Efficient Inhibition of Collagen-Induced Platelet Activation and Adhesion by LAIR-2, a Soluble Ig-Like Receptor Family Member

LAIR-1 (Leukocyte Associated Ig-like Receptor -1) is a collagen receptor that functions as an inhibitory receptor on immune cells. It has a soluble family member, LAIR-2, that also binds collagen and can interfere with LAIR-1/collagen interactions. Collagen is a main initiator for platelet adhesion and aggregation. Here, we explored the potential of soluble LAIR proteins to inhibit thrombus formation in vitro. LAIR-2/Fc but not LAIR-1/Fc inhibited collagen-induced platelet aggregation. In addition, LAIR-2/Fc also interfered with platelet adhesion to collagen at low shear rate (300 s−1; IC50 = 18 µg/ml) and high shear rate (1500 s−1; IC50 = 30 µg/ml). Additional experiments revealed that LAIR-2/Fc leaves interactions between collagen and α2β1 unaffected, but efficiently prevents binding of collagen to Glycoprotein VI and von Willebrand factor. Thus, LAIR-2/Fc has the capacity to interfere with platelet-collagen interactions mediated by Glycoprotein VI and the VWF/Glycoprotein Ib axis

Varicella vaccination in HIV-1-infected children after immune reconstitution

BACKGROUND: HIV-1-infected children have an increased risk of severe chickenpox. However, vaccination is not recommended in severely immunocompromised children. OBJECTIVE: Can the live-attenuated varicella zoster virus (VZV) Oka strain be safely and effectively given to HIV-1-infected children despite previously low CD4 T-cell counts? METHODS: VZV vaccine was administered twice to 15 VZV-seronegative HIV-1-infected children when total lymphocyte counts were greater than 700 lymphocytes/microl, and six HIV-negative VZV-seronegative siblings. Weekly clinical follow-up and sampling were performed. RESULTS: None of the children developed any clinical symptom or serious adverse reaction after immunization. Only nine (60%) of the HIV-1-infected children had VZV-specific antibodies after two immunizations, whereas 100% of the siblings seroconverted. Age at baseline was negatively correlated with the VZV IgG titre at 6 weeks after the second vaccination in HIV-1-infected children. VZV-specific antibody titres after two immunizations were at a similar level to those found after wild-type infection in non-vaccinated HIV-1-infected patients, but significantly lower than in HIV-negative siblings. Importantly, VZV-specific T-cell responses increased after vaccination and were comparable in both groups over time. Documented wild-type VZV contact in three vaccinated patients did not result in breakthrough infections. CONCLUSION: VZV vaccination of previously immunocompromised HIV-1-infected children was safe. Vaccination induced specific immune responses in some of the vaccinated HIV-1-infected children, suggesting that previously immunocompromised individuals are protected against severe forms of varicell

Shift of CMV-specific CD4+ T-cells to the highly differentiated CD45RO-CD27- phenotype parallels loss of proliferative capacity and precedes progression to HIV-related CMV end-organ disease

To identify factors related to progression to CMV end-organ disease, cytokine production, proliferative capacity and phenotype of CMV-specific CD4(+) T-cells were analysed longitudinally. Numbers of IFNgamma(+)CD4(+) and IFNgamma(+)IL-2(+)CD4(+) T-cells tended to decrease in individuals progressing to AIDS with CMV end-organ disease (AIDS-CMV), whereas they remained detectable in long-term asymptomatics (LTAs) and progressors to AIDS with opportunistic infections (AIDS-OI). In parallel, CMV-specific proliferative capacity was lost in AIDS-CMV. Initially, the majority of the CMV-specific IFNgamma(+)CD4(+) T-cells were of the CD45RO(+)CD27(-) subset, but during progression to AIDS-CMV a shift in phenotype to the CD45RO(-)CD27(-) subset was observed. Our data indicate that a decrease in CMV-specific cytokine production and proliferative capacity precedes progression to AIDS-CMV. Accumulation of CD4(+) T-cells with a CD45RO(-)CD27(-) phenotype suggests that persistent antigen exposure drives differentiation of CMV-specific CD4(+) T-cells towards a poorly proliferating, and highly differentiated "effector" subset, which eventually fails to produce IFNgamma in patients developing AIDS-CM

LAIR-2/Fc inhibits adhesion of platelets to collagen under flow conditions.

<p><i>Panel</i> A: Collagen type III-coated coverslips were perfused with whole blood in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. Representative pictures are shown. Perfusion was performed at a shear rate of 300 s⁻¹ (upper panels) or 1500 s⁻¹ (lower panels). Percentages below figures indicate percentage surface-coverage for each individual photo. <i>Panels</i> B & C: Quantitative representation of surface coverage of collagen type III-coated coverslips that were perfused in the absence or presence of 100 µg/ml soluble LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc at 300 s⁻¹ (<i>panel</i> B) or 1500 s⁻¹ (<i>panel</i> C). Data represent mean±SD of three independent perfusions. <i>Panel</i> D: Dose dependent inhibition of surface platelet coverage in the presence of LAIR-2/Fc at shear rates of 300 s⁻¹ (open symbols) or 1500 s⁻¹ (closed symbols). *: <i>p</i><0.005.</p

LAIR-2/Fc interferes with VWF binding to collagen.

<p>Microtiter wells were coated with 50 µg/ml collagen I (<i>panel</i> A) or collagen III (<i>panel</i> B) and subsequently incubated with 0.1 µg/ml purified plasma-derived VWF in the presence of increasing concentrations of LAIR-1/Fc (open circles), LAIR-2/Fc (closed circles) or SIRL-1/Fc (squares). VWF binding was detected with horseradish-conjugated polyclonal anti-human VWF antibodies and 3-3′-5-5′-tetramethylbenzidine. Data are representative of 3 independent experiments.</p

The CD200-CD200 receptor inhibitory axis controls arteriogenesis and local T lymphocyte influx.

The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd200-/- and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd200-/- mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd200-/- mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd200-/- compared to wildtype. CD200R agonist treatment was performed in male C57Bl/6J mice to validate the role of the CD200-CD200R axis in arteriogenesis. CD200R agonist treatment after unilateral femoral artery ligation resulted in a significant decrease in vessel geometry, perfusion recovery and T lymphocyte influx at day 7 compared to isotype treatment. In this study, we show a causal role for the CD200-CD200R inhibitory axis in arteriogenesis in a murine hindlimb ischemia model. Lack of CD200R signaling is accompanied by increased T lymphocyte recruitment to the collateral vasculature and results in enlargement of preexisting collateral arteries

Platelets do not express LAIR-1 or LAIR-2.

<p><i>Panel</i> A: Flowcytometric analysis of washed platelets (2×10E5/µl), unstimulated or stimulated for 5 min at RT with 10 µM TRAP or 5000 nM PMA. Upper panels represent staining for CD62L, lower panels represent LAIR-1 staining. <i>Panel</i> B: Western blots containing whole platelet lysates and controls were incubated with antibodies against LAIR-1 (<i>lanes 1–3</i>), LAIR-2 (<i>lanes 4–8</i>) or GpVI (<i>lane 9</i>). <i>Lane 1:</i> purified LAIR-1/Fc; <i>lane 2:</i> purified LAIR-2/Fc; <i>lane 3:</i> whole platelet lysate; <i>lane 4:</i> purified LAIR-1/Fc; <i>lane 5:</i> purified LAIR-2/Fc; <i>lane 6:</i> conditioned medium of non-transfected human 293T cells; <i>lane 7:</i> conditioned medium of human 293T cells secreting LAIR-2; <i>