Tdrd3-null mice show post-transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity.

The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex that can alter both DNA and RNA topology in animals. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impaired cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal short-term memory and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we generated a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, olfaction, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive and psychiatric disorders.

Tmem161a regulates bone formation and bone strength through the P38 MAPK pathway.

Bone remodeling is an extraordinarily complex process involving a variety of factors, such as genetic, metabolic, and environmental components. Although genetic factors play a particularly important role, many have not been identified. In this study, we investigated the role of transmembrane 161a (Tmem161a) in bone structure and function using wild-type (WT) and Tmem161a-depleted (Tmem161aGT/GT) mice. Mice femurs were examined by histological, morphological, and bone strength analyses. Osteoblast differentiation and mineral deposition were examined in Tmem161a-overexpressed, -knockdown and -knockout MC3T3-e1 cells. In WT mice, Tmem161a was expressed in osteoblasts of femurs; however, it was depleted in Tmem161aGT/GT mice. Cortical bone mineral density, thickness, and bone strength were significantly increased in Tmem161aGT/GT mice femurs. In MC3T3-e1 cells, decreased expression of alkaline phosphatase (ALP) and Osterix were found in Tmem161a overexpression, and these findings were reversed in Tmem161a-knockdown or -knockout cells. Microarray and western blot analyses revealed upregulation of the P38 MAPK pathway in Tmem161a-knockout cells, which referred as stress-activated protein kinases. ALP and flow cytometry analyses revealed that Tmem161a-knockout cells were resistant to oxidative stress. In summary, Tmem161a is an important regulator of P38 MAPK signaling, and depletion of Tmem161a induces thicker and stronger bones in mice.

Radiocesium mobility in different parts of the two major tree species in Fukushima.

Radiocesium (137Cs) released in the Fukushima Dai-ichi Nuclear Power Plant accident is still cycling in the forest ecosystem. We examined the mobility of 137Cs in the external parts-leaves/needles, branches, and bark-of the two major tree species in Fukushima, Japanese cedar (Cryptomeria japonica) and konara oak (Quercus serrata). This variable mobility will likely lead to spatial heterogeneity of 137Cs and difficulty in predicting its dynamics for decades. We conducted leaching experiments on these samples by using ultrapure water and ammonium acetate. In Japanese cedar, the 137Cs percentage leached from current-year needles was 26-45% (ultrapure water) and 27-60% (ammonium acetate)-similar to those from old needles and branches. In konara oak, the 137Cs percentage leached from leaves was 47-72% (ultrapure water) and 70-100% (ammonium acetate)-comparable to those from current-year and old branches. Relatively poor 137Cs mobility was observed in the outer bark of Japanese cedar and in organic layer samples from both species. Comparison of the results from corresponding parts revealed greater 137Cs mobility in konara oak than in Japanese cedar. We suggest that more active cycling of 137Cs occurs in konara oak.

Tdrd3-null mice show post-transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity.

The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex in animals that can alter the topology of both DNA and RNA. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impairments in cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal shorten-memory and learning, and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we built a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive impairment and psychiatric disorders.

A gain-of-function mutation in microRNA 142 is sufficient to cause the development of T-cell leukemia in mice.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression. MicroRNA expression levels fluctuate, and point mutations and methylation occur in cancer cells; however, to date, there have been no reports of carcinogenic point mutations in miRNAs. MicroRNA 142 (miR-142) is frequently mutated in patients with follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia (CLL), and acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). To understand the role of miR-142 mutation in blood cancers, the CRISPR-Cas9 system was utilized to successfully generate miR-142-55A>G mutant knock-in (Ki) mice, simulating the most frequent mutation in patients with miR-142 mutated AML/MDS. Bone marrow cells from miR-142 mutant heterozygous Ki mice were transplanted, and we found that the miR-142 mutant/wild-type cells were sufficient for the development of CD8+ T-cell leukemia in mice post-transplantation. RNA-sequencing analysis in hematopoietic stem/progenitor cells and CD8+ T-cells revealed that miR-142-Ki/+ cells had increased expression of the mTORC1 activator, a potential target of wild-type miR-142-3p. Notably, the expression of genes involved in apoptosis, differentiation, and the inhibition of the Akt-mTOR pathway was suppressed in miR-142-55A>G heterozygous cells, indicating that these genes are repressed by the mutant miR-142-3p. Thus, in addition to the loss of function due to the halving of wild-type miR-142-3p alleles, mutated miR-142-3p gained the function to suppress the expression of distinct target genes, sufficient to cause leukemogenesis in mice.

