Cytokines (Chapter 12, a little of 13 and 3, "Immunology": sixth edition)CYTOKINES, PROPERTIES AND FUNCTION (underlined comments are not

in the textbook, all greek characters are spelled-out) Properties of cytokines: Cytokines are low molecular weight (generally under 30 kDa) typically functioning as intercellular (between cells) messengers, mediating their effect via specific receptors on target cells. They may also occur in membrane-bound forms which still bind to the receptor following contact with another cell. Cytokines, despite being antigen nonspecific, regulate the intensity and duration of the inflammatory/immune response by stimulating/inhibiting activation, proliferation and/or differentiation and migration of multiple cell types and by regulating the synthesis and secretion of immunoglobulins and other cytokines. [examples in Table 12-1] Most cytokines are single polypeptide chains, although these may be in aggregated forms in biological fluids, for example, tumor necrosis factor (TNF)-alpha circulates as a homotrimer. Exceptions to this rule are IL-12 and IL-23 which are comprised of two different polypeptide chains (heterodimers) Cytokines act through specific receptors, binding with high affinity and are therefore extremely potent, often having effects at picomolar concentrations. As a result the production of cytokines is tightly regulated. 1. cytokines may act in an autocrine (binds to receptors on same cell as secreted the cytokine), paracrine (binds to receptors on a nearby cell, a variation on paracrine is "juxtacrine" meaning binds a neighbouring cell) or, in some cases, endocrine fashion (binds to receptors on distant target cells) [Figure 12-1] 2. cytokines may have various attributes: a cytokine may have different biological effects on different target cells (pleiotropy). Two or more cytokines may have the same effect on a target cell (redundancy, eg., IL-2 and IL-15). Cytokines may also synergize (an effect greater than the additive effect of each cytokine used alone) with each other or may antagonize each other [Figure 12-2]. Finally, cytokines often stimulate other cytokines, as in a cascade, forming cytokine networks [Figure 12-5] 3. the term cytokine encompasses several more specific terms: lymphokines (secreted by lymphocytes), monokines (secreted by macrophages and monocytes), chemokines (cytokines with chemoattractant properties), interferons, tumor necrosis factors and interleukins (IL), so named because of their role in communication between leukocytes. There are multiple members in each grouping, for example there are presently 25 interleukins described and cloned and over 50 chemokines. 4. cytokines are classified into four groups based on structure: hematopoietin family (examples, IL-2, IL-4); interferon family (example, interferon-beta); tumor necrosis factor family (example, TNFalpha) and chemokine family (examples, IL-8, MCP-1). How do nonspecific cytokines maintain the specificity of the immune response? 1. limited production of the cytokine: often cytokines are only made following some stimulus, for example a macrophage phagocytosing bacteria, a T cell engaging the T cell antigen receptor, or some other cell-cell or pathogen-cell contacts 2. if only one chain of a heterodimer is made the cytokine may not be effective. 3. limited radius of effectiveness, i.e., only those cells in physical contact with or in the immediate vicinity of the cytokine-secreting cell will be exposed to an effective concentration of the cytokine 4. short half lives mean that cytokines can only act for a limited period of time
3115 cytokine lecture notes 1

