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Departments of
* Cytokine Biology and
Immunology, ZymoGenetics, Inc., Seattle, Washington
Correspondence: Donald C. Foster, Department of Cytokine and Receptor Biology, ZymoGenetics, Inc., 1201 Eastlake Ave. E., Seattle, WA 98102. E-mail: fosterd{at}zgi.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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chain of the IL-2/15 receptor, which forms a heterodimeric receptor complex with IL-21R. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of natural killer cells. The elucidation of the unique biological effects of IL-21 represents an intense area of interest in current cytokine biology.
Key Words: cytolytic T cells • functional cloning • innate immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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-helical secondary and tertiary structure, in many cases they share only very limited homology in their primary sequences. In contrast, their receptors have primary structural motifs that are highly conserved [3
, 4
]. The class I cytokine receptor family includes the receptors for interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, EPO, G-CSF, GM-CSF, leukemia-inhibitory factor, oncostatin M, cardiotrophin-I, ciliary neurotrophic factor, TPO, and leptin. Members of this family of receptors are readily identified by a conserved, cytokine-binding domain of about 200 amino acid residues in the extracellular portion of the receptor. This cytokine-binding domain is comprised of two fibronectin type-III domains of about 100 amino acids each, and the N-terminal domain contains conserved cysteine residues and the highly conserved Trp-Ser-X-Trp-Ser (WSXWS) sequence motif present in the C-terminal domain. This cytokine-binding domain is duplicated in some of the receptors, and other receptors contain additional structural domains in their extracellular regions, such as classical fibronectin type-III repeats or immunoglobulin (Ig)-like domains.
As members of the class I cytokine receptor family share highly conserved, structural regions, it is possible to clone new members of this family on the basis of primary amino acid sequence homology. The receptors can then be used to identify their cognate ligands through functional cloning or binding assays. For example, the c-mpl receptor led to identification of TPO using functional cloning [5 ], and a soluble receptor-binding-based assay was used in the identification of angiopoietin [6 ].
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THE IL-21 RECEPTOR (IL-21R) |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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(the ligand-binding component of the IL-4R; ref. [14
]), EPO-R, and growth hormone receptor (GH-R); like these three receptors, it has only a single copy of the cytokine-binding domain, followed by a transmembrane domain and a relatively long cytoplasmic domain.
IL-21R gene structure and chromosomal localization
The IL-21R gene is located on human chromosome 16p11, near IL-4RA. Comparison of our IL-21R cDNA sequence with the genomic sequence (AC002303) reveals that the gene is organized into nine exons, spans about 20 kb of genomic DNA, and lies 65 kb from the IL-4RA gene [7
]. Ozaki et al. [8
] identified two alternative IL-21R 5' exons, which they term exons 1a and 1b. Both contain 5'-untranslated region (UTR) sequence and are about 27 kb upstream of exon 2. Thus, the spacing between IL-4RA and IL-21R is reduced to about 39 kb and is unlikely to contain genes for additional receptors. Reporter assays in COS-7 cells demonstrate that the human IL-21R promoter region from -60 to +127 (relative to the start of exon 1a) is essential for expression, and the larger promoter region -789 to +195 gives the highest levels of transcription [15
].
A translocation t(3;16)(q27;p11) found in two cases of diffuse, large B cell lymphoma was recently characterized in detail [15 ]. These translocations result in fusion of the promoter, exons 1a/1b, and some intronic sequence of IL-21R to the BCL6 gene at 3q27. At least in these lymphomas, exon 1b is preferentially spliced out of the messages derived from the normal and the fusion genes. When expressed in COS-7 cells, this fusion gene leads to increased abundance of Bcl-6 protein relative to that seen with the endogenous BCL6 promoter, and a cell line derived from one of the lymphoma cases does express Bcl-6. The authors conclude that this enhanced accumulation of Bcl-6 protein likely contributes to the pathogenesis of these cases of DLBCL [15 ].