SARS-CoV-2 Omicron BA.2.75 Variant May Be Much More Infective than Preexisting Variants Based on In Silico Model.

Previously, we developed a mathematical model via molecular simulation analysis to predict the infectivity of six SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 based on our previous research. We subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the variants that we had analyzed in our previous research. As a result, BA.2.75 has both the highest docking affinity (ratio per Wuhan variant) and the longest evolutionary distance of the S gene from the Wuhan variant. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.

EPLINß Is Involved in the Assembly of Cadherin-catenin Complexes in Osteoblasts and Affects Bone Formation.

Epithelial protein lost in neoplasm (EPLIN) is an actin-associated cytoskeletal protein that plays an important role in epithelial cell adhesion. EPLIN has two isoforms: EPLINα and EPLINß. In this study, we investigated the role of EPLINß in osteoblasts using EPLINß-deficient (EPLINßGT/GT ) mice. The skeletal phenotype of EPLINßGT/GT mice is indistinguishable from the wildtype (WT), but bone properties and strength were significantly decreased compared with WT littermates. Histomorphological analysis revealed altered organization of bone spicules and osteoblast cell arrangement, and decreased alkaline phosphatase activity in EPLINßGT/GT mouse bones. Transmission electron microscopy revealed wider intercellular spaces between osteoblasts in EPLINßGT/GT mice, suggesting aberrant cell adhesion. In EPLINßGT/GT osteoblasts, α- and ß-catenins and F-actin were observed at the cell membrane, but OB-cadherin was localized at the perinuclear region, indicating that cadherin-catenin complexes were not formed. EPLINß knockdown in MC3T3-e1 osteoblast cells showed similar results as in calvaria cell cultures. Bone formation markers, such as RUNX2, Osterix, ALP, and Col1a1 mRNA were reduced in EPLINß knockdown cells, suggesting an important role for EPLINß in osteoblast formation. In conclusion, we propose that EPLINß is involved in the assembly of cadherin-catenin complexes in osteoblasts and affects bone formation.

LincRNA-p21 exon 1 expression correlates with Cdkn1a expression in vivo.

LincRNA-p21 is a long intergenic non-coding RNA (LincRNA) gene reported to activate the transcription of the adjacent Cdkn1a (p21) gene in cis. The importance of the enhancer elements in the LincRNA-p21 gene region has also been reported; however, the involvement of the LincRNA-p21 transcripts in regulating Cdkn1a in vivo is still unclear. In this study, we used a LincRNA-p21-trapped mouse line (LincRNA-p21Gt ) in which ßgeo was inserted into intron 1, and all enhancer elements were retained. In LincRNA-p21Gt/Gt mice, the transcription of LincRNA-p21 was repressed due to the ßgeo sequence, and the expression of exon 1 of LincRNA-p21 was restored through its deletion or replacement by another sequence, and Cdkn1a expression was also upregulated. Furthermore, regardless of the full-length transcripts, the expression of Cdkn1a correlated with the transcription of the exon 1 of LincRNA-p21. This result indicates that the LincRNA-p21 transcripts are not functional, but the transcriptional activity around exon 1 is important for Cdkn1a expression.

Gene trapping reveals a new transcriptionally active genome element: The chromosome-specific clustered trap region.

Nearly half of the human genome consists of repetitive sequences such as long interspersed nuclear elements. The relationship between these repeating sequences and diseases has remained unclear. Gene trapping is a useful technique for disrupting a gene and expressing a reporter gene by using the promoter activity of the gene. The analysis of trapped genes revealed a new genome element-the chromosome-specific clustered trap (CSCT) region. For any examined sequence within this region, an equivalent was found using the BLAT of the University of California, Santa Cruz (UCSC) Genome Browser. CSCT13 mapped to chromosome 13 and contained only three genes. To elucidate its in vivo function, the whole CSCT13 region (1.6 Mbp) was deleted using the CRISPR/Cas9 system in mouse embryonic stem cells, and subsequently, a CSCT13 knockout mouse line was established. The rate of homozygotes was significantly lower than expected according to Mendel's laws. In addition, the number of offspring obtained by mating homozygotes was significantly smaller than that obtained by crossing controls. Furthermore, CSCT13 might have an effect on meiotic homologous recombination. This study identifies a transcriptionally active CSCT with an important role in mouse development.

