatory response and has a critical role in orchestrating the local inflammatory response through cell activation and initiation of a cytokine cascade. TNF also activates diverse homeostatic or pathogenic signaling pathways that mediate a wide range of downstream effects that include cell-cell communication, proliferation, differentiation, and cell death. ^, TNF synthesis is triggered by multiple stimuli, including PAMPs, DAMPs, viruses, ischemia/hypoxia, trauma, cytokines, and TNF itself. ^{, ,} Monocytes and tissue macrophages are the primary producer cells for TNF. TNF is also produced by lymphocytes, natural killer (NK) cells, neutrophils, mast cells, endothelial cells, keratinocytes, smooth muscle cells, and astrocytes. TNF is synthesized as a 26-kDa transmembrane protein, which is processed to the mature soluble 17-kDa form following cleavage by a membrane-bound disintegrin metalloproteinase, TNF-converting enzyme (TACE/ADAM 17). ^, Both the soluble (sTNF) and membrane associated form of TNF (mTNF) are active, with mTNF regulating local inflammatory responses.

Tumor Necrosis Factor Receptors

TNF binds to two structurally related receptors. TNFR1, a 55- to 60-kDa receptor, is expressed on most cell types, while TNFR2, a 75- to 80-kDa receptor, is expressed principally on immune and endothelial cells ( Fig. 121.1 ). TNFR1 binds both sTNF and mTNF, while TNFR2 is preferentially activated by mTNF. TNF signaling is tightly regulated through ubiquitination and phosphorylation, which produce distinct signaling complexes that control different functional outcomes, such as inflammation, cell survival, apoptosis, and necroptosis. ^, TNFR1 activation leads to the assembly of complex I or II (see Fig. 121.1 ). Complex I is largely responsible for proinflammatory and anti-apoptotic cellular responses through activation of mitogen-activated protein kinases and transcription factors such as nuclear factor κB (NFκB) and activator protein. ^{, , ,} Complex I regulates inflammation, host defense, and cell survival. ^{, , ,} Other complexes can also be formed, termed complex IIa, IIb , and IIc , which lead to cell death. Formation of complex IIa or IIb leads to apoptosis, while formation of complex IIc results in necroptosis and inflammation. Factors such as cell type and presence of other cytokines and inflammatory mediators may shift TNF-mediated signaling either towards or against cell death. In contrast, TNFR2, which is activated primarily by mTNF, does not contain a death domain and cannot elicit cell apoptosis or necroptosis. TNFR2 is important for cell–cell communication and assembles complex I that induces cell survival, proliferation, and tissue regeneration. ^{, ,}

Tumor Necrosis Factor in the Fetus and Neonate

TNF is produced by human fetal Kupffer cells, as well as placental mononuclear cells, in response to stimulation by the PAMP LPS. ^, Although pregnancy is generally biased away from T helper 1 (T _H 1) responses and rejection of the fetus, TNF can be found in amniotic fluid during the second and third trimesters, with levels rising throughout pregnancy. Further, elevated TNF levels have been related to pregnancy complications including growth restriction, preeclampsia, and preterm birth. Although animal studies show conflicting effects on neonatal immune development, there remains a paucity of human clinical data on the safety of anti-TNF biologic agents for the growing fetus. ^,

Several (but not all) investigators have demonstrated significantly decreased TNF production by umbilical cord blood monocytes. In addition, preterm infants born before 30 weeks’ gestation demonstrated significantly diminished LPS-stimulated TNF secretion when compared with neonates of later gestational ages or adults. When analyzed in culture, monocytes and macrophages derived from newborn infants also secreted diminished amounts of TNF when compared with adult cells. ^, Kwak and colleagues examined TNF production by umbilical cord blood mononuclear cells in response to several stimuli. In contrast with the immediate response to LPS (in which TNF, IL-1β, IL-6, and IL-8 appeared almost simultaneously), stimulation by group B streptococci resulted in increased TNF production but a delayed appearance of the other cytokines. Additional investigations by Levy and colleagues demonstrated that impaired perinatal TNF production was mediated by the purine metabolite, adenosine. TNFR expression may also be diminished. Using flow cytometry and a human recombinant TNF that binds to the 75- and 55-kDa receptors, Chheda and colleagues demonstrated reduced expression of TNFRs on umbilical cord blood monocytes when compared with that of adult cells. Thus abnormalities of TNF production, release, and dissociation from the production of other cytokines may protect the fetus from excessive inflammatory responses. On the other hand, excessive TNF production, such as in the setting of a fetal inflammatory response to chorioamnionitis, may mediate oligodendrocyte injury at a vulnerable period in development.