5. regulation of cytokine receptor expression. For example, only lymphocytes which have interacted with antigen may express particular cytokine receptors 6. antagonism, includes shedding receptors which may occupy a cytokine in solution preventing it from binding another receptor. Examples of shed receptors becoming antagonists are IL-1, IL-2, IL-4, -6, and -7, IFN-alpha, -gamma, TNF-beta and leukemia inhibitory factor (LIF). In addition, there is a form of IL-1 receptor (IL-1RII) that even in the transmembrane form does not transmit a signal so it is also an antagonist or "decoy", preventing IL-1 from binding to the receptor that does transmit a signal (the IL-1RI). Some cytokines have specific antagonists which are not receptors. The IL-1 receptor antagonist (IL-1Ra) binds to the IL-1RI but the receptor fails to transmit a signal. IL-18 has a specific "IL-18 binding protein" that bind the cytokine preventing it from binding its receptor. Chemokines [Table 13-2, pg. 330; Chapter 13 pages 329-331] Chemokines are small polypeptides that selectively control the adhesion, chemotaxis (movement) and activation of leukocytes. Some are constitutively expressed and likely are involved in homeostatic or developmental roles. Others are expressed only following stimulation of the cell. There are four classes of chemokines based on the position of two of four conserved cysteines (C) 1. CXC, also known as alpha chemokines, where "X" could be any amino acid. Two further subclasses are distinguished: with an ELR motif are neutrophil chemoattractants, without the ELR motif are mononuclear chemoattractants 2. CC, mononuclear cell chemoattractants, also called the beta chemokines 3. C, two members, also referred to as the gamma chemokines 4. CX3C, a single member, fractalkine, is a neutrophil chemoattractant and is membrane-bound, also called the delta chemokines Cytokine secretion by Th1 and Th2 cells: [Table 12-4] 1. T helper cells (CD4+ lymphocytes) can be divided into Th1 and Th2 subsets, each with distinct cytokine secretion profiles. Th1 make IL-2 and IFN-gamma (involved in cell-mediated immunity); Th2 make IL-4, IL-5, IL-10 (involved in B cell activation and antibody responses). 2. the Th1 subset is involved in responses to intracellular pathogens including the production of opsonizing antibodies. Th1 cytokines promote the differentiation of CD8+ cells to become cytotoxic. The Th2 subset mediates the responses to extracellular pathogens, including eosinophil activation and promoting production of IgM and IgE and noncomplement-activating IgG isotypes, much of which contributes to allergic reactions. 3. cytokines produced by Th1 and Th2 cells exhibit cross-regulation. IFN-gamma inhibits Th2 proliferation while IL-10 indirectly (by acting on antigen presenting cells) downregulates IFNgamma and IL-2 production by Th1 cells (required for Th1 proliferation). IL-4 directly antagonizes IFN-gamma activity. Both subsets arise from a common precursor cell, Th0, which appears capable of making cytokines from both subsets. The differentiation of this cell is determined by the cytokine environment during antigen activation [Figure 12-12]. IL-4 is essential for development of Th2 while IFN-gamma, IL-12 and IL-18 are important for the development of Th1. IL-12 and IL-18 may be derived from the antigenpresenting cell. The influence of IL-4 is predominant. A cytokine that promotes differentiation of one helper subset may also suppress the development of the alternate subset, an effect known as cross-regulation. Cross-regulation explains the inverse relationship between classical cell-mediated and antibody responses leading to allergy.
3115 cytokine lecture notes 2