The lncRNA Caren antagonizes heart failure by inactivating DNA damage response and activating mitochondrial biogenesis.

In the past decade, many long noncoding RNAs (lncRNAs) have been identified and their in vitro functions defined, although in some cases their functions in vivo remain less clear. Moreover, unlike nuclear lncRNAs, the roles of cytoplasmic lncRNAs are less defined. Here, using a gene trapping approach in mouse embryonic stem cells, we identify Caren (short for cardiomyocyte-enriched noncoding transcript), a cytoplasmic lncRNA abundantly expressed in cardiomyocytes. Caren maintains cardiac function under pathological stress by inactivating the ataxia telangiectasia mutated (ATM)-DNA damage response (DDR) pathway and activating mitochondrial bioenergetics. The presence of Caren transcripts does not alter expression of nearby (cis) genes but rather decreases translation of an mRNA transcribed from a distant gene encoding histidine triad nucleotide-binding protein 1 (Hint1), which activates the ATM-DDR pathway and reduces mitochondrial respiratory capacity in cardiomyocytes. Therefore, the cytoplasmic lncRNA Caren functions in cardioprotection by regulating translation of a distant gene and maintaining cardiomyocyte homeostasis.

MicroRNA-142 Critically Regulates Group 2 Innate Lymphoid Cell Homeostasis and Function.

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.

Tamoxifen feeding method is suitable for efficient conditional knockout.

The Cre-driver system is used to generate conditional knockout mice. Tamoxifen inducible Cre-driver mice can be used for spatiotemporal knockout by administration of the drug. A major tamoxifen administration is performed by intraperitoneal administration or oral administration. However, these forced administrations may be damaging to mice. Herein, we have demonstrated an improved method of administering tamoxifen with powdered food to mice. A mouse line expressing the tamoxifen-inducible Cre gene was used ubiquitously in this experiment to evaluate the efficiency of Cre recombination in the whole body. Our method also achieved efficient recombination without causing injury to mice. The X-gal staining intensity of the feeding method was equivalent to that of the intraperitoneal administration method. Furthermore, this method can be used for recombination before birth, or during the fetal period. We recommend researchers to employ this feeding method to administer tamoxifen to minimize the risk of injury to mice.

Vertical distribution of radiocesium concentrations among crown positions and year-to-year variation in four major tree species after the Fukushima Daiichi Nuclear Power Plant accident.

To evaluate the distribution of radiocesium (137Cs) among crown positions in trees after the Fukushima Daiichi Nuclear Power Plant accident, we collected foliage and branch samples from different crown positions of four major tree species (Chamaecyparis obtusa, Cryptomeria japonica, Pinus densiflora, and Quercus serrata) from 2011 to 2019 in northeast Japan. We divided the samples into current-year and more than 1-year-old groups (called old foliage and old branches), which sometimes included directly contaminated parts. The 137Cs activity concentration in dry foliage and branches was measured using a germanium semiconductor detector. There were complex differences in the relative 137Cs activity concentration among species and organ types (i.e., foliage and branches) among crown positions. The relative 137Cs activity concentration in current-year foliage was higher in the upper crowns of C. obtusa, but higher in lower crown positions in C. japonica. No differences among crown positions were observed in P. densiflora and Q. serrata. In current-year branches, the relative 137Cs concentration in Q. serrata was similar among crown positions but higher in the upper crown in P. densiflora. The concentrations in old foliage and old branches in all species tended to be higher in the lower crown. The factors causing these interspecific and organ type differences among crown positions may be related to the organ turnover rate, dilution effect due to different growth rates, and potassium distribution within the crown. No year-to-year variation was observed in most foliage and branches in all species, except for current-year branches of Q. serrata, old foliage in C. japonica and P. densiflora, and old branches in P. densiflora. Our long-term data on the interspecific and inter-organ patterns of contamination, focusing on variation among crown positions and year-to-year trends, might help to improve the estimation of 137Cs deposition and dynamics in polluted forest ecosystems.

The lysophospholipase D enzyme Gdpd3 is required to maintain chronic myelogenous leukaemia stem cells.

Although advanced lipidomics technology facilitates quantitation of intracellular lipid components, little is known about the regulation of lipid metabolism in cancer cells. Here, we show that disruption of the Gdpd3 gene encoding a lysophospholipase D enzyme significantly decreased self-renewal capacity in murine chronic myelogenous leukaemia (CML) stem cells in vivo. Sophisticated lipidomics analyses revealed that Gdpd3 deficiency reduced levels of certain lysophosphatidic acids (LPAs) and lipid mediators in CML cells. Loss of Gdpd3 also activated AKT/mTORC1 signalling and cell cycle progression while suppressing Foxo3a/ß-catenin interaction within CML stem cell nuclei. Strikingly, CML stem cells carrying a hypomorphic mutation of Lgr4/Gpr48, which encodes a leucine-rich repeat (LRR)-containing G-protein coupled receptor (GPCR) acting downstream of Gdpd3, displayed inadequate disease-initiating capacity in vivo. Our data showing that lysophospholipid metabolism is required for CML stem cell maintenance in vivo establish a new, biologically significant mechanism of cancer recurrence that is independent of oncogene addiction.

Phosphorylation of the Anaphase Promoting Complex activator FZR1/CDH1 is required for Meiosis II entry in mouse male germ cell.

FZR1/CDH1 is an activator of Anaphase promoting complex/Cyclosome (APC/C), best known for its role as E3 ubiquitin ligase that drives the cell cycle. APC/C activity is regulated by CDK-mediated phosphorylation of FZR1 during mitotic cell cycle. Although the critical role of FZR1 phosphorylation has been shown mainly in yeast and in vitro cell culture studies, its biological significance in mammalian tissues in vivo remained elusive. Here, we examined the in vivo role of FZR1 phosphorylation using a mouse model, in which non-phosphorylatable substitutions were introduced in the putative CDK-phosphorylation sites of FZR1. Although ablation of FZR1 phosphorylation did not show substantial consequences in mouse somatic tissues, it led to severe testicular defects resulting in male infertility. In the absence of FZR1 phosphorylation, male juvenile germ cells entered meiosis normally but failed to enter meiosis II or form differentiated spermatids. In aged testis, male mutant germ cells were overall abolished, showing Sertoli cell-only phenotype. In contrast, female mutants showed apparently normal progression of meiosis. The present study demonstrated that phosphorylation of FZR1 is required for temporal regulation of APC/C activity at meiosis II entry, and for maintenance of spermatogonia, which raised an insight into the sexual dimorphism of FZR1-regulation in germ cells.

Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs.

Post-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48-50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48-50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model.

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Vertical and seasonal variations in temperature responses of leaf respiration in a Chamaecyparis obtusa canopy.

Leaf respiration (R) is a major component of carbon balance in forest ecosystems. Clarifying the variability of leaf R within a canopy is essential for predicting the impact of global warming on forest productivity and the potential future function of the forest ecosystem as a carbon sink. We examined vertical and seasonal variations in short-term temperature responses of leaf R as well as environmental factors (light and mean air temperature) and physiological factors [leaf nitrogen (N), leaf mass per area (LMA), and shoot growth] in the canopy of a 10-year-old stand of hinoki cypress [Chamaecyparis obtusa (Sieb. et Zucc.) Endl.] in Kyushu, Japan. Leaf respiration rate adjusted to 20 °C (R20) exhibited evident vertical gradients in each season and was correlated with light, LMA and leaf N. In contrast, the temperature sensitivity of leaf R (Q10) did not vary vertically throughout the seasons. Seasonally, Q10 was higher in winter than in summer and was strongly negatively correlated to mean air temperature. A negative correlation of R20 with mean air temperature was also observed for each of the three canopy layers. These results clearly indicate that leaf R was able to adjust to seasonal changes in ambient temperature under field conditions and down-regulate during warmer periods. We also found that the degree of thermal acclimation did not vary with canopy position. Overall, our results suggest that vertical and seasonal variations in temperature responses of leaf R within a hinoki cypress canopy could be predicted by relatively simple parameters (light and temperature). There was an exception of extremely high R20 values in April that may have been due to the onset of shoot growth in spring. Understanding thermal acclimation and variations in leaf R within forest canopies will improve global terrestrial carbon cycle models.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.