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Conventional dendritic cells (cDCs) include functionally and phenotypically diverse populations, such as cDC1s and cDC2s. The latter population has been variously subdivided into Notch-dependent cDC2s, KLF4-dependent cDC2s, T-bet + cDC2As and T-bet - cDC2Bs, but it is unclear how all these subtypes are interrelated and to what degree they represent cell states or cell subsets. All cDCs are derived from bone marrow progenitors called pre-cDCs, which circulate through the blood to colonize peripheral tissues. Here, we identified distinct mouse pre-cDC2 subsets biased to give rise to cDC2As or cDC2Bs. We showed that a Siglec-H + pre-cDC2A population in the bone marrow preferentially gave rise to Siglec-H - CD8α + pre-cDC2As in tissues, which differentiated into T-bet + cDC2As. In contrast, a Siglec-H - fraction of pre-cDCs in the bone marrow and periphery mostly generated T-bet - cDC2Bs, a lineage marked by the expression of LysM. Our results showed that cDC2A versus cDC2B fate specification starts in the bone marrow and suggest that cDC2 subsets are ontogenetically determined lineages, rather than cell states imposed by the peripheral tissue environment., (© 2024. The Author(s).)

Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Plasmacytoid dendritic cells (pDCs) are the main source of type I interferon (IFN-I) during viral infections. Their other functions are debated, due to a lack of tools to identify and target them in vivo without affecting pDC-like cells and transitional DCs (tDCs), which harbor overlapping phenotypes and transcriptomes but a higher efficacy for T cell activation. In the present report, we present a reporter mouse, pDC-Tom, designed through intersectional genetics based on unique Siglech and Pacsin1 coexpression in pDCs. The pDC-Tom mice specifically tagged pDCs and, on breeding with Zbtb46 GFP mice, enabled transcriptomic profiling of all splenic DC types, unraveling diverging activation of pDC-like cells versus tDCs during a viral infection. The pDC-Tom mice also revealed initially similar but later divergent microanatomical relocation of splenic IFN + versus IFN - pDCs during infection. The mouse models and specific gene modules we report here will be useful to delineate the physiological functions of pDCs versus other DC types., (© 2023. The Author(s).)

Objectives: To better understand how immune responses may be harnessed against breast cancer, we investigated which immune cell types and signalling pathways are required for spontaneous control of a mouse model of mammary adenocarcinoma., Methods: The NOP23 mammary adenocarcinoma cell line expressing epitopes derived from the ovalbumin model antigen is spontaneously controlled when orthotopically engrafted in syngeneic C57BL/6 mice. We combined this breast cancer model with antibody-mediated depletion of lymphocytes and with mutant mice affected in interferon (IFN) or type 1 conventional dendritic cell (cDC1) responses. We monitored tumor growth and immune infiltration including the activation of cognate ovalbumin-specific T cells., Results: Breast cancer immunosurveillance required cDC1, NK/NK T cells, conventional CD4 + T cells and CD8 + cytotoxic T lymphocytes (CTLs). cDC1 were required constitutively, but especially during T-cell priming. In tumors, cDC1 were interacting simultaneously with CD4 + T cells and tumor-specific CTLs. cDC1 expression of the XCR1 chemokine receptor and of the T-cell-attracting or T-cell-activating cytokines CXCL9, IL-12 and IL-15 was dispensable for tumor rejection, whereas IFN responses were necessary, including cDC1-intrinsic signalling by STAT1 and IFN-γ but not type I IFN (IFN-I). cDC1 and IFNs promoted CD4 + and CD8 + T-cell infiltration, terminal differentiation and effector functions. In breast cancer patients, high intratumor expression of genes specific to cDC1, CTLs, CD4 + T cells or IFN responses is associated with a better prognosis., Conclusion: Interferons and cDC1 are critical for breast cancer immunosurveillance. IFN-γ plays a prominent role over IFN-I in licensing cDC1 for efficient T-cell activation., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN-I). What other functions pDCs exert in vivo during viral infections is controversial, and more studies are needed to understand their orchestration. In the present study, we characterize in depth and link pDC activation states in animals infected by mouse cytomegalovirus by combining Ifnb1 reporter mice with flow cytometry, single-cell RNA sequencing, confocal microscopy and a cognate CD4 T cell activation assay. We show that IFN-I production and T cell activation were performed by the same pDC, but these occurred sequentially in time and in different micro-anatomical locations. In addition, we show that pDC commitment to IFN-I production was marked early on by their downregulation of leukemia inhibitory factor receptor and was promoted by cell-intrinsic tumor necrosis factor signaling. We propose a new model for how individual pDCs are endowed to exert different functions in vivo during a viral infection, in a manner tightly orchestrated in time and space.