Cytokine receptors: Most cytokine receptors are grouped into 5 major families on the basis of conserved structural features, often involving the position of cysteines. Recall there were four classes of cytokines. The receptor classes are: (i) immunoglobulin superfamily receptors (ii) Class I, or hematopoietin receptor family (includes most cytokine receptors involved in immune function) (iii) Class II, or interferon receptor family (iv) TNF receptor family (v) the chemokine receptor family [Figure 12-6] Receptors may be composed of more than one polypeptide subunit; for example, a heterodimer may contain a cytokine-specific polypeptide and a signal-transducing peptide subunit. Subfamilies of hematopoietin class of receptors may even share the signaling polypeptide. The IL-2R, consisting of 3 subunits (alpha, beta and gamma), is among the best characterized cytokine receptor belonging to the hematopoietin receptor family. IL-2R may be present in 3 forms; low affinity monomeric IL-2R (alpha chain only); intermediate affinity dimeric IL-2R (beta and gamma chains); and a high affinity trimeric IL-2R (alpha,beta,gamma chains). The affinity of the receptor for IL-2 increases with increasing numbers of polypeptides but signal transduction requires both the beta and gamma chains [Figure 12-9]. Gamma chain expression is constitutive by T cells. Expression of alpha and beta chains by T cells is enhanced following antigenic stimulation, ensuring these cells become capable of binding IL-2 with high affinity only if activated. NK cells express the dimeric IL-2R constitutively and bind IL-2 with intermediate affinity. The idea of heterodimer receptors is taken to another level in some cases in which multiple cytokine receptor types utilize the same polypeptide unit, often as the intracellular signaling unit. This explains the redundancy and antagonism between some cytokines. Continuing with the IL-2R example, the gamma chain of this receptor is also used in the IL-4, -7, -9, and IL-15 receptors [Figure 12-7]. Each of these receptors has a unique low affinity alpha chain which is responsible for the specificity of the receptor to the particular cytokine; however, the common gamma chain implies that the transmembrane signal is similar for the different receptors. The alpha chain associates with the cytokine then non-covalently with the signal transducing chain. The dimeric receptor also exhibits increased affinity for the cytokine. In fact, the alpha units (for GM-CSF in the textbook) may compete for associating with beta signaling units in the plasma membrane [Figure 12-8]. The extreme in sharing is seen with the signaling chain of IL-6, pg130. This polypeptide acts as the signal transducing chain of the IL-6, IL-11, ciliary neurotrophic factor (CNTF), LIF and Oncostatin M receptors. It is no surprise that these cytokines display overlapping activities. An interesting twist in the utilization of cytokine receptors was the discovery in 1996 that certain chemokine receptors acted as co-receptors (with CD4) for HIV infection. Approximately 90% of HIV strains are thought to infect using CCR5 (macrophage tropic), the virus can mutate to use CXCR4 (T cell tropic) as patients develop AIDS. Neither G protein signaling nor internalization of the receptor is necessary. The ligands for these receptors (RANTES, MIP-1alpha, MIP-1beta) can acts as antagonists to HIV infection.

3115 cytokine lecture notes 3

Soluble Cytokine Receptors Receptors may occur in soluble forms which typically retain high affinity for the cytokine and thus are capable of binding the cytokine in solution. One of two main mechanisms results in solublization: 1. proteolytic cleavage of the extracellular domain, releasing the receptor from the cell membrane. This is often a result of some specific activation event acting on the cell; for example the M-CSF receptor is cleaved from the cell surface by a protease induced by the activation of protein kinase C. Also, the TNF receptors, p55 and p75 are solublized by this mechanism. In fact it appears that solublization of p75 can occur following binding of TNF to p55. 2. splicing out of the transmembrane encoding exon of the primary RNA transcript resulting in a mRNA that encodes a protein that is secreted, and not anchored in the plasma membrane. Examples of cytokine receptors that are solublized by this mechanism includes IL-1, IL-4, IL-7, some of which appear to occur constitutively, and perhaps not due to specific activation signals. Mechanism/roles of soluble receptors include [Figure from Blood 87:847-857, 1996]: 1. receptor down-regulation; the receptor can no longer serve as the signaling molecule to the cell, limiting the response of the cell to the cytokine ligand 2. the soluble receptor may become a binding protein that protects the ligand from degradation or clearance in the extracellular space. The receptor now has no role in signaling but facilitates the delivery of the ligand to additional membrane-bound receptors. 3. the soluble receptor binds to the cytokine preventing it from binding further membrane-bound receptors- becoming a direct antagonist. Examples include the IL-1, IL-4 and TNF receptors. 4. Receptor families consisting of multichain receptors, such as the IL-6R family, binding of the soluble alpha receptor chain to the ligand can confer sensitivity to another cell which may have only the beta chain (gp130). This greatly expands the number and types of cells sensitive to the soluble receptor/ligand complex. Cytokine Receptor signaling The receptor is responsible for transmitting a signal into the cell upon binding the appropriate ligand. Many of the class I and class II cytokine receptors lack intrinsic tyrosine kinase domains. Yet to a great extent this is achieved by phosphorylation of proteins already present in the cytoplasm which results in a rapid pattern of alterations in multiple proteins. The present unifying model to explain signaling (using Class I and II receptors) is as follows [Figure 12-10]:

! the receptor is composed of multiple chains: an alpha chain binds the cytokine while a beta chain is
necessary for signal transduction (but still may play a role in binding the cytokine, already discussed)

! different inactive protein tyrosine kinases are associated with different subunits of the receptor. The
alpha chain is associated with the "Janus kinase" or JAK, even in the absence of the cytokine ligand. However, in the absence of cytokine the JAK lacks protein tyrosine kinase activity

! cytokine binding induces the association of the two separate cytokine receptor subunits and
activation of the associated JAKs by each other

! activated JAKs create docking sites for the signal transducers and activators of transcription or
"STAT" transcription factors by phosphorylation of specific tyrosine residues on the receptors. This
3115 cytokine lecture notes 4

docking is between the "SH2" domain of the STAT and the phophorylated tyrosine on the receptor. In turn, the JAK then phosphorylate the docked STAT

! after phosphorylation the STATs translocate from the receptor as dimers, to the nucleus to initiate
the transcription of specific genes. Which genes are transcribed is determined by specific DNA sequences to which monomeric or dimeric STATs bind in the promotor region of the gene. There are multiple JAKs and STATs acting in different permutations [Table 12-2] Ultimately the specificity of a cytokine effect is due to three factors: 1) the particular JAK/STAT pathway; 2) STAT specific sequences in the promotor regions of genes; 3) only certain target genes can be activated in a particular cell type. In any given cell type only a subset of the potential target genes of a particular STAT may be permitted expression. Th cell cytokine cross -regulation can be explained at the level of intracellular signaling [Figure 12-13]. The expression of the transcription factor T-Bet drives the cell to Th1 differentiation and suppresses Th2 differentiation. Expression of the transcription factor GATA-3 promotes development of Th2 but inhibits development of Th1. Heterodimerization of STATs can be accomplished by the simultaneous activation of different cytokine receptors that result in phosphorylation of different STATS, e.g. IL-6 results in STAT3 and interferongamma results in STAT1 phosphorylation and when both cytokines bind receptors on the same cell, then STAT1/STAT3 heterodimers can result. [Figure in class] In marked contrast to the Type 1 and Type II receptors, the chemokine family of receptors signals through an entirely different mechanism [Figure 13-2, pg. 331]. Chemokine receptors are coupled with heterotrimeric large G proteins. The signal transduction process generates second messengers such as cAMP, IP3, iCa2+ and activated small G proteins. Yet even chemokine receptors are reportedly able to signal through JAK-STAT phosphorylation events which seem contingent on dimerization of chemokine receptors. The dimerization can include homodimers or heterodimers. IL-1 and TNF signal through NF-kappaB activation [Chapter 3, page 69 last paragraph and Figure 3-14 on page 70] IL-1 and TNF signal through another mechanism which also involves serial serine/threonine (not tyrosine) phosphorylation of different proteins but ultimately the activation of NFkappaB. Working backwards up the pathway, nuclear factor kappa B (NFkappaB), a heterodimer, is sequestered in the cytoplasm by another molecule, IkappaB (also a heterodimer). IkappaBalpha becomes phosphorylated by IKK (IkappaB kinase) and upon phosphorylation is released from NFkappaB. The phosphorylated IkappaB becomes ubiquinated which targets it for destruction by the proteosome. Meanwhile the liberated NFkappaB is free to translocate to the nucleus where it direct gene transcription similar to the STATs. The range of genes activated by NFkappaB is wide and thus this transcription factor has proven important in efforts to control the inflammatory response, and is considered by many as the "Master regulator" of inflammation [Figure and Table from Amer.J.Physiology.Cell Physiology 278:C451, 2000].

3115 cytokine lecture notes 5