Cytokines

The term cytokine, or immunocytokines , was used initially to separate a group of immunomodulatory proteins, called alsoimmunotransmitters , from otherGrowth factors that modulate the proliferation and bioactivities of non-immune cells. However, this terminology suggesting aclear-cut distinction cannot be maintained and may not be meaningful altogether. Some Cytokines are produced by a rather limited number of different cell types while others are produced by almost the entire spectrum of known cell types.

The initial concept of "one producer cell -one cytokine -one target cell" has been falsified for almost every cytokine investigated more closely. A definition of these factors on the basis of their producer or target cells is therefore also problematic.

The same applies to classifications based upon identical or shared biological activities of cytokines especially with broad definitions (see, for example: BCDF (B-cell differentiation factors ), BCGF (B-cell growth factors ), Motogenic cytokines , Chemotactic cytokines (see: Chemokines ), CSF (colony stimulating factors ), angiogenesis factors, or TRF (T-cell replacing factors )) ( for some personal views on aspects of nomenclature see also: Some personal remarks ).

Designations such as HBGF (heparin-binding growth factors ) take into account some biochemical shared by a variety of cytokines but are also problematic.

Today the term cytokine is used as a generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment (for some mechanistic concepts underlying cytokine actions see also: autocrine , paracrine , juxtacrine , retrocrine ). Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death (see: Apoptosis ).

In many respects the biological activities of cytokines resemble those of classical hormones produced in specialized glandular tissues. Some Cytokines also behave like classical hormones in that they act at a systemic level, affecting, for example, biological phenomena such as Inflammation , Septic shock , and Acute phase reaction , Wound healing , and the neuroimmune network .

In general Cytokines act on a wider spectrum of target cells than hormones. Perhaps the major feature distinguishing Cytokines from mediators regarded generally as hormones is the fact that, unlike hormones, Cytokines are not produced by specialized cells which are organized in specialized glands, i. e. there is not a single organ source for these mediators. The fact that cytokines are secreted proteins also means that the sites of their expression does not necessarily predict the sites at which they exert their biological function.

Some Cytokines have been found, upon determination of their primary structures, to be identical with classical enzymes (see, for example: ADF (adult T-cell leukemia-derived factor ), nm23 , PD-ECGF (platelet-derived endothelial cell growth factor ), neuroleukin ). Cytokines normally do not possess enzymatic activities although there is a growing list of exceptions.

The biological activities of cytokines can be measured by a variety of Bioassays employing, among other things, Factor-dependent cell lines , or by other assays using, for example, antibodies (see also: Cytokine assays , WHO cytokine standardization ). RT-PCR quantitation of cytokines employs modern techniques of molecular biology and detects the presence of mRNA encoding specific cytokines.

In the more restricted sense Cytokines comprise Interleukins , initially thought to be produced exclusively by leukocytes, Lymphokines , initially thought to be produced exclusively by lymphocytes, Monokines , initially thought to be produced exclusively by monocytes, interferons (see: IFN ), initially thought to be involved in antiviral responses, colony stimulating factors (see: CSF ), initially thought to support the growth of cells in semisolid media (see also: Colony formation assay ), Chemokines , thought to be involved in Chemotaxis , and a variety of other proteins.

The termType-1 cytokines refers to cytokines produced by Type-1 helper cells whileType-2 cytokines are those produced by Type-2 helper cells. Type-1 cytokines include IL2 , IFN-gamma , IL12 and TNF-beta , while Type-2 cytokines include IL4 , IL5 , IL6 , IL10 , and IL13 (see: TH1/TH2 cytokines ).

It has been suggested that the generic termPeptide regulatory factors ( abbr. PRF ) be used for all these factors to avoid the general difficulties with the nomenclature (see also: Some personal remarks ). This term has the advantage that it includes also a number of low molecular mass peptides which are generally not regarded as Cytokines although they have many activities of cytokines. Some of these low molecular weight proteins and peptides have been referred to asMinicytokines .

