RIP1-mediated regulation of lymphocyte survival and death responses

Abstract

RIP1 is an adaptor serine/threonine kinase associated with the signaling complex of death receptors (DRs) including Fas, TNFR1, and TRAIL-Rs which can initiate apoptosis. While DRs are dispensable throughout development, RIP1 deletion results in perinatal lethality. The developmental defect caused by absence of RIP1 remains unexplained. In previous studies, RIP1-deficient hematopoietic progenitors failed to reconstitute the T cell compartment and our recent data indicate a new role for RIP1 in TCR-induced activation of the pro-survival NF-κB pathway. Here, we show that RIP1 is also critical for B cell development. In addition, RIP1(-/-) B cells stimulated through LPS/TLR4 are impaired in NF-κB activation but have no major defect in the Akt pathway. Recently, RIP1 has also emerged as a critical player in necrosis-like death, necroptosis, in various cell lines. We have demonstrated that RIP1 deficiency can reverse the embryonic and T cell proliferation defects in mice lacking FADD, a caspase adaptor protein, which indicates a potential role for RIP1 in mediating in vivo necroptosis. We provide an overview and discussion of the accumulating data revealing insights into the diverse functions of RIP1 in survival and death signaling in lymphocytes.

Fig 1. RIP1 protein structure and expression

A, A diagram of the human RIP1 protein is shown, which consists of an amino terminal serine/threonine kinase domain (KD) and carboxy terminal death domain (DD). The intermediate domain between the KD and DD contains a RIP homotypic interaction motif (RHIM), a caspase 8 cleavage site (D324), and a ubiquitinylation site (K377). B, Western blotting analysis shows the expression of the RIP1 protein in wild type E17.5 mouse embryos, adult mouse tissues, and purified lymphocytes. The RIP1^−/− mutant embryo was used as a control.

Fig. 2. RIP1 deficiency results in a time-dependent decline in the size of the peripheral B cell pool

RIP1^−/− fetal liver cells were adoptively transferred to immunodeficient NSG recipient mice irradiated (200 RAD), as described previously (25, 77). At 7 or 12 weeks post transfer, the resulting hematopoietic chimeras were used to prepare single cell suspensions from the specified lymphoid organs. Reconstitution of the peripheral B cell compartment was determined by flow cytometric analysis of the IgM⁺ and/or IgD⁺ populations in the indicated organs. Gates were set to indicate immature (IgD^loIgM⁺) and mature (IgD⁺IgM⁺) B cell populations. The numbers are percentages of the gated populations. Data are representative of 5 independent experiments. RIP1^+/+ fetal liver cell-transferred recipients were used as a control.

Fig. 3. RIP1 deficiency leads to impaired B lineage cell development in the bone marrow

At 7 or 12 weeks post transfer of RIP1^−/− or RIP1^+/+ fetal liver cells, bone marrow cells were isolated from NSG recipients and were stained for B220, CD43, IgM, and IgD, and analyzed by flow cytometry. Gates were set to indicate pro-B (CD43⁺B220^lo), pro/pre-B (B220^loIgM⁻), immature B (IgM⁺IgD⁻), and recirculating mature B cell (IgM⁺IgD⁺) populations in NSG recipients transferred with RIP1^−/− mutant and RIP1^+/+ wild type control fetal liver cells. The percentages of each gated populations are shown. The data are representative of 5 independent experiments.

Fig. 4. Selective defects in TLR-induced proliferation in RIP1^−/− B cells

B cells were isolated from the spleen and lymph nodes of NSG recipients transferred with RIP1^+/+ (open bars) or RIP1^−/− (filled bars) fetal liver cells, and stimulated with the indicated agonists. Proliferation was determined by the incorporation of radioactivity of [³H] thymidine into activated B cells (cpm, counts per minute). Error bars are ± SEM of triplicates. The data are representative of 4 independent experiments.

Fig. 5. LPS-induced signaling in RIP1^−/− B cells

Peripheral B cells purified from NSG recipients transferred with RIP1^−/− or RIP1^+/+ fetal liver cells (A and B) or MEFs of the indicated genotypes (C) were stimulated with LPS (10 μg/ml), and western blotting analysis was performed using Abs specific for p-p65 NF-κB, p-IκB, IκB, and p-Akt. D, The CD69 and CD86 activation markers on B cells were analyzed by flow cytometry 12 hours post stimulation with LPS (1 μg/ml). Unstimulated B cells were used as a control. Data shown are representative of at least 4 independent experiments.