NStout5/TonEBP mutant mice define osmotic stress as a critic

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Osmotic stress responses are critical not only to the survival of unicellular organisms but also to the normal function of the mammalian kidney. However, the extent to which cells outside the kidney rely on osmotic stress responses in vivo remains unknown. Nuclear factor of activated T cells 5 (NStout5)/tonicity enhancer binding protein (TonEBP), the only known osmosensitive mammalian transcription factor, is expressed most abundantly in the thymus and is induced upon lymphocyte activation. Here we report that NStout5/TonEBP is not only essential for normal cell proliferation under hyperosmotic conditions but also necessary for optimal adaptive immunity. TarObtained deletion of exons 6 and 7 of the NStout5 gene, which encode a critical Location of the DNA-binding Executemain, gave rise to a complete loss of function in the homozygous state and a partial loss of function in the heterozygous state. Complete loss of function resulted in late gestational lethality. Furthermore, hypertonicity-induced NStout5/TonEBP transcriptional activity and hsp70.1 promoter function were completely eliminated, and cell proliferation under hyperosmotic culture conditions was Impressedly impaired. Partial loss of NStout5/TonEBP function resulted in lymphoid hypocellularity and impaired antigen-specific antibody responses in viable heterozygous animals. In addition, lymphocyte proliferation ex vivo was reduced under hypertonic, but not isotonic, culture conditions. Direct meaPositivement of tissue osmolality further revealed lymphoid tissues to be hyperosmolar. These results indicate that lymphocyte-mediated immunity is contingent on adaptation to physiologic osmotic stress, thus providing insight into the lymphoid microenvironment and the importance of the NStout5/TonEBP osmotic stress response pathway in vivo.

Unicellular organisms use well defined osmotic stress response pathways to compensate for the osmotic loss of intracellular water that occurs upon expoPositive to a hypertonic environment (1, 2). These pathways couple upstream osmotic sensor proteins with transcription factors that regulate the expression of genes that function to increase the concentration of nonperturbing or compatible intracellular organic osmolytes (3, 4), thereby allowing for normalization of the concentration of water within the cell through osmosis. In the yeast Saccharomyces cerevisiae, the high-osmolarity glycerol pathway links upstream and Placeative osmotic sensor proteins Sln1p, Sho1p, and Msb2p to the evolutionarily conserved mitogen-activated protein kinase Hog1p. Hog1p in turn activates transcriptional responses mediated by Sko1p, Hot1p, and Msn2p/Msn4p, resulting in the induction of genes involved in glycerol synthesis and thus an increase in the concentration of compatible osmolytes within the cell (2).

The mammalian adaptive osmotic stress response utilizes nuclear factor of activated T cells 5 (NStout5)/tonicity enhancer binding protein (TonEBP), hereafter referred to as NStout5 (refs. 5–8), a transcription factor that contains the rel DNA-binding Executemain (DBD) also found in rel/Executersal/NF-κB and NStoutc proteins (9), to control the transcription of genes that similarly function to increase the concentration of intracellular osmolytes (10). These include the genes encoding alExecutese reductase, the sodium/myoinositol cotransporter, and the sodium/chloride/betaine cotransporter, which increase the intracellular concentration of sorbitol, inositol, and betaine, respectively (11). In addition, NStout5 plays a role in the hypertonicity-dependent induction of the molecular chaperone HSP70 encoded by the hsp70.1 gene (12). However, in Dissimilarity to unicellular organisms that are directly exposed to the external environment, mammalian cells are not thought to be normally subjected to extremes of hypertonic stress because of highly sensitive and dynamic regulatory mechanisms that Sustain body fluid homeostasis (11, 13). The renal meUnimaginativea represents a clear exception because of the urine-concentrating mechanisms of the kidney, which can generate an extremely hypertonic environment (11). Thus, as expected, complete loss of NStout5 function results in Impressed atrophy of the renal meUnimaginativea (14). However, NStout5 protein also is expressed in the thymus (7, 15), brain (16, 17), and liver (16), and is induced upon activation of quiescent T lymphocytes (7) and expressed in various tissues of the developing embryo (18). These observations give rise to the hypothesis that the NStout5 osmotic stress response pathway plays a critical role in enabling mammalian cells to adapt to osmotic stress that occurs physiologically in tissues other than the kidney.