Sequencing of 12 Mb of the chromosomal region containing the IL-21R gene revealed the presence of multiple copies of large, duplicated segments originating in other regions of the genome [16 ]. These duplicated segments, or duplicons, are interspersed throughout the pericentromeric regions of several chromosomes and can predispose their target regions to additional duplications or deletions [17 ]. The 16p11 region appears to be one of the most prone to accumulate these duplicated segments, as more than half of the identified segments are represented at least once [17 ]. It is unclear whether these repeats mediate chromosomal abnormalities that disrupt the IL-21R locus.
Murine Il-21r
Murine Il-21r cDNAs were identified from mouse splenocyte [7
] and thymus [8
] libraries. The deduced amino acid sequences from both sources are identical to each other and are 62% identical to the human sequence. Subsequent work in our laboratory comparing Il-21r sequences derived from C57Bl/6 (the source of our splenocyte library), C3H/HeJ, Balb/c, and DBA/2J identified two polymorphisms in the Balb/c mice, and the sequences from C3H/HeJ and DBA/2J were identical to the C57Bl/6 sequence. Both are conservative substitutions (R69K and V200M); their functional relevance is not known.
Tissue distribution of IL-21R
The tissue distribution of a cytokine receptor offers a strong indication of the potential sites of action of the corresponding ligand. We performed northern analysis using fragments from the coding region of human IL-21R, revealing transcripts of about 3 kb and 5 kb in human spleen, thymus, lymph node, and peripheral blood leukocytes [7
]. Further northern analysis using blots containing lymphoid subsets [18
] and human cancer cell lines (Clontech, Palo Alto, CA) revealed strong expression in a mixed lymphocyte reaction and in the Burkitt lymphoma Raji. The two monocytic cell lines, THP-1 [19
] and U937 [20
], were negative (our unpublished observations). Flow cytometric analysis using biotinylated IL-21 showed receptor expression on resting B cells as well as on cell lines IM-9 (B lymphoma), natural killer (NK)-92 (NK-like), and Jurkat (T cell) [7
]. Similarly, Ozaki et al. [8
] performed northern analysis showing IL-21R expression in phytohemagglutinin-activated, human peripheral blood mononuclear cells (PBMC), as well as in cell lines YT (NK-like) and MT-2 (T cell). Western analysis confirmed that IL-21R protein was expressed in each of these sources, as well as in cell lines HUT-102B2 (T cell) and Raji. Negative cell lines were CEM, Molt4, and K562 [8
]. Ueda et al. [15
] showed robust IL-21R expression in 8 of 10 lymphoma cell lines. Taken together, these results indicate expression of IL-21R in resting peripheral B cells, activated PBMC, and in some but not all cell lines of T, B, and NK origin. By reverse transcriptase-polymerase chain reaction (RT-PCR), IL-21R message also can be detected in human bone marrow (BM), in fresh peripheral NK cells (ref. [8
]; our unpublished observations), as well as in CD3+ T cells and CD19+ B cells (see below).
In situ hybridization to human lymph node sections revealed a high density of cells expressing IL-21R in the germinal center (GC). This region of the lymph node contains large numbers of activated, proliferating B lymphocytes as well as smaller numbers of activated T lymphocytes [21 ]. Reciprocal interactions between B and T cells through CD40 and CD40 ligand have been shown to be crucial to GC formation and memory B cell production [22 , 23 ]. As IL-21 costimulates proliferation in B cells in synergy with anti-CD40 stimulation (see below), high expression of IL-21R in the GC suggests that this receptor/ligand pair may play a role in the antigen-dependent immune response and maintenance of immunity.
To assess the possible regulation of IL-21R expression during lymphoid cell activation, we isolated CD3+ T cells (95% pure) and CD19+ B cells (96% pure) from peripheral blood and tonsil, respectively. The cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and RNA was prepared at 0 h (resting) and at 4 h and 24 h of activation. Quantitative RT-PCR was carried out using IL-21R-specific primers. The results show that IL-21R is present in tonsillar B cells and is up-regulated during activation, and it is expressed in resting T cells at low levels and is up-regulated on activation (our unpublished observations).