Most Cytokines are unrelated in terms of sequence although some can be grouped into families (see: Gene family ; see also: Cytokine receptor families ) or are classified into categories according to the types of secondary and tertiary structure.

IFN-alpha , IFN-beta , IFN-omega , IL2 , IL3 , IL4 , IL5 , IL6 , IL7 , IL9 , G-CSF , M-CSF , GM-CSF , and PDGF , for example, have an alpha-spiral secondary structure. Beta-structural cytokines include IL1-alpha , IL1-beta , TNF-alpha , TNF-beta , and FGF . Composite structures (alpha +beta) are observed, among other things, with IL8 , IFN-gamma , IP-10 , PF4 , GRO , 9E3 . According to the type of tertiary structure, alpha-spiral proteins can be grouped further into IFN like and IL2 like families, and beta-structural proteins can be grouped into IL1 like and TNF like families. For other aspects of biochemistry see also: Recombinant cytokines , Muteins , Peptide mimetics .

Most Cytokines are glycoproteins which are secreted by cells using classical secretory pathways (see also: signal sequence ). Many genes encoding cytokines can give rise to a variety of variant forms of cytokines by means of alternative splicing, yielding molecules with slightly different but biologically significant bioactivities. In many cases the expression patterns of different forms of cytokines or of members of a cytokine family are overlapping only partially, suggesting a specific role for each factor.

Membrane-bound forms have been described also for many Cytokines, and some may be associated also with the extracellular matrix . It is likely that the switching between soluble and membrane forms of cytokines is an important regulatory event (see also: Autocrine , paracrine , juxtacrine , retrocrine ). In some cases membrane forms of a cytokine have been found to be indispensable for normal development, with soluble forms being unable to entirely substitute for them.

Most Cytokines are generally not stored inside cells (exceptions are, for example TGF-beta and PDGF which are stored in platelets). The expression of most Cytokines is strictly regulated, i. e. these factors are usually produced only by activated cells (see also: Cell activation ) in response to an induction signal. Expression is normally transient and can be regulated at all levels of Gene expression (see also: ARE (AU-rich element )). However, constitutive expression has been observed also. The expression of many Cytokines also seems to be regulated differentially, depending on cell type and developmental age.

Most Cytokines were detected initially in functional tests in vitro as biochemically undefined activities or as distinct factors with distinct biological activities. This also explains, at least in part, the plethora of different names for some of the Cytokines. In many instances these activities were named after a particular biological activity observed in an in vitro assay (see also: Bioassays and Cytokine assays for alternatives) or after cells that were found to elaborate these factors (for techniques allowing identification of cytokine genes, cytokine receptor genes, and other relevant genes without prior knowledge of their activities see: Gene library ). One should be aware of the fact that at this moment in time the relevance of many in vitro activities of cytokines to their endogenous functions within an intact organism is not clearly defined.

Almost all Cytokines are pleiotropic effectors showing multiple biological activities. In addition, multiple cytokines often have overlapping activities and a single cell frequently interacts with multiple cytokines with seemingly identical responses (cross-talk). One of the consequences of this functional overlap is the observation that one factor may frequently functionally replace another factor altogether or at least partially compensate for the lack of another factor. Since most Cytokines have ubiquitous biological activities, their physiologic significance as normal regulators of physiology is often difficult to assess.

Studies of gene functions in experimental transgenic animals in which a cytokine gene has been functionally inactivated by gene targeting (see also: Knock-out ) are of particular importance in research on cytokines because, unlike in vitro studies, they provide information about the true in vivo functions of a given cytokine by highlighting the effects of their absence. In many instances these studies have shown that null mutations of particular cytokine genes do not have the effects in vivo expected from their activities in vitro. If information about loss-of-function studies is available for a given cytokine or its receptor it can be found in this information universe of cytokines as a special subentry (Transgenic/Knock-out/Antisense studies) for each particular cytokine.