Molecular Mechanisms

The IL-1 ligand superfamily includes IL-1α (or IL-1F1), IL-1β (or IL-1F2), IL-18, IL-33, IL-36α, IL-36β, IL-36γ (agonists), IL-1 receptor antagonist (IL-1ra; or IL-1F3), IL-36 receptor antagonist, IL-38 (see “Inhibitors of Proinflammatory Cytokines”), and IL-37 (antiinflammatory cytokine). IL-1α and IL-1β are important mediators of local and systemic inflammation. The two molecules share little sequence homology (22% to 26%) but have similar tertiary structures, bind to the same receptors, and share biologic activities.

IL-1α and IL-1β are synthesized as 31-kDa precursor proteins (proIL-1α and proIL-1β). ^, IL-1α is constitutively expressed in healthy individuals in multiple cell types including epithelial and mesenchymal cell types. Most of IL-1α remains in the cell cytosol in the pro-form, where it can function in an autocrine fashion or be transported to the cell surface and participate in cell-to-cell communication. IL-1α is also biologically active when it is cleaved to the 17.5-kDa mature protein by membrane-associated cysteine protease calpains and then released from the cell. IL-1α is rarely observed in the circulation and appears systemically only during severe disease, where it is released following cell necrosis and functions as a DAMP. ^,

By contrast, IL-1β is produced principally by cells of the innate immune system such as monocytes, macrophages, and dendritic cells. IL-1β is active only in its cleaved mature form, and after secretion, it is the principal mediator of the systemic effects of IL-1 and is an important regulator of host defense. Activation and release of the mature biologically active 17-kDa form is tightly regulated and requires cleavage by caspase 1 following inflammasome activation. ^, Non–inflammasome-mediated activation of IL-1β can also occur, mediated by proteases derived from neutrophils or pathogens themselves. IL-1 synthesis is triggered by many of the same stimuli that activate TNF production (i.e., PAMPs and DAMPs).

Interleukin-1 in the Fetus and Neonate

Similar to TNF, IL-1 is produced by fetal Kupffer cells in response to LPS. IL-1 is present in amniotic fluid and increases with premature rupture of membranes. In the context of preterm labor or prolonged rupture of membranes, fetal IL-1 initiates a robust systemic inflammatory response that may progress to multiorgan system damage and death.

Production of IL-1 by umbilical cord blood monocytes is comparable with that of adult controls, even in monocytes obtained from preterm infants. ^{, ,} However, in preterm infants with bacterial sepsis, monocyte IL-1 secretion is lower during acute infection but improves significantly during convalescence. When studying the role of IL-1 signaling, Benjamin found that IL-1α rather than IL-1β determined murine sepsis related mortality. After stimulation with LPS or TNF, Contrino and colleagues demonstrated significantly increased expression of IL-1 by umbilical cord blood neutrophils compared with cells from adults suggesting that neutrophil IL-1 secretion may be important in the amplification of the early inflammatory response. IL-1 signaling in mice also stimulates production of neutrophil chemoattractants and recruitment to sites of group B streptococcal infection. Presicce and colleagues further reported that IL-1 blockade decreases concentrations of proinflammatory mediators, secretion of neutrophil chemoattractants, and accumulation of neutrophils at sites of inflammation. Recent evidence from a murine model suggests that antenatal exposure to IL-1 is associated with preterm birth and increased neonatal mortality. Surviving neonates demonstrated elevated levels of multiple proinflammatory cytokines in placental and fetal tissues and morphologic alterations in brain, lung, and intestine.