Plasmacytoid dendritic cells (pDC) are the major source of type I interferons (IFN-I) during viral infections, in response to triggering of endosomal Toll-like receptors (TLRs) 7 or 9 by viral single-stranded RNA or unmethylated CpG DNA, respectively. Synthetic ligands have been used to disentangle the underlying signaling pathways. The adaptor protein AP3 is necessary to transport molecular complexes of TLRs, synthetic CpG DNA, and MyD88 into endosomal compartments allowing interferon regulatory factor 7 (IRF7) recruitment whose phosphorylation then initiates IFN-I production. High basal expression of IRF7 by pDC and its further enhancement by positive IFN-I feedback signaling appear to be necessary for robust cytokine production. In contrast, we show here that in vivo during mouse cytomegalovirus (MCMV) infection pDC produce high amounts of IFN-I downstream of the TLR9-to-MyD88-to-IRF7 signaling pathway without requiring IFN-I positive feedback, high IRF7 expression, or AP3-driven endosomal routing of TLRs. Hence, the current model of the molecular requirements for professional IFN-I production by pDC, established by using synthetic TLR ligands, does not strictly apply to a physiological viral infection., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.

Type I interferons (IFNs) are central to antiviral defense, but how they orchestrate immune cell function is incompletely understood. We determined that IFNs produced during murine cytomegalovirus (MCMV) infection differentially affect dendritic cells (DCs) and natural killer (NK) cells. IFNs induce cell-intrinsic responses in DCs, activating antiproliferative, antiviral, and lymphocyte-activating gene networks, consistent with high activity of the transcription factor STAT1 in these cells. By comparison, NK cells exhibit lower STAT1 expression and reduced IFN responsiveness. Rather, IFNs indirectly affect NK cells by inducing IL-15, which activates the transcription factor E2F and stimulates genes promoting cell expansion. IFN cell-intrinsic responses are necessary in DCs, but not NK cells, for MCMV resistance. Thus, sensitivity to IFN-induced cytokines and differences in IFN receptor signaling program immune cells to mount distinct responses that promote viral control., (Copyright © 2012 Elsevier Inc. All rights reserved.)

As initially demonstrated with murine cytomegalovirus (MCMV), plasmacytoid dendritic cells (pDCs) are the major source of IFN-alpha/beta in response to a variety of viruses in vivo. However, contradictory results have been obtained pertaining to the mechanisms promoting IFN-alpha/beta production by pDCs in response to MCMV. In this study we show that TLR7 and TLR9 exert redundant functions for IFN-alpha/beta, IL-12p40, and TNF-alpha production by pDCs in vivo during MCMV infection. In contrast, we confirm that systemic production of IL-12p70 strictly depends on TLR9. The combined loss of TLR7 and TLR9 recapitulates critical features of the phenotype of MyD88-deficient mice, including a dramatic decrease in systemic IFN-alpha/beta levels, an increase in viral load, and increased susceptibility to MCMV-induced mortality. This is the first demonstration of the implication of TLR7 in the recognition of a DNA virus.

