Targeted disruption of the inosine 5'-monophosphate dehydrogenase type I gene in mice

Abstract

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I(-/-) HPRT(-/0) splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

FIG. 1.

Targeted disruption of the IMPDH type I gene. Shown are a schematic diagram of the IMPDH type I gene with exons 2 to 14 (open boxes), the targeting construct with exons 3 to 9 and exons 9 to 13 cloned adjacent to the neo gene, and the expected result of homologous recombination. The 2.3-kb XbaI fragment used as a probe for Southern analysis and oligonucleotides (E8F, NeoR, and E10R) used for PCR are indicated.

FIG. 3.

Breeding strategy to generate double-knockout mice. Boxed genotypes indicate animals used for experiments.

FIG. 2.

Analysis of homologous recombination. (A) A representative Southern hybridization demonstrating the 9.3-kb (wild-type allele) and 8.3-kb (knockout allele) HindIII fragments. (B) PCR analysis demonstrating a 1.2-kb fragment (wild-type allele) or 0.6- and 2.4-kb fragments (knockout allele). Lane 3, wild type; lanes 1, 2, and 6, homozygous; lanes 4 and 5, heterozygous; lane 7, molecular weight markers consisting of lambda/HindIII and φX174/HeaIII digests.

FIG. 4.

Expression of IMPDH type I, IMPDH type II, and HPRT mRNAs and IMPDH proteins in wild-type and double-knockout mice. (A) Northern hybridizations were performed on 20 μg of total RNA from tissues of wild-type (WT) and double-knockout mice. Lanes: 1, thymus; 2, liver; 3, lung; 4, testis; 5, brain. (B) Total RNA (10 μg) from resting (R) or ConA-activated (CA) splenocytes from wild-type or double-knockout mice. Both blots were probed with IMPDH type I, type II, or HPRT cDNA probes, as indicated. Levels of 28S RNA were used for quantitation. (C) (Upper panel) Expression of IMPDH type I and type II proteins in tissues from wild-type and double-knockout mice. Lanes containing tissue types are as in panel A. (Lower panel) Expression of IMPDH type I and type II proteins in thymuses (lanes a through d) of wild-type (lanes a and b) and type I^−/− HPRT^−/0 (lanes c and d) mice, lungs (lanes e through h) of wild type (lanes e and f) and type I^−/− (lanes g and h) mice, and resting (lanes i and k) and ConA-activated (lanes j and l) lymphocytes from wild-type (lanes i and j) and type I^−/− (lanes k and l) mice. Lower arrow, type I proteins; upper arrow, type II proteins. Asterisk indicates a high-molecular-weight variant of IMPDH I in the thymuses and lungs of wild-type mice. Total-cell lysates (10 μg) were separated on SDS-polyacrylamide gel electrophoresis gels and probed with an anti-IMPDH specific antibody.

FIG. 5.

Intracellular GTP levels. Splenocytes from wild-type, IMPDH type I^−/−, and double-mutant (type I^−/− type II^+/− or type I^−/− HPRT^−/0) mice were stimulated with anti-CD3 plus anti-CD28 antibodies for 48 h. GTP levels in resting and antibody-activated wild-type splenocytes were 0.2 ± 0.05 and 1.8 ± 0.3 nmol per 10⁷ cells, respectively (n = 11). Values from activated splenocytes (IMPDH type I^−/− [n = 4], type I^−/− type II^+/− [n = 3], and type I^−/− HPRT^−/0 [n = 4]) were plotted as percentages of wild-type levels (means ± standard deviations).

FIG. 6.

IMPDH and HPRT enzymatic activities in mutant mice. (A) IMPDH enzyme activity was measured in tissues of single- and double-knockout mice. Total-cell lysates of 20 μg (thymus), 50 μg (liver, brain, and testis), and 60 μg (lung) were used for each assay. Activities from mutant mice (IMPDH type I^−/− [n = 4], type I^−/− type II^+/− [n = 5], and type I^−/− HPRT^−/0 [n = 5]) were plotted as percentages of activities obtained from wild-type tissues (n = 14). (B) IMPDH enzyme activities were measured on 10 μg of splenocyte lysates from mice with an IMPDH type I^−/− or type II^+/− genotype before and after ConA activation and plotted as percentages of wild-type activities. (C) HPRT activities were measured from type I^−/− type II^+/− double-knockout mice (n = 4) and plotted as percentages of wild-type activity. Values are means ± standard deviations, with each determination performed in duplicate.

FIG. 7.

Effects of loss of IMPDH type I, IMPDH type II, and HPRT activities on splenocyte proliferation. (A) Splenocytes from wild-type (100%), IMPDH type I-deficient, or double-knockout (type I^−/− type II^+/− or type I^−/− HPRT^−/0) mice were stimulated with ConA, LPS, PMA plus ionomycin (PI), or anti-CD3 plus anti-CD28 antibodies (Ab) as indicated for 48 h. Proliferation was measured by [³H]thymidine incorporation. Data are means ± standard deviations from three experiments performed in triplicate. (B) Splenocytes from wild-type and type I^−/− type II^+/− mice were stimulated with antibodies for 48 h, and cell cycle analysis was performed after PI labeling. The sub-G₁ population was quantitated. Results of an experiment representative of three determinations are shown. (C) Cell cycle analysis of activated splenocytes from paired wild-type and type I^−/− type II^+/− littermates. Cells were stimulated with anti-CD3 plus anti-CD28 antibodies for 48 h. Data are percentages of cells in each stage of the cell cycle.

FIG. 8.

(A and B) Levels of IL-2 and IL-4 secreted by splenocytes of wild-type and type I^−/− HPRT ^−/0 mice. Splenocytes from two wild-type (wt-1 and wt-2) and two IMPDH type I^−/− HPRT^−/0 (Mut-1 and Mut-2) animals were stimulated with anti-CD3 plus anti-CD28 antibodies. Culture supernatants were collected at the indicated times and measured for the presence of IL-2 (A) and IL-4 (B). Enzyme-linked immunosorbent assays were performed in duplicate. (C and D) Splenocytes from wild-type (wt) mice were activated by antibodies in the absence (solid circles) or presence of 0.2 μM MPA (M0.2) (open circles) or 0.5 μM MPA (M0.5) (solid inverted triangles), and levels of IL-2 (C) and IL-4 (D) secretion were determined in duplicate.

FIG. 9.

Effects of loss of IMPDH and HPRT activities on the cytolytic activities of cytotoxic T lymphocytes. The functions of cytotoxic T lymphocytes from wild-type (100%), IMPDH type I^−/− (n = 3) (A), type I^−/− HPRT^−/0 (n = 5) (B), or type I^−/− type II^+/− (n = 6) (C) mice were analyzed in ⁵¹Cr release assays. Data are means ± standard deviations. Assays were performed in triplicate.