In the process of identifying a source of murine Il-21r, we performed RT-PCR on a number of RNA samples. Message was detected in mouse spleen, dendritic cells, neonatal skin, BM, and lung, as well as in the cell lines BaF3 (pre-B) and EL4 (thymoma; unpublished observations). Northern analyses showed strong expression in thymus and spleen, as well as low-level expression in lung and small intestine (ref. [8 ]; our unpublished observations). Our in situ hybridizations to human lung and small intestine showed clear receptor expression in duodenal lamina propria lymphocytes and in lymphocytic infiltrates within fibrotic lung sections; Ozaki et al. [8 ] also suggested that their observed lung and small intestine expression could be a result of lymphocytes present in these tissues.
Signaling capability
To determine whether the cytoplasmic domain of IL-21R is able to transduce a signal in a homodimeric configuration, chimeric receptors of two types were constructed. We fused the extracellular and transmembrane domains of c-Mpl, the TPOR, with the intracellular domain of IL-21R [7
]. Ozaki et al. [8
] designed a similar chimeric receptor using the extracellular domain of the EPOR. c-Mpl and EPOR signal as homodimers on stimulation by their respective ligands. Both chimeric receptors were functional in the factor-dependent cell line BaF3 [24
], although the proliferative signal was weak in both cases; the EPOR/IL-21R chimera was also tested in 32D and cytolytic T lymphocyte CTLL-2 cells, with negative results. The structural similarity of IL-21R to IL-4R and its sequence similarity to IL-2Rß and IL-9R are very intriguing; each of these receptors forms a functional complex that includes the common chain of IL-2R (c) [25
]. It has been demonstrated [26
] that IL-4R is capable of signaling as a homodimer, although the true functional receptor complex for IL-4 is a heterodimer of IL-4R with c. The data obtained with IL-21R chimeric receptors suggested that although IL-21R can transduce a signal as a homodimer, its natural complex likely includes other subunits.
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FUNCTIONAL CLONING OF IL-21 |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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Structural homologies
The sequence of the human IL-21 cDNA contains an open reading frame that encodes a polypeptide precursor of 162 amino acids. The mature polypeptide has a predicted molecular weight of 15 Kd and consists of a 131 amino acid four-helix-bundle cytokine domain with highest sequence and structural homology to IL-15 and IL-2 [7
].
Using the Stanford G3 Radiation Hybrid Panel (Research Genetics, Inc., Huntsville, AL), the human IL-21 gene was mapped to 4q26-q27, approximately 180 kb from the IL-2 gene. The IL-15 gene lies distal to IL-2 and IL-21. The presence of these three highly related genes in the same chromosomal region suggests that they may have arisen by gene duplication. To more fully understand the phylogenetic relationship of IL-21 to IL-2 and IL-15, we compared the structures of the three genes. Although the IL-2 gene has only four exons [27 , 28 ], its overall structure is quite similar to that of IL-21 (Fig. 1A ). The first three introns in both genes are in identical positions relative to the protein structure and have identical phasing. In addition, both genes have a short 5' UTR start codon within the first exon, a very short first intron, and much longer second and third introns. Although the IL-21 gene is larger overall, the only true structural difference between the two genes is the presence of a very small fourth intron in IL-21.
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When the mature amino acid sequences of the three cytokines are aligned, the positions of IL-15 introns V, VI, and VII very closely parallel the positions of introns I, II, and III in IL-2 and IL-21 (Fig. 1B) . The positions of the helices relative to the introns appear to be well conserved except for intron IV in IL-21, which interrupts helix D (Fig. 1B) . Thus, although the overall structure of the IL-15 gene is quite different from those of IL-2 and IL-21, all three genes show conservation of intron placement within their structurally significant coding regions.