Background: Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8alpha in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes., Results: We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively., Conclusion: Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

Plasmacytoid dendritic cells (pDCs) are an important source of IFN-alpha/beta in response to a variety of viruses in vivo, including murine cytomegalovirus (MCMV). However, the respective contributions of various infected organs, and within these of pDCs, conventional dendritic cells and other cells, to the systemic production of IFN-alpha/beta or other innate cytokines during viral infections in vivo is largely unknown. Whether a specialization of pDC subsets in the production of different patterns of innate cytokines exists in vivo in response to a viral infection has not been investigated. Here, by analyzing for the first time directly ex vivo, at the single-cell level, the simultaneous production of up to three cytokines in pDCs isolated from MCMV-infected mice, we show that (i) pDCs are the quasi-exclusive source of IFN-alpha/beta, IL-12 and tumor necrosis factor (TNF)-alpha, early during MCMV infection, in two immunocompetent mouse lines and with two viral strains, (ii) pDC activation for IFN-alpha/beta production is organ specific and (iii) a significant proportion of pDCs simultaneously produce IFN-alpha/beta, TNF-alpha and IL-12, although TNF-alpha and IFN-alpha/beta appear more often co-expressed with one another than each of them with IL-12. Altogether, these results show a broad and non-redundant role of pDCs in early innate detection of, and defense against, viral infection. The data also show differences in the responsiveness of pDCs from different tissues and suggest distinct molecular requirements for pDC production of various cytokines. These observations must be taken into account when designing new antiviral vaccination strategies aimed at harnessing pDC responses.

Understanding the mechanisms that help promote protective immune responses to pathogens is a major challenge in biomedical research and an important goal for the design of innovative therapeutic or vaccination strategies. While natural killer (NK) cells can directly contribute to the control of viral replication, whether, and how, they may help orchestrate global antiviral defense is largely unknown. To address this question, we took advantage of the well-defined molecular interactions involved in the recognition of mouse cytomegalovirus (MCMV) by NK cells. By using congenic or mutant mice and wild-type versus genetically engineered viruses, we examined the consequences on antiviral CD8 T cell responses of specific defects in the ability of the NK cells to control MCMV. This system allowed us to demonstrate, to our knowledge for the first time, that NK cells accelerate CD8 T cell responses against a viral infection in vivo. Moreover, we identify the underlying mechanism as the ability of NK cells to limit IFN-alpha/beta production to levels not immunosuppressive to the host. This is achieved through the early control of cytomegalovirus, which dramatically reduces the activation of plasmacytoid dendritic cells (pDCs) for cytokine production, preserves the conventional dendritic cell (cDC) compartment, and accelerates antiviral CD8 T cell responses. Conversely, exogenous IFN-alpha administration in resistant animals ablates cDCs and delays CD8 T cell activation in the face of NK cell control of viral replication. Collectively, our data demonstrate that the ability of NK cells to respond very early to cytomegalovirus infection critically contributes to balance the intensity of other innate immune responses, which dampens early immunopathology and promotes optimal initiation of antiviral CD8 T cell responses. Thus, the extent to which NK cell responses benefit the host goes beyond their direct antiviral effects and extends to the prevention of innate cytokine shock and to the promotion of adaptive immunity.

The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.

APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.

The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.

We have determined the sequence of the human immunodeficiency virus type 1 (HIV-1) vif genes from a cohort of 42 long-term nonprogressors (LTNP) and compared these sequences to those of 8 late progressors. The coding potential of the vif open reading frame directly derived by nested PCR from uncultured peripheral blood mononuclear cell DNA was conserved in all 50 individuals. The nucleotide distances between vif sequences were not significantly different between LTNP and late progressors, indicating similar selections of viruses within both types of long-term HIV-1-infected subjects. However, a statistically significant correlation between an amino acid signature at position 132 of Vif and the viral load was found within LTNP. Namely, amino acid Ser was associated with low viral load and amino acid Arg with high viral load. This signature was also observed when LTNP with low viral load were compared to progressors. The Ser132 signature was introduced in place of Arg132 present in the HIV-1 YU-2 Vif prototype into chimeric viruses to assess the impact of Vif signature on the virus. While the replication properties in the SupT1 cell line were unmodified, the mutagenized virus revealed a fivefold decreased replication in activated PBMC, suggesting a possible role of this Vif signature for viral production in vivo., (Copyright 2000 Academic Press.)