Molecular Mechanisms

IL-6 is a pleiotropic cytokine with a wide range of biologic functions and is an important regulator of inflammation, immune responses, oncogenesis, and hematopoiesis. IL-6 is a potent inducer of the acute-phase response, as well as specific cellular and humoral immune responses, including B-cell differentiation and T-cell activation, including T helper 17 (T _H 17) cells. IL-6 is also an important regulator of the transition from acute to chronic inflammation and of the shift from neutrophil to mononuclear cell infiltration. IL-6 is not expressed constitutively, and its production is triggered in response to infection and tissue injury, as well as other cytokines, such as TNF, IL-1β, and interferon (IFN)-γ. IL-6 is produced by a variety of cells, including monocytes, macrophages, endothelial cells, fibroblasts, B and T cells, and some tumor cells. ^,

Interleukin-6 Receptors

The IL-6 receptor system consists of two components: a ligand-binding molecule IL-6R and a nonligand binding signal transducer glycoprotein 130 (gp130) ( Fig. 121.3 ). ^, GP130 is also the common signal-transducing subunit for other members of the cytokine receptor family, including IL-11, oncostatin M, ciliary neurotrophic factor, cardiotrophin 1, leukemia inhibitory factor, IL-27, and IL-31, leading to redundancy in cytokine activity. ^{, ,} Although the membrane IL-6R is expressed on hepatocytes, monocytes, macrophages, neutrophils, and some types of lymphocytes, a naturally occurring soluble form of IL-6R (sIL-6R) is also present in most body fluids. IL-6 binding to sIL-6R triggers the association with the signal-transducing gp130 component in cells that do not express the membrane-bound form of IL-6R. Thus, unlike most other soluble cytokine receptors, sIL-6R can act as an agonist and through trans signaling can activate cell types that do not express IL-6R. A third type of IL-6 signaling, termed IL-6 transpresentation, occurs in the context of antigen specific interactions of dendritic cells with T cells. ^, IL-6 bound to IL-6R on dendritic cells is presented to membrane bound gp130 on T cells leading to the development of pathogenic Th17 cells.

Interleukin-6 in the Fetus and Neonate

After stimulation with LPS, IL-6 is produced by human fetal Kupffer cells as early as 13 weeks after conception. This cytokine is now recognized as an important marker of the fetal inflammatory response syndrome and subsequent neonatal morbidity and mortality. In a cohort of pregnancies between 34 and 37 weeks gestation, Musilova found that intraamniotic infection and microbial invasion was associated with the highest cord blood IL-6 levels compared to controls. Furthermore, elevated levels of IL-6 in amniotic fluid were predictive of brain injury in preterm infants. Hadley and colleagues reported an increase in IL-6, IL-8, TNF, and IL-1β in term labor, providing evidence of their role in promoting labor with antiinflammatory cytokines notably absent. In addition, Mir recently demonstrated that elevations of IL-6 and Il-8 during parturition were most likely of fetal origin and modulated by placental clearance. Recent studies also noted the effectiveness of a point of care IL-6 determination in the identification of intra-amniotic inflammation. ^,

Several studies have evaluated IL-6 production by monocytes from newborn infants. ^{, , ,} Liechty and others demonstrated equivalent IL-6 production by fetal and maternal mononuclear cells after stimulation with IL-1. Angelone and colleagues demonstrated robust LPS-stimulated umbilical cord blood IL-6 production and higher IL-6 to TNF ratios than in adult peripheral blood. However, IL-6 production was reduced in preterm neonates, perhaps contributing to their enhanced susceptibility to overwhelming bacterial infection. ^{, ,} Using flow cytometry and LPS stimulation, Schultz and colleagues demonstrated that both term and preterm monocytes demonstrated enhanced synthesis of IL-6 compared to monocytes from adults, suggesting an enhanced inflammatory response. A recent study by Reuschel and colleagues investigated cytokine profiles of cord blood monocytes stimulated with LPS from several gram negative species. IL-6 and IL-8 responses were greatest following stimulation with Escherichia coli and Enterobacter aerogenes, while stimulation with Pseudomonas aeruginosa showed the weakest response.