Murine Il-21
A search of available nucleic acid databases using the human IL-21 sequence revealed only a single, partial, unspliced expressed sequence tag (EST; EST1483966) representing murine Il-21. A cDNA clone was obtained by screening a library constructed from mouse CD90+ activated splenocytes using a probe designed from the partial EST sequence. The predicted amino acid sequence shows 57% identity to the predicted human protein. It is interesting that as with human IL-21, no properly spliced mouse Il-21 ESTs were found in any database. This likely reflects the exquisite transcriptional control of this message (see below) and the relative under-representation of libraries prepared from activated immune cells in EST databases.
The 3'-UTR of murine Il-21 contains nine iterations of an "AUUUA" motif, including one "UUAUUUAUU" nonamer. The 3'-UTR of human IL-21 was deduced from genomic sequence; this sequence also contains nine "AUUUA" motifs, one of which is an imperfect nonamer "UUAUUUAUG." These sequences are present in mRNAs of several hematopoietic cytokines and have been shown to be involved with message instability; such mRNAs are stabilized by treatment of cells with calcium ionophores [34 35 36 37 ]. The "UUAUUUAUU" nonamer has been shown to be the key element in mediating c-fos mRNA degradation [38 ]. The presence of multiple instability elements in IL-21 may explain our finding that its message level is essentially zero in all resting cell types and is markedly increased by ionomycin treatment (see below).
Functionally important IL-21 residues
The amino acid sequences of human and murine IL-21 are especially well-conserved in the regions predicted to encode helices A and D, indicating that these regions are likely involved with receptor activation. Human and murine IL-21 have an acidic amino acid in position D33 (human numbering from initiation Met), corresponding to E9 in mature IL-4, which has been shown to be involved in the primary high-affinity interaction between IL-4 and IL-4R [39
]. Human and murine IL-21 also contain a conserved Q145, equivalent to Q141 of IL-2 that has been implicated in the interaction between IL-2 and c [40
]. Mutations Q145D, I148D, and I148Stop within helix D of IL-21 produce proteins with reduced signaling capability but with wild-type receptor-binding characteristics. Combination mutants Q145D/I148D and Q145D/I148Stop can still bind to cells expressing IL-21R/c but completely fail to activate the receptor, further demonstrating the importance of these residues in IL-21 function [41
]
IL-21 tissue distribution
In our initial search for a ligand source, we found that the few positive conditioned media were all collected from activated T cells (monkey and human). The absence of properly spliced message from all EST databases provides further evidence of the tight regulation of this transcript. Our subsequent real-time PCR data provide definitive evidence that IL-21 is expressed exclusively by activated CD4+ T cells [7
]. General activation using PMA and ionomycin enhances message levels, but higher-level expression is seen in cells stimulated with anti-CD3 monoclonal antibody (mAb). IL-21 expression is increased to an even greater extent by treatment with a combination of anti-CD3 and anti-CD28 mAb, indicating that this message is likely up-regulated during T cell activation in vivo.
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THE FUNCTIONAL IL-21R COMPLEX |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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c
The IL-2R has been studied in detail and is composed of an -ß-c heterotrimer. The ß and c subunits are essential for signal transduction and are members of the hematopoietin receptor superfamily [3
], whereas the subunit appears to primarily be involved in high-affinity binding conversion and is structurally distinct from the hematopoietin receptor family. The c subunit has been shown to participate in forming the receptors for IL-4, IL-7, IL-9, and IL-15, in addition to IL-2 (for review, see ref. [42
]), and null mutations in the c gene result in X-linked, severe-combined immunodeficiency (X-SCID) [43
]. BaF3 cells express endogenous c, which could explain how IL-21 functions in our cloning assay. As the cytokines most closely related to IL-21 use c, we designed assays to test whether the IL-21R also associates with c.