Many Cytokines show stimulating or inhibitory activities and may synergise or antagonize also the actions of other factors. A single cytokine may elicit reactions also under certain circumstances which are the reverse of those shown under other circumstances. The type, the duration, and also the extent of cellular activities induced by a particular cytokine can be influenced considerably by the micro-environment of a cell, depending, for example, on the growth state of the cells (sparse or confluent), the type of neighboring cells, cytokine concentrations, the combination of other Cytokines present at the same time, and even on the temporal sequence of several Cytokines acting on the same cell.

The fact that every cell type may have different responses to the same growth factor can be explained, at least in part, by different spectrums of genes expressed in these cells and the availability and levels of various transcription factors that drive Gene expression . The responses elicited by Cytokines are therefore contextual and the "informational content", i. e. the intrinsic activities of a given cytokine may vary with conditions. Although a variety of cytokines are known to share at least some biological effects the observations that single cells usually show different patterns of gene expression in response to different cytokines can be taken as evidence for the existence of cytokine receptor-specific signal transduction pathways. Shared and different transcriptional activators that transduce a signal from a cytokine receptor to a transcription regulatory element of DNA are involved in these processes (for some examples see: STAT proteins , Janus kinases , IRS ).

It has been observed, for example, that bFGF is a strong mitogen for fibroblasts at low concentrations and a chemoattractant at high concentrations (see also: Chemotaxis ). bFGF has been shown also to be a biphasic regulator of human hepatoblastoma-derived HepG2 cells, depending upon concentration. The interferon IFN-gamma can stimulate the proliferation of B-cells prestimulated with Anti-IgM, and inhibits the activities of the same cells induced by IL4 . On the other hand, IL4 activates B-cells and promotes their proliferation while inhibiting the effects induced by IL2 in the same cells. The activity of at least two Cytokines (IL1-alpha and IL1-beta ) is regulated by an endogenous receptor antagonist, the IL1 receptor antagonist (see: IL1ra ). Several Cytokines, including TNF , IFN-gamma , IL2 and IL4 , are inhibited by soluble receptors (see also: Receptor shedding , Cytokine inhibitors , retrocrine ). Several Cytokines, including IL10 and TGF-beta , act to inhibit other Cytokines.

The processes responsible for the regulation of cytokines are not well understood. Cells utilize distinct biochemical pathways converging on mediator release and these can be probed, among other things, by employing a variety of substances mimicking or inhibiting the actions of cytokines (see, for example: Bryostatins , Calcium ionophore , Genistein , H8 , Herbimycin A , K-252a , Lavendustin A , Phorbol esters , Okadaic acid , Staurosporine , Suramin , Tyrphostins , Vanadate ).

Frequently one observes a hierarchical order of cytokine actions with some early Cytokines preactivating cells so that they then can respond to late-acting Cytokines (see also: Cell activation ). Many Cytokines induce the synthesis of novel gene products once they have bound to their respective receptors (see: ERG , Early response gene ). Some of the novel products are themselves Cytokines (see: Chemokines , for example). In addition, there are a variety of biological response modifiers that function as Anti-cytokines .

Cytokine mediators can be transported quickly to remote areas of a multicellular organism. They can address multiple target cells and can be degraded quickly. Concentration gradients can be used to elicit specific responses. These possibilities by far exceed the possibilities provided by mere cell-to-cell contacts within a multicellular organism. It can be assumed that Cytokines play a pivotal role in all sorts of cell-to-cell communication processes although many of the mechanisms of their actions have not yet been elucidated in full detail.

A close examination of the physiological and pathological effects of the regulated or deregulated (see: Transgenic animals) expression of cytokines in complex organisms has shown that these mediators are involved in virtually all general systemic reactions of an organism (see also: CytokineTopics ), including such important processes as the regulation of immune responses (see, for example: BCDF , B-cell growth and differentiation factors; BCGF , B-cell growth factors ; TRF , T-cell replacing factors ; Isotype switching ), inflammatory processes (see: Inflammation ), Hematopoiesis (see also: Hematopoietins ), and Wound healing .