Molecular Mechanisms

IL-8 (also termed CXCL8 ) is a member of the chemokine supergene family, which is composed of approximately 50 members. Chemokines are critical regulators of immune cell activation and trafficking. ^, Chemokines also play an important role in homeostasis, proliferation, differentiation, and survival. ^, CXCL8 is the prototype chemokine that orchestrates the recruitment of leukocytes to sites of inflammation. CXCL8 is produced by monocytes, macrophages, fibroblasts, endothelial cells, epithelial cells, hepatocytes, keratinocytes, synovial cells, chondrocytes, and some tumor cells. LPS, TNF, and IL-1, but not IL-6, trigger production of CXCL8. IL-3, granulocyte-macrophage colony-stimulating factor, lectins, immune complexes, and phagocytosis also stimulate CXCL8 production.

The chemokine family is divided into four structural groups characterized by the number and spacing of conserved cysteine residues: CXC, CC, C, and CX ₃ C. The CXC chemokine group contains one amino acid between the first two cysteine residues. The CXC chemokine subgroup is further divided into two groups based on the presence or absence of a Glu-Leu-Arg (ELR) motif located before the first cysteine residue and termed ELR ⁺ CXC and ELR ⁻ CXC chemokines . ELR ⁺ CXC chemokines act primarily on neutrophils, whereas ELR ⁻ CXC chemokines interact with mononuclear leukocytes. The ELR ⁺ CXC family includes several chemokines with biologic activity similar to that of CXCL8 such as CXCL1 (GRO-α), CXCL2 (GRO-β), CXCL3 (GRO-γ), CXCL7 (neutrophil-activating peptide 2), CXCL5 (epithelial cell–derived neutrophil-activating protein 78), and CXCL6 (granulocyte chemotactic protein 2).

CC chemokines—the second structural group—contain adjacent cysteine residues. CC chemokines act principally on monocytes, basophils, eosinophils, and some lymphocyte subpopulations. ^, The best-characterized CC chemokine is CCL2 (monocyte chemotactic protein 1). Other members of the CC chemokine family include CCL8 (monocyte chemotactic protein 2), CCL7 (monocyte chemotactic protein 3), CCL5 (RANTES), CCL3 (macrophage inflammatory protein 1α), and CCL4 (macrophage inflammatory protein 1β).

The C chemokine structural group has a single amino-terminal cysteine and contains two highly related chemokines, XCL1 (lymphotactin) and XCL2 (single cysteine motif 1β). The last subgroup, CX ₃ C, contains a single member, CX3CL1 (fractalkine), which is characterized by separation of the first two cysteine residues by three amino acids. CX3CL1, as well as CXCL16, is present in two forms: a membrane-bound form, which acts as a leukocyte adhesion receptor, and a cleaved form, which is secreted and acts as a soluble chemoattractant. ^,

Chemokine Receptors

Chemokine receptors are a family of G protein–coupled receptors composed of at least 10 CC chemokine receptors, six CXC chemokine receptors, one CX ₃ C chemokine receptor, and one C chemokine receptor. ^, An unusual feature of this receptor family is the ability to bind more than one chemokine within a particular subclass. An exception is Duffy antigen receptor for chemokines, located on erythrocytes and endothelial cells, which will bind both CC and CXC chemokines. Engagement of this receptor does not appear to activate signal transduction, and Duffy antigen receptor for chemokines may limit leukocyte activation by serving as a chemokine sink.

Two distinct CXCL8 receptors, CXCR1 and CXCR2, have been characterized. ^, CXCR1 binds CXCL8 with high affinity and CXCL1 and CXCL7 with low affinity, whereas CXCR2 binds most ELR ⁺ CXC chemokines. Binding of CXCL8 to neutrophil CXCR1 and CXCR2 triggers degranulation and cell migration, but only activation of CXCR1 induces reactive oxygen species (ROS). CXCL8 binding also triggers receptor phosphorylation and desensitization, a process thought to be important for continued cellular ability to detect chemotactic gradients.