The ORIGEN electrochemiluminescence technology (Igen International, Inc., Gaithersburg, MD) provides a method for measuring dimerization of two differentially labeled proteins in the presence of an unlabeled third. A ruthenium metal chelate (Ru) label on one protein emits luminescence when brought near an electric field through dimerization with a biotinylated protein that is held in place via streptavidin-coated magnetic beads. In this assay, ligand-mediated dimerization of Ru-IL-21R with bio-IL-21R or of Ru-c with bio-IL-21R is measured. The results of this assay show that IL-21R and c dimerize in the presence of IL-21 but not IL-2, IL-4, or IL-15; hence, the dimerization is specific to IL-21. A homodimer of IL-21R does not form in the presence of IL-21 or any other cytokine tested (Jim West, ZymoGenetics Inc., Seattle, WA, unpublished observations).
To determine whether the dimerization of IL-21R and c is involved in signal transduction, neutralizing mAb to c were used in proliferation assays with normal, murine, splenic B cells. The addition of the anti-c antibodies partially blocked proliferation induced by IL-21 and anti-CD40, suggesting that c plays a role in IL-21 signal transduction in B cells (ref. [44
]; our unpublished observations).
Using cell lines derived from X-SCID patients lacking functional c, Asao et al. [45
] and Habib et al. [44
] provided definitive demonstrations of the involvement of c for IL-21 function. Both groups demonstrated an absence of signal transduction in response to IL-21 in the parental cell lines and reconstitution of signaling capability in the same cell lines transfected with full-length c. Thus, three different lines of evidence support involvement of c as a component of the IL-21R complex. Asao and coworkers [45
] also expressed several different mutant forms of c to test their functionality. One of these was a single-point mutant derived from a patient with atypical NK-positive X-SCID; this receptor was partially functional as a component of the IL-21R, supporting normal levels of phosphorylation of STAT3 but reduced STAT1 activation [45
]. The heterodimeric IL-21R/c receptor complex transduces signals through Jak1, Jak3, Stat1, Stat3, and Stat5 [44
, 45
].
Species specificity
In in vitro proliferation assays, murine IL-21 is active on T cells and B cells isolated from human or mouse (see below for detailed discussion of activities). Likewise, human IL-21 is active on human B cells and T cells and on mouse B cells. Oddly, human IL-21 appears to be a very poor stimulator of mouse T cells, despite its activity on mouse B cells (Stacey Dillon, ZymoGenetics Inc., Seattle, WA, unpublished observations). The activation threshold may be greater for T cells than for B cells, such that a slight preference for murine versus human IL-21 is more apparent in this cell type. Alternatively, there may be an additional, species-specific receptor complex in mouse T cells.
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IL-21 BIOLOGY |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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c plays a preeminent role in immunology. These proteins regulate lymphocyte development and control a broad spectrum of activities that shape innate and acquired immune responses. The discovery of IL-21 and IL-21R has added a new member to this family, and there is a significant interest in characterizing the novel immune functions that these molecules regulate. Efforts to describe these activities have focused on the cell types known to express IL-21R, notably T cells, B cells, and NK cells.
T cells
IL-21 is a product of activated CD4+ T cells, indicating that one of its primary functions may be associated with T helper immune responses. The methods used to activate IL-21 gene expression have been limited to cross-linking CD3/CD28 and treatment with PMA + ionomycin. It remains to be determined how the various costimulatory pathways that trigger T cell activation affect IL-21 expression. IL-21R is expressed by CD4+ and CD8+ T cells following activation. The very low levels of this receptor on naïve T cells argues that IL-21 may not be involved in the preimmune phase of T cell biology but modifies T cell responses downstream of antigen activation. The autocrine effects of IL-21 on T cell proliferation have been confirmed using cultured mouse and human thymocytes as well as peripheral T cells that were costimulated with anti-CD3 antibodies [7
]. IL-21 also enhances the proliferative effects of IL-2, IL-15, or IL-7 on peripheral T cells, even in the absence of anti-CD3 [7
].