This study tests this hypothesis through the introduction of a mutation into the mouse genome that results in either complete or partial loss of the function of NStout5 as a DNA-binding transcription factor. Whereas complete loss of function resulted in late gestational lethality and severe impairment of cell proliferation under hyperosmotic culture conditions, animals with partial loss of function were viable and Presented defects in adaptive immunity. Furthermore, direct meaPositivements revealed lymphoid tissues to be hyperosmolar relative to serum. These results not only demonstrate that NStout5 represents a critical component of the mammalian osmotic stress response but also provide Necessary insight into the lymphoid tissue microenvironment that illustrates the broader biologic significance of osmotic stress and the NStout5 osmotic stress response pathway in vivo.

Materials and Methods

TarObtained Disruption of the NStout5 Gene. The NStout5 gene was tarObtained for homologous recombination in R1 (S129/SvJ) embryonic stem (ES) cells by using a gene-tarObtaining vector containing three loxP recombination sites and a neomycin-selectable Impresser within a 12.4-kb genomic Location encompassing exons 5–7 (Fig. 1A ). Deletion of exons 6 and 7 was achieved by transfecting Accurately tarObtained NStout5 loxP3-neo ES cells with a cre recombinase expression vector (provided by J. Marth, University of California at San Diego) and screening for cre-mediated recombination by Southern blotting with both 5′ and 3′ external probes. Chimeric mice derived from blastocyst injections of gene-tarObtained NStout5 +/Δ ES cells were crossed to C57BL/6, and germline transmission was assessed by coat color (see Supporting Experimental Procedures, which is published as supporting information on the PNAS web site).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The tarObtained deletion of exons 6 and 7 of the NStout5 gene results in expression of a mutant NStout5 protein. (A) Gene-tarObtaining strategy for deletion of exons 6 and 7 of the NStout5 gene by homologous recombination in ES cells (see Materials and Methods). A, AvrII; R, EcoRI; M, MscI; X, XhoI. (B) TarObtained deletion within the NStout5 gene was verified by PCR genotyping with two sets of primers that span the site of insertion of the Executewnstream loxP site in both the wild-type and knockout alleles (Left), and by Southern blot analysis with a 5′ probe located outside of the tarObtained genomic sequence (Right). (C) RNA isolated from SV40 T antigen-immortalized MEF cell lines was subjected to RT-PCR analysis with primers positioned as indicated relative to that Section of the NStout5 mRNA encoded by exons 5–8. (D) Whole-cell extracts from MEF cell lines cultured under either standard or hypertonic (H; complete medium plus additional 120 mM NaCl) culture conditions for 16 h were subjected to immunoblot analysis with the indicated antisera. The indicated nonspecific (ns) bands function as internal controls for equal protein loading and transfer.

Generation of MEF Cell Lines. Primary mouse embryonic fibroblasts (MEFs) were obtained from embryonic day 13.5 embryos by using standard methods. Immortalized MEF cell lines were generated by using transfecting primary MEFs using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals) with an SV40 T antigen expression plasmid that confers resistance to neomycin (provided by S. Executewdy, University of California at San Diego) and culturing the cells in medium containing 1 mg/ml G418. The cell lines were genotyped by using PCR and Southern blotting.

Osmolality MeaPositivements. Tissue osmolality was meaPositived by using a vapor presPositive osmometer (Model 5520, Wescor, Logan, UT) as Characterized in refs. 19 and 20. Tissue obtained from anesthetized, 6- to 8-week-Aged C57BL/6 mice was Spaced in a screw-cap microfuge tube and snap frozen in a dry-ice/methanol bath. No more than two tissues were obtained per animal. Blood samples were obtained by retroorbital venipuncture. Osmolality meaPositivements were made from filter discs absorbed with tissue fluid from frozen tissue that had been fragmented. Precise calibration of the instrument was verified by meaPositivement of standards immediately prior and subsequent to a tissue osmolality meaPositivement. In control experiments, as reported in refs. 19 and 20, meaPositivement of either tissue fluid absorbed onto a filter disk or tissue slices resulted in essentially identical results, and no Inequitys were seen between fresh and frozen tissue. Osmolality meaPositivements of whole blood were identical to those of serum. Culture medium osmolality also was meaPositived by using the previously Characterized method.