Cytokines are important mediators involved in embryogenesis and organ development (see also: Angiogenesis ) and their activities in these processes may differ from those observed postnatally. In addition they play a key role in neuroimmunological, neuroendocrinological, and neuroregulatory processes (see: Neuroimmune network ). Cytokines are important positive or negative regulators of mitosis (see also: Cell cycle ), differentiation, migration (see also: Chemotaxis , Chemokines),cell survival and cell death (see also: Apoptosis ), and transformation (see also: Oncogene ). It has been shown that a number of viral infectious agents exploit the cytokine repertoire of organisms to evade immune responses of the host. Virus-encoded factors appear to affect the activities of cytokines in at least four different ways: by inhibiting the synthesis and release of cytokines from infected cells; by interfering with the interaction between cytokines and their receptors; by inhibiting signal transmission pathways of cytokines; and (d) by synthesizing virus-encoded cytokines that antagonize the effects of host cytokines mediating antiviral processes (see: Viroceptor , Virokine ). Bacteria and other micro-organisms also appear to produce cytokine-like substances which they utilize to subvert host responses (see: Bacteriokine , Microkine ).

Cytokines themselves rarely are related closely among each other in terms of primary sequences. Some appear to have some common three-dimensional features and some of them can be grouped into families. For example, the TNF ligand superfamily members (with the exception of LT-alpha ) are type II membrane glycoproteins with homology to TNF in the extracellular domain (overall homologies, 20percent. The HBNF family includes members of the group of fibroblast growth factors . Another group of diverse factors with conserved sequence features are the Chemokines . The analysis of crystal structures of several cytokines with very little sequence homology has revealed a common overall topology that is not deducible from sequence comparisons (see: Cystine knot growth factor family ).

The biological activities of cytokines are mediated by specific membrane receptors which can be expressed on virtually all cell types known. Their expression is also subject to several regulatory mechanisms (see: Receptor transmodulation ) although some receptors are expressed also constitutively.

Cytokine receptor proteins have been shown to share a number of characteristics. Many receptors are members of cytokine receptor families . Many receptors are multi-subunit structures that bind ligands and at the same time possess functions as signal transducers due to their intrinsic tyrosine kinase activity (see also: Autophosphorylation ; see also: PTK ; protein tyrosine kinase ). Many receptors often share common signal transducing receptor components in the same family (see also: Cytokine receptor families ), which explains, at least in part, the functional redundancy of cytokines. This and the ubiquitous cellular distribution of certain cytokine receptors has hampered attempts to define critical responsive cell populations and the physiologically important cell-specific functions of cytokines in vivo. Many receptors are associated with special signal transducing proteins in the interior of the cell (see, for example Janus kinases , STAT proteins ). Some receptors may bind more than one cytokine. Several cytokine receptors have been shown to be converted into soluble binding proteins that regulate ligand access to the cell by specific proteolytic cleavage of receptor ectodomains.

The many specific activities of individual Cytokines have been the basis for current concepts of therapeutical intervention, in particular of the treatment of hematopoietic malfunctions and tumor therapy. Applications involve the support of chemo- and radiotherapy, bone marrow transplantation, and general immunostimulation (see also: Adoptive immunotherapy , LAK cells , TIL , Cytokine gene transfer , Cytokine fusion toxins ).

Although some recombinant cytokines are now in clinical use, and and attempts are made to develop hybrid molecules from known cytokines (see: Muteins ) which possess the advantages of the respective factors, but not their disadvantages, one must be aware of the fact that current knowledge is still limited. Cytokines are powerful two-edged weapons that can trigger a cascade of reactions, and may show activities that often go beyond the single highly specific property which it is hoped they possess. New factors are being discovered constantly and they extend our knowledge about the Cytokine network .

Nevertheless it can be stated that our new (and growing) understanding of the biological mechanisms governing cytokine actions are an important contribution to medical knowledge. The biochemistry and molecular biology of cytokine actions explain some well-known and sometimes also some of the more obscure clinical aspects of diseases. Knowledge that Cytokines create regulatory hierarchies and provide independent and/or interrelated regulatory mechanisms that can confer distinct and interactive developmental functions lays a solid, albeit rather complicated foundation, for current and future clinical experiences.

Status: date of last revision: 05/01/1999

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