Evidence is accumulating that IL-21 enhances primary T cell responses and effector cell differentiation. Kasaian et al. [46
] have recently reported that IL-21 significantly increased alloantigen stimulation of purified murine T cells, resulting in increased CTL activity. This effect appeared similar to that achieved with IL-2, IL-15, and IL-12. In addition, IL-21 was able to enhance interferon- (IFN-) production by T cells, alone or in combination with IL-2 or IL-15 [46
]. IL-21 has also been shown to increase phosphoantigen-dependent proliferation of human 
T cells [47
]. Collectively, these data indicate that IL-21 can enhance primary immune responses. In addition, they suggest that IL-21 may be important for the development of a T helper cell type 1 (Th1) response and for stimulating cell-mediated effector functions.
In addition to enhancing a primary antigen response, IL-21 may also modulate memory T cell functions. Recently, it has been shown that IL-21 prevents the IL-15-mediated proliferation of murine CD44+CD8+ memory T cells and the subsequent up-regulation of cytokine receptors for IL-2, IL-15, and IFN- [46
]. This result suggests that IL-15-induced proliferation of memory CD8+ cells is T cell receptor-independent and that these T cells display functional characteristics akin to innate immunity (see ref. [46
] for discussion). The apparent inhibition of these cells by IL-21, combined with the abrogation of some NK cell responses (see below), has been interpreted to mean that IL-21 promotes the transition between innate and adaptive immunity.
B cells
The IL-21R is readily detectable on human peripheral B cells [7
] and mouse splenic B cells. Within mouse BM, IL-21R is present on B220+IgM+ cells but not on B220+IgM- cells, thus indicating that IL-21R is expressed relatively late during B cell development. The effects of IL-21 on peripheral B cell proliferation vary markedly depending on the type of costimulus provided to the B cells. For instance, IL-21 inhibits the proliferation of murine B cells stimulated with anti-IgM antibodies or with lipopolysaccharides (our unpublished observations), and it inhibits the proliferation of human B cells treated with anti-IgM and IL-4 [7
]. Thus, IL-21 may down-modulate T-independent B cell proliferation that is associated with innate immunity. In contrast, IL-21 dramatically enhances B cell mitogenesis following incubation with an activating CD40 antibody [7
]. This result argues that IL-21 enhances B cell function following T:B cell interactions. It was reported recently that the circulating IgE titers of IL-21R-/- mice were threefold higher than those of control mice [46
]. Similarly, an elevated IgE response was observed in IL-21-/- animals relative to controls following immunization (our unpublished observations). This suggests that IL-21 may antagonize IgE production.
In addition to normal B cell function, IL-21 may regulate aspects of B cell tumorigenesis. IL-21R is not expressed on acute B cell leukemia cell lines, but as described above, it is readily detectable on many B cell lymphoma cell lines. IL-21 inhibits the in vitro proliferation of a subset of IL-21R-expressing B cell lymphoma cell lines (our unpublished observations). Conversely, IL-21 appears to be a growth and survival factor for myeloma cell lines and some myeloma specimens, which are cancers derived from terminally differentiated B lymphocytes [48 ]. It will be interesting to further assess the relative roles that IL-21 plays during the various stages of B cell maturation and during the subsequent processes of transformation.
NK cells
IL-21 exhibits positive and negative effects on NK cells. The types of responses elicited in cultured cells appear to vary depending on the maturational stage of the cells, as well as the concentrations of costimulatory factors. As reported by Kasaian et al. [46
], IL-21 has an inhibitory effect on the IL-15-mediated expansion of naïve mouse NK cells; it fails to stimulate the cytolytic activity of freshly isolated mouse NK cells; and it antagonizes the viability of mouse NK cells previously activated with polyriboinosinic polyribocytidylic acid or IL-15. In contrast to these inhibitory effects, IL-21 was shown to stimulate cytotoxicity and IFN- production in previously activated NK cells and to enhance these responses in combination with IL-15. Somewhat different effects have been observed with human NK cells. For instance, IL-21 alone does stimulate the cytolytic activity of freshly isolated, peripheral human NK cells [7
]. Moreover, the combination of IL-21 plus IL-15 can stimulate a dramatic expansion of CD56+CD16+ NK cells from cultures derived from BM [7
] or peripheral blood (our unpublished observations). These cells appear to have enhanced effector cell activity relative to the typical CD56+CD16- cells that arise following exposure to IL-15 alone [49
].
One explanation for the apparent species difference in NK cell responses is the relative naïve nature of laboratory mouse NK cells compared with human NK cells, which are exposed to significantly more environmental antigens. Thus, the differential effects observed among naïve mouse NK cells, activated mouse NK cells, and human NK cells could be a result of differences in IL-21R expression and/or states of maturation. Another difference that might explain the inhibitory effects observed with IL-21 on mouse NK cell proliferation versus the stimulatory effect of IL-21 on human NK cells relates to the relative concentrations of IL-21 and IL-15 that were used in the cultures. Although a direct comparison of conditions is difficult, it appears that the experiments described on mouse NK cells [46 ] were performed using relatively higher doses of IL-21 and lower doses of IL-15 than those we performed on human NK cells [7 ]. Recent experiments have uncovered a biphasic effect of IL-21 on the growth of mouse NK cells (Colin Brooks, Newcastle Medical School, Newcastle, U.K., personal communication). At low doses, IL-21 caused a pronounced, synergistic effect in combination with IL-2, whereas at high doses, it inhibited IL-2-mediated NK cell proliferation. The activity of IL-21 on murine and human NK cells in fact may be similar when dose, stage of maturation, and activation state are matched.
Kasaian et al. [46 ] have suggested that to accelerate the transition between innate and adaptive immunity, IL-21 enhances the effector functions of NK cells and CD8+ T cells (see above) but also limits the expansion of resting and activated NK cells. It is also possible that the IL-21 effect on NK cells may vary depending on the timing and magnitude of the T cell response and the subsequent concentrations of IL-21. Thus, antigen activation of relatively few T cells may promote NK cell expansion and effector cell function, whereas a larger number of activated T cells may actually down-regulate NK cell expansion and function.
Nonlymphoid cells
The cytokines that use c are known to mediate a variety of responses, not just on lymphoid cells but also on cells of various myeloid lineages such as monocytes/macrophages, neutrophils, mast cells, basophils, and eosinophils, as well as a variety of nonhematopoietic tissues (reviewed in ref. [50
]). IL-21R is not readily detectable on nonlymphoid cell types, although its expression may be regulated acutely depending on stages of maturation or activation. Transgenic mice overexpressing IL-21 show inflammatory infiltrates in multiple tissues consisting of macrophages and neutrophils, as well as increased numbers of myeloid cells in spleen and BM (our unpublished observations). Although it is unclear whether this is a primary or secondary effect, it seems possible that IL-21 might play some role in the regulation of normal, nonlymphoid cell development and/or activity.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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c as a component of its receptor complex. The lymphoid-restricted tissue distribution of IL-21R suggests that the primary activities of IL-21 are immunomodulatory, an assumption that is being borne out by in vitro and in vivo analyses. As a product of activated helper T cells, IL-21 shares characteristics of a Th1 cytokine. IL-21 is a potent stimulator of IFN- production and cytolytic activity for NK cells and CD8+ T cells, and it synergizes with IL-2 and IL-15 in regulating these responses. Additionally, IL-21 can down-regulate IL-15-induced NK cell expansion and prevent B cell proliferation induced by T-independent antigens. Collectively, the modulatory effects of IL-21 provide a link between innate and adaptive immunity that may help focus the strength and duration of cell-mediated immune responses.
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ACKNOWLEDGEMENTS |
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Received June 5, 2002; accepted June 30, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONTHE IL-21 RECEPTOR (IL-21R)FUNCTIONAL CLONING OF IL-21THE FUNCTIONAL IL-21R COMPLEXIL-21 BIOLOGYCONCLUSIONSREFERENCES |
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