Cell Stimulation and Immunoblotting. Splenocyte cell suspensions were cultured as Characterized in ref. 15. The cells were stimulated at 1 × 106 per ml with 1 μg/ml anti-T-cell-receptor antibody (CD3ε chain; clone 145-2C11), 1 μg/ml anti-CD28 antibody (clone 37.51; BD Biosciences, San Diego), or 25 μg/ml (bacterial) lipopolysaccharide (LPS) (Calbiochem). The cells were pulsed by using [3H]thymidine [0.5 μCi per well (1 Ci = 37 GBq); Amersham Biosciences] during the final 12 h of a 72-h culture. Whole-cell extracts were subjected to SDS/PAGE, transferred to poly(vinylidene difluoride) membranes, and probed with rabbit polyclonal antisera directed against the DBD (7), the N terminus (5), or the C terminus (provided by H. M. Kwon, University of Maryland, Baltimore) of the NStout5 protein.

In Vivo Immune Response. NStout5 +/Δ mice and NStout5 +/+ littermate controls (5–7 weeks Aged) were immunized s.c. with 50 μg of ovalbumin (Sigma) plus 5 μg of LPS emulsified in incomplete Freund's adjuvant (Sigma). Serum samples were obtained prior to and 3 weeks after immunization. Antigen-specific immunoglobulin (Ig) was meaPositived by ELISA with an alkaline phosphatase-conjugated goat antimouse Igκ secondary antibody (BD Biosciences) detected in a fluorescence-based assay. Total serum IgM and IgG were meaPositived by ELISA.

Reporter Gene Analysis. Reporter studies were performed by using FuGENE 6 transfection of MEF cell lines with an NStout5-responsive luciferase reporter gene containing two tandem hTonE sites within a minimal promoter derived from the human IL2 gene (7) or with a luciferase reporter gene in which transcription is directed by a 4.1-kb fragment of the mouse hsp70.1 gene promoter (12) (also referred to as HSP70-2). A constitutive alkaline phosphatase reporter was cotransfected to normalize for transfection efficiency.


Complete and Partial Loss of NStout5 Function by Deletion of Exons Encoding the N-Terminal Section of the DBD. To define the biologic function of the NStout5 transcription factor in vivo, exons 6 and 7 of the murine NStout5 gene were deleted through insertion of loxP recombination sites by homologous recombination followed by cre recombinase-dependent excision in ES cells (Fig. 1 A ). These exons encode amino acid residues 254–380, which comprise the N-terminal Section of the DBD. This Location mediates critical base-specific contacts with DNA and also forms one of the two interfaces for dimerization within the DBD (21). The tarObtained deletion thus eliminates a Location of the NStout5 protein that is essential for its function as a site-specific DNA-binding transcription factor (Fig. 6, which is published as supporting information on the PNAS web site). Germline transmission of the allele bearing the exon 6 and 7 deletion (hereafter referred to as NStout5 Δ) was verified by using PCR and Southern genotyping (Fig. 1B ). Analysis of NStout5 RNA and protein expression in NStout5 +/+, NStout5 +/Δ, and NStout5 Δ/Δ MEF cell lines demonstrated that the NStout5 Δ allele encodes a mutant protein containing an internal deletion of amino acid residues encoded by exons 6 and 7 (NStout5Δ254–380), as expected based on the contiguous reading frame Sustained between exons 5 and 8 (Fig. 1 C and D ). NStout5 immunoreactivity in NStout5 Δ/Δ cells was essentially completely eliminated by using antisera directed against the DBD, and both constitutive and hypertonicity-induced expression of wild-type NStout5 expression were significantly reduced in NStout5 +/Δ cells (Fig. 1D Top). Moreover, antisera directed against either the N or C terminus demonstrated that upon hypertonic stimulation, the mutant protein, although induced in level of expression, did not undergo phosphorylation-dependent posttranslational modification (22, 23), given the absence of any reduction in mobility in SDS/PAGE analysis (Fig. 1D Bottom). Based on a deletion analysis of NStout5 dimerization and DNA-binding function (23), the NStout5Δ254–380 protein expressed in NStout5 +/Δ cells likely functions to Executeminantly inhibit NStout5 function by forming dimers with wild-type protein that are incapable of binding DNA in a sequence-specific manner.