It has been shown previously that proteinase 3 (PR3), a neutrophil intracellular protease that is the main antigen of antineutrophil cytoplasm (ANCA) autoantibodies, is present on the plasma membrane of a subset of freshly isolated neutrophils. This study shows that the size of this subset of membrane PR3-positive (mPR3+) neutrophils is a stable feature of a given individual, most likely genetically controlled. It ranges from 0 to 100% of neutrophils and allows us to define a new polymorphism in the healthy population, with three discrete phenotypes corresponding respectively to less than 20% mPR3 + neutrophils (mPR3low) or to a mean percentage of 47% (mPR3intermediate) and 71.5% (mPR3high) mPR3+ neutrophils. The frequency of the mPR3high phenotype was significantly increased in patients with ANCA-associated vasculitis (85% versus 55% in healthy subjects). The percentage of mPR3+ neutrophils was not affected by disease activity, relapses, or therapy, and did not reflect in vivo cell activation. In addition, mPR3+ phenotypes were normally distributed in cystic fibrosis patients, indicating that infection and/or inflammation per se do not lead to a high percentage of mPR3+ neutrophils. The frequency of the mPR3high phenotype was not related to anti-PR3 autoimmunization, since it was increased in vasculitic patients regardless of the ANCA specificity (anti-PR3, anti-myeloperoxidase, or unknown). Interestingly, the frequency of the mPR3high phenotype was also increased in patients with rheumatoid arthritis. It was normal in type I-diabetes, a T cell-dependent autoimmune disease. It is proposed here that a high proportion of membrane PR3-positive neutrophils could favor the occurrence or the progression of chronic inflammatory diseases.

Blood phagocyte opsonin receptor CR1 (CD35) and CR3 (CD11b) functions were examined in cystic fibrosis (CF) patients with endobronchial Staphylococcus aureus or Pseudomonas aeruginosa chronic infection, CF patients without infection, heterozygous, non-CF patients with chronic pulmonary infection, and healthy controls. Circulating and platelet-activating factor (PAF)-primed phagocyte luminol luminescence responses to complement-opsonized zymosan were increased in both groups of infected CF and non-CF children relative to uninfected CF children and healthy control children and adults. The ratio between circulating and PAF-primed phagocyte responses was significantly elevated in all children with CF, and in these, the ratio could serve as an indicator of response to antibiotic treatment. The ratios of circulating and PAF-primed phenotypic expression for CR1, CR3, and FcgammaRIII (CD16), but not FcgammaRII (CD32), correlated with the functional ratios. Phagocyte opsonin receptor response capacity might be used for evaluation of inflammation and infection in CF patients.

This study demonstrates (by flow cytometry and immunoprecipitation after cell surface radiolabeling and by using monoclonal antibodies to alpha v, beta 3, and alpha v beta 3 and alpha v beta 5 complexes) that alpha v beta 3, the vitronectin receptor, and alpha v beta 5 are expressed in vitro on cultured human mesangial cells (HMC) of the 5th to 8th passages. Antibodies to alpha v, beta 3 and alpha v beta 3 respectively precipitated an alpha beta heterodimer with molecular weights of 140 and 97 kDa. We analyzed the role of the various integrins in HMC interactions with vitronectin, and with fibronectin and von Willebrand factor (vWf), which are synthetized respectively by mesangial and endothelial cells. Cell adhesion increased in a dose dependent manner with the concentration of plastic-coated matrix protein and vWf. Inhibition of cell attachment with monoclonal antibodies to integrins indicated that HMC adhesion to vWf primarily involves alpha v beta 3, and that alpha v beta 5 may also contribute to cell binding to vWf. Adhesion to vitronectin involves both alpha v beta 3 and alpha v beta 5 complexes. In contrast, adhesion to fibronectin was not affected by monoclonal antibodies to alpha v beta 3 and alpha v beta 5 complexes. We propose that integrins alpha v beta 3 and alpha v beta 5, present on HMC, could mediate an interaction between mesangial and endothelial cells by binding to vWf, released at the basal site of endothelial cells.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