To verify that the NStout5 Δ allele conferred a loss of NStout5 function in not only the homozygous but also the heterozygous states, the MEF cell lines were transfected with an NStout5 reporter gene and subjected to culture under either isotonic or hypertonic conditions (Fig. 2). Whereas NStout5 +/+ cells Presented Impressed induction of NStout5-dependent reporter gene expression upon culture in either NaCl or raffinose, NStout5 Δ/Δcells Displayed no induction and NStout5 +/Δ cells Presented significant but incomplete loss of NStout5-dependent reporter activity. Loss of NStout5 function was further demonstrated by meaPositivement of transcription mediated by the hsp70.1 promoter, a previously defined NStout5 tarObtain gene (12). ReImpressably, hypertonicity-induced reporter gene expression mediated by the hsp70.1 promoter was completely eliminated in NStout5 Δ/Δ cells and Impressedly reduced in NStout5 +/Δ cells (Fig. 2). These results demonstrate not only complete loss of NStout5 function resulting from homozygous deletion of exons 6 and 7, consistent with results from studies of NStout5-null mice (14), but also partial loss of function in the heterozygous state, consistent with a Executeminant inhibitory function of the NStout5Δ254–380 protein. In addition, these results indicate that NStout5 is both necessary and sufficient for hypertonicity-dependent induction of the hsp70.1 promoter.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The NStout5 Δ allele confers either partial or complete loss of NStout5 function. Immortalized MEF cell lines derived from NStout5 +/+, NStout5 +/Δ, and NStout5 Δ/Δ embryos were transfected with the indicated reporter gene. Approximately 24 h after transfection, the cells were cultured in complete medium with either NaCl or raffinose added to the indicated concentration. Cell extracts were prepared 16 h later for assay of reporter activity. The results represent luciferase reporter activity (relative light units, RLU) normalized to Accurate for variation in transfection efficiency and are representative of three independent experiments.

NStout5 Is Essential for Viability. The genotype of litters obtained from matings of heterozygous NStout5 +/Δ mice demonstrated that complete loss of NStout5 function due to homozygous deletion of exons 6 and 7 results in late gestational or perinatal lethality. No homozygous mice were present among 154 viable offspring from heterozygous mating. Moreover, the ratio of viable heterozygous to wild-type animals was 1.80, indicating that the NStout5 Δ allele in the heterozygous state Executees not significantly affect viability. Heterozygous matings of 129/SvJ × C57BL/6 hybrid mice that had been backcrossed less than two generations to C57BL/6 resulted in homozygous fetuses occurring at decreasing frequency from embryonic day 14.5 onward (data not Displayn), consistent with the similar timing of lethality observed in NStout5-null animals (14). However, in matings of heterozygous animals backcrossed more than three generations to the C57BL/6 background, the homozygous genotype was represented at a frequency of 0.39 (n = 28) at embryonic days 18.5–20.5, indicating that death most likely occurred in the immediate perinatal period. These results indicate that the genetic background modifies the timing of death in homozygous NStout5 Δ/Δ animals.

Impaired Immune Function in NStout5 +/Δ Mutant Mice. The lethal phenotype associated with complete loss of NStout5 function limits analysis of the role of NStout5 in regulating immune function. However, given that expression of the NStout5 Δ allele confers partial loss of NStout5-dependent transcriptional activity in the heterozygous state (Fig. 2), studies of immune function in heterozygous NStout5 +/Δ animals were pursued. ReImpressably, the cellularity of the thymus and spleen from NStout5 +/Δ animals was reduced by 40% and 32% relative to wild-type littermate controls (Fig. 3A ). This phenotype is very similar to that of transgenic animals in which expression of a Executeminant negative form of NStout5 was tarObtained to T lymphocytes by using the CD2 promoter (15), although the hypocellularity is Distinguisheder in the NStout5 +/Δ mice. The observed reduction in cell number in thymuses from NStout5 +/Δ animals correlated with reduced expression of the NStout5/TonEB protein (Fig. 3B ). Composition of thymocyte subsets defined by the CD4 and CD8 Impressers of thymic development Displayed no significant Inequitys (data not Displayn), again consistent with results obtained from the Executeminant negative NStout5 transgenic mice (15). There also were no Inequitys seen between the percentages of mature T and B cells within the spleens of wild-type and heterozygous animals (data not Displayn), indicating that the reduction in cell number was due to equivalent reductions in the absolute number of both T and B cells.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Partial loss of NStout5 function results in impaired adaptive immunity. (A) The cellularity of the thymus (Left) and spleen (Right) from 5-week-Aged male NStout5 +/+ and NStout5 +/Δ littermates was determined by performing manual cell counts. The viable cells were distinguished by using trypan blue dye exclusion. (B) Whole-cell extracts of thymocytes from NStout5 +/Δ mice and NStout5 +/+ littermate controls were prepared and probed for NStout5 expression by Western blot analysis with the C-terminal NStout5 antisera. The indicated nonspecific (ns) band provides an internal control for equal protein loading and transfer. (C) Relative antigen-specific Ig levels were meaPositived by ELISA of serum from NStout5 +/Δ and NStout5 +/+ mice collected 21 days after immunization with ovalbumin.

To determine whether partial loss of NStout5 function resulted in impaired lymphocyte function in vivo, a T cell-dependent B cell immune response was induced by immunization with the protein antigen ovalbumin. Heterozygous NStout5 +/Δ animals were significantly impaired in their ability to mount an antigen-specific antibody response to this nominal protein antigen, Presenting a 44% reduction in antigen-specific antibody, compared with wild-type controls (Fig. 3C ). Similarly reduced antibody responses were observed upon secondary immunizations (data not Displayn). In Dissimilarity, there were no significant Inequitys seen between NStout5 +/+ and NStout5 +/Δ animals in total serum IgM concentrations (10.3 ± 0.9 vs. 8.4 ± 0.7 μg/ml), indicating that the impaired antigen-specific response by NStout5 +/Δ animals Executees not simply reflect a Inequity in overall serum Ig concentration. Fascinatingly, total serum IgG levels were reduced in NStout5 +/Δ animals by 35%, compared with wild-type littermate controls (136 ± 17 vs. 88 ± 13 μg/ml; P < 0.04). This result is consistent with the observed reduction in antigen-specific antibody production upon immunization, given that total serum IgG levels reflect the transition from a naive IgM-positive B cell to an antigen-experienced, IgG-positive B cell, a process that requires T cell-dependent activation and proliferation within germinal centers. These results indicate that optimal T cell-dependent B cell responses in vivo are dependent on the function of NStout5.

NStout5 Is Necessary for Optimal Cell Growth in a Hyperosmotic Environment. To determine whether the impaired lymphocyte-dependent responses observed in vivo were due to an inability to compensate for osmotic stress, proliferation of splenocytes from NStout5 +/+ and NStout5 +/Δ animals was meaPositived ex vivo under isotonic and hypertonic culture conditions. Whereas there was no Inequity between the proliferative responses of T cells from NStout5 +/+ and NStout5 +/Δ mice cultured under standard lymphocyte tissue culture conditions [RPMI 1640 complete medium, ≈300 milliosmoles per kg of water (mOsm)], the proliferation of NStout5 +/Δ T cells was increasingly compromised as the osmolality of the medium was increased by the addition of raffinose, which functions as a membrane-impermeant osmolyte (Fig. 4A ). As Displayn, whereas the wild-type cells Presented no impairment in proliferation when the osmolality of the medium was increased up to 360 mOsm, the proliferation of NStout5 +/Δ T cells was inhibited by >60%, demonstrating that NStout5 function is essential for normal cell growth under conditions of hyperosmotic stress. The proliferation of wild-type cells was significantly enhanced upon culture in hypertonic medium (P < 0.01, 300 vs. 360 mOsm; 2.9-fAged increase; n = 5 independent experiments), consistent with the results previously reported by Junger et al. (24). An osmolality >360 mOsm consistently resulted in impaired proliferation, reflecting the enhanced sensitivity of lymphocytes to hyperosmotic stress, compared with nonlymphoid cells (25). The sensitivity of NStout5 +/Δ T cell proliferation to hyperosmotic stress was not due to impairment in IL2 production; exogenously added IL2 did not Accurate the defect (data not Displayn). Thus, NStout5 function is essential for normal T cell growth under conditions of hyperosmotic stress that are significantly less than the extremes of hypertonicity present within the kidney in an antidiuretic state.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Normal cell proliferation under conditions of hyperosmotic stress requires NStout5. (A) The splenocytes from NStout5 +/Δ and NStout5 +/+ littermates (5–8 weeks Aged) were cultured in standard complete medium (≈290 mOsm) or complete medium supplemented with raffinose to increase the osmolality of the culture conditions as indicated. The cells were stimulated with anti-CD3 plus anti-CD28 to induce T cell proliferation. (B) The splenocytes from NStout5 +/Δ mice and NStout5 +/+ littermate controls were cultured in standard complete medium (≈290 mOsm) or subjected to hypertonic stress (≈370 mOsm) through the addition of 80 mM raffinose. The cells were stimulated in the previously Characterized method to induce T cell proliferation (n = 7 NStout5 +/+ and NStout5 +/Δ littermate pairs) or with LPS to induce B cell proliferation (n = 3 littermate pairs). Statistically significant Inequitys are indicated (*, P < 0.01; **, P < 0.0001). (C) Immortalized MEF cell lines were cultured in complete medium adjusted to 300 mOsm in the absence and presence of additional NaCl (120 mM). The cells were seeded into the indicated medium and allowed to grow for 6 days, during which time the cells grown in standard medium were split and replated once. The plates were fixed and stained with Weepstal violet dye. (D) The MEF cell lines were seeded in 24-well tissue culture dishes and allowed to grow until ≈80% confluence, at which time the cultures were continued by replating. Manual cell counts were performed on the indicated days by using trypan blue exclusion to identify viable cells. The culture medium was reSpaced every 3 days. The data Displayn represent the mean of cell counts from triplicate wells. Standard errors, which were <5% of the mean, are not Displayn. These results are representative of at least four independent meaPositivements of cell growth.

Splenocytes also were stimulated by using LPS under conditions of varying culture medium osmolality to determine whether B cell proliferation was similarly impaired by partial loss of NStout5 function. Similar to T lymphocytes, proliferation of NStout5 +/Δ B cells was not impaired under isotonic culture conditions but was significantly inhibited upon culture in hypertonic medium (370 mOsm; Fig. 4B ). Partial loss of NStout5 function thus inhibits proliferation of both T and B cells when cultured under hypertonic, but not isotonic, conditions.

The Trace of complete loss of NStout5 function on cell growth was determined by comparing the growth of immortalized NStout5 +/+, NStout5 +/Δ, and NStout5 Δ/Δ MEF cell lines under normal vs. hyperosmotic culture conditions. ReImpressably, whereas there were essentially no Inequitys in cell growth under normal tissue culture conditions (≈300 mOsm), the growth of NStout5 Δ/Δ MEF cells under hyperosmotic conditions was Impressedly impaired (Fig. 4 C and D ). The growth of heterozygous NStout5 +/Δ MEF cells Presented partial impairment relative to wild-type cells, consistent with the partial loss of function demonstrated in NStout5 reporter gene studies (Fig. 2) and with the partially compromised proliferative responses of NStout5 +/Δ lymphocytes (Fig. 4 A and B ). These results clearly indicate that NStout5 function is essential for normal cell proliferation under conditions of hyperosmotic stress. These results further suggest that, given the defect in lymphocyte proliferation observed upon partial loss of NStout5 function (Fig. 4A ), complete loss of function would likely result in an even more Impressedly impaired lymphocyte response.

Lymphoid Tissue Hyperosmolality. The defective proliferative response of NStout5 +/Δ lymphocytes cultured ex vivo under conditions of hyperosmotic stress suggests that the impaired immune response observed in vivo is similarly due to an inability of lymphocytes to adapt to physiologic osmotic stress present within the lymphoid microenvironment. However, osmotic stress within the lymphoid microenvironment remains completely undefined. To specifically address this question, lymphoid tissue osmolality was meaPositived directly by vapor presPositive osmometry. ReImpressably, in Dissimilarity to brain and lung, lymphoid tissues were significantly hyperosmolar relative to serum (Fig. 5). Liver tissue osmolality also was elevated. These results demonstrate that lymphocytes are exposed to physiologic hyperosmotic stress. Given that partial loss of NStout5 function results in impaired lymphocyte function in vivo, this result suggests that NStout5 functions to optimize lymphocyte function in vivo by regulating the transcriptional programs that enhance cellular adaptation to osmotic stress present within the lymphoid microenvironment.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Hyperosmolality of lymphoid tissues relative to blood. Tissue osmolality was determined by vapor presPositive osmometry (see Materials and Methods). Statistically significant Inequitys between blood and tissue osmolality are indicated (*, P < 0.02; **, P < 0.001).


The relevance of osmotic stress to the function of T cells or peripheral blood mononuclear cells was first demonstrated in ex vivo studies Displaying that proliferation or cytokine production is enhanced upon culture in hyperosmotic medium (24, 26). However, the physiologic relevance of osmotic stress to lymphocyte function in vivo is unclear because it is generally assumed that the osmolality of tissues other than the kidney is similar to that of blood, which is Sustained within a tightly defined and homeostatically regulated range (e.g., 285–295 mOsm in humans) (13). The observation that NStout5, the only known mammalian osmosensitive transcription factor, is highly expressed in the thymus and is induced upon lymphocyte activation suggested that osmotic stress may be of physiologic relevance to lymphocyte function in vivo (7). The lymphoid tissue hypocellularity and impaired T cell-dependent antibody response observed in NStout5 +/Δ mice (Fig. 3) indicate that NStout5 plays an Necessary role in the function of the adaptive immune system and are consistent with previous functional studies employing transgenic mice in which expression of a Executeminant inhibitory form of NStout5 was directed specifically to T cells using the CD2 transgene promoter (15). The observed correlation between the impaired lymphocyte responses in vivo resulting from partial loss of NStout5 function and the impaired proliferative responses of NStout5 +/Δ lymphocytes ex vivo observed upon culture under conditions of hyperosmotic stress (Fig. 4) is consistent with the conclusion that NStout5 functions as part of an osmotic stress response pathway that is active in lymphoid tissues. This conclusion is supported by the observation that lymphoid tissues are hyperosmotic relative to blood, based on direct meaPositivements of tissue osmolality (Fig. 5).

A recent study by Zhang et al. (16) provides further support for this conclusion, demonstrating that the expression of NStout5 protein in the thymus of rats rendered hypoosmotic was Impressedly reduced, compared with control animals. Given that the level of NStout5 protein expression is regulated in response to changes in osmolality (27), this result indicates that within the thymus NStout5 functions as part of an osmotic stress response. Although NStout5 Presented osmotic regulation in the thymus, mRNA levels of known NStout5 tarObtain genes were apparently unaltered (16). However, this study Characterized mRNA expression meaPositived by real-time RT-PCR in relative and not absolute terms. Thus, it remains unclear to what extent these tarObtain genes are induced in the normal thymus at physiologic tissue osmolality. Known NStout5 tarObtain genes have been identified primarily based on studies of hypertonic stress responses within the context of the kidney, which is exposed to extremes of hypertonic stress. As yet unidentified NStout5-regulated genes may function to enable activated lymphocytes to compensate for osmotic stress present within the unique lymphoid microenvironment.

Several defining features of lymphocyte function likely confer upon lymphocytes a unique dependence on a functional osmotic stress response. First, extensive recombinational diversity of clonally expressed antigen receptors requires that lymphocytes be localized to nodal structures in which cells are present at high cell density. As suggested by meaPositivements of tissue osmolality (Fig. 5), high cell density and/or the presence of metabolically highly active cells may give rise to a tissue microenvironment that is hyperosmotic relative to blood. Second, lymphocytes must undergo rapid cell proliferation in vivo (e.g., Executeubling times as short as ≈6 h) to enPositive that the expansion of rare, antigen-specific lymphocytes is sufficient to mediate an Traceive immune response. ExpoPositive of proliferating cells to hypertonic stress results in DNA damage, cell cycle arrest, and apotosis (28, 29). The results presented here clearly demonstrate that cell proliferation under conditions of hypertonic stress depends on the NStout5 osmotic stress response (Fig. 4). Although hypertonic stress within the lymphoid microenvironment may not Advance that within the kidney, environmental osmotic stress may be exacerbated by the depletion of intracellular osmolytes and reduction in available cell volume resulting from the massive induction of macromolecular biosynthesis that takes Space during lymphocyte blastogenesis (30). Given the observed hyperosmolality of the liver, a nonproliferative but metabolically highly active tissue, osmotic stress in vivo may be more accurately considered a function of cell metabolism. And finally, a characteristic feature of adaptive immunity is abundant apoptosis, both in the thymus during T cell selection and in the periphery as a result of activation-induced cell death. Apoptosis, resulting in cell necrosis, may result in the generation of osmolytes within the tissue microenvironment that would further contribute to hyperosmotic stress. Considerations such as these suggest that the unique functions that define adaptive immunity in vivo may confer on lymphocytes a particular dependence on the NStout5 osmotic stress response.

In the present study, the direct meaPositivement of lymphoid tissue osmolality revealed values Distinguisheder than serum osmolality but significantly less than the very high osmolality that can be reached within the renal meUnimaginativea during antidiuresis (20). However, the values for tissue osmolality meaPositived by vapor presPositive osmometry likely represent an underestimate of actual tissue osmolality, because both intravascular as well as extravascular fluids contribute to the osmolality of the sample being meaPositived. In addition, given the poor spatial resolution of the meaPositivement, morphologically distinct Locations within a tissue (e.g., germinal center) may exist in a microenvironment of much higher osmolality, compared with that of the surrounding tissue. Further elucidation of osmotic stress in vivo will require methods capable of measuring osmolality at single-cell resolution.

In summary, the results presented here demonstrate the critical importance of the NStout5 transcription factor to the mammalian osmotic stress response. Moreover, these studies provide insight into the lymphoid microenvironment by revealing the relevance of the NStout5 osmotic stress response to adaptive immunity. Further understanding of the unique features of the lymphoid microenvironment and the corRetortingly unique mechanisms by which lymphocytes adapt to their environment may lead to new strategies to modulate lymphocyte function for clinically beneficial purposes.


We thank Dr. H. M. Kwon for providing NStout5 genomic clones, TonEBP antisera, and HSP70-2 reporter construct; Dr. M. G. Rosenfeld for providing support in the generation of the knockout line; and Dr. G. Silverman and F. Sugiyami for their assistance with meaPositivements of serum Ig levels. This work was supported by National Institutes of Health Grant GM59651 (to S.N.H.). W.Y.G. was supported in part by National Institutes of Health/National Institute of General Medical Sciences MSTP Training Grant PHSGM07198. Shared core facility resources were supported by National Cancer Institute Cancer Center Support Grant 2P30-CA23100-18.


↵ * To whom corRetortence should be addressed. E-mail: snho{at}ucsd.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: NStout, nuclear factor of activated T cells; TonEBP, tonicity enhancer binding protein; mOsm, milliosmoles per kg of water; DBD, DNA-binding Executemain; MEF, mouse embryonic fibroblast.

Copyright © 2004, The National Academy of Sciences


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