Leukosialin (CD43) is a sialic acid-rich molecule with a relative molecular mass (M(r)) of 140,000 highly represented on polymorphonuclear neutrophils (PMN) and on most leukocytes. One of its functions may be to prevent nonspecific cell-to-cell interactions through negative charge repulsions. As tested by immunofluorescence, neutrophil CD43 membrane expression was shown to decrease by up to 80% upon cell activation by phorbol myristate acetate (10 ng/ml) or by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 10(-6) M) in the presence of cytochalasin B. The kinetic of this decrease paralleled that of CD11b up-regulation. FMLP alone, tumor necrosis factor (TNF-alpha), lipopolysaccharide and granulocyte macrophage colony-stimulating factor had moderate or insignificant effects, while inducing striking CD11b up-regulation. Cell priming with TNF-alpha followed by FMLP stimulation resulted in up to 40% decrease of CD43 expression. Anti-CD43 mAb immunoprecipitated three fragments of M(r) 130,000, 49,000 and 34,000 from the cell-free supernatant of activated neutrophils, suggesting that CD43 is released from the membrane by proteolysis. Indeed, the decrease in CD43 expression was inhibited by phenylmethanesulfonylfluoride (PMSF). Homotypic aggregation of activated PMN was also inhibited by PMSF and could result, at least in part, from the shedding of CD43. The shedding of such a strongly anionic and major membrane protein should drastically modify PMN surface charge and may allow previously hindered interactions by exposing new adhesion molecules.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.

A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.

Acetyl-CoA acetyltransferase (1-O-alkyl-sn-glycero-3-phosphocholine) is a key enzyme in paf-acether biosynthesis. Its immunological activation as related to paf-acether formation was investigated in mast cells derived from mouse bone marrow. The action of ketotifen, a prophylactic anti-asthma drug, on the antigen-induced activation of acetyltransferase and on the release of paf-acether and beta-hexosaminidase was studied in mast cells. Mast cells were sensitized with dinitrophenyl-specific monoclonal IgE and preincubated for 15 min at 37 degrees C with various concentrations of ketotifen or vehicle prior to challenge with dinitrophenyl coupled to bovine serum albumin (40 ng/ml). Acetyltransferase activity and mediator formation and release were measured. Ketotifen inhibited dose dependently the antigen-induced paf-acether formation and release, beta-hexosaminidase release and acetyltransferase stimulation. The IC50 values were 20.0 +/- 4.4, 11.8 +/- 6.2, 8.8 +/- 3.8 and 20.5 +/- 3.4 microM (mean +/- S.E.M., n = 3) respectively. Mast cells were preincubated with 50 microM ketotifen for 15 min at 37 degrees C then washed prior to antigen challenge. The release of paf-acether and beta-hexosaminidase and the stimulation of acetyltransferase were inhibited by 90.0 +/- 15.0, 91.0 +/- 15.0 and 88.0 +/- 11.0% (n = 3) respectively. In addition, Ca2+ entry was inhibited by 100% as assessed from Quin-2 fluorescence. Thus, the release of a preformed granular enzyme beta-hexosaminidase is inhibited by ketotifen together with the enzymatic formation of a newly formed mediator.(ABSTRACT TRUNCATED AT 250 WORDS)

It is well documented that ether-lipids, especially the 1-0-alkyl-2-0-acyl-sn-glycero-3-phosphocholine, can serve as precursors in paf-acether (platelet-activating factor) biosynthesis. This study was undertaken to determine the amount of these compounds in two cell types which are good producers of paf-acether: human neutrophils and rabbit platelets. The method of phospholipid analysis was based on selective destruction of diacyl molecules by lipase from guinea pig pancreas and of plasmalogens by acidolysis; phospholipids were then separated by bidimensional thin-layer chromatography. In platelets as in neutrophils, the major phospholipids were choline (37%) and ethanolamine (30 and 32%, respectively) phosphoglycerides and sphingomyelin (18 and 16%, respectively). In rabbit platelets the 1-alkyl molecules represented 15% of the choline class (0.7 nmol/10(7) cells). Neutrophils differed strikingly from platelets by the high level of these molecules which constituted 50% of the choline class (16.3 nmol/10(7) cells). It therefore appears that in the two cell types, the amount of 1-0-alkyl-2-0-acyl-sn-glycero-3-phosphocholine largely exceeded that would be necessary for paf-acether biosynthesis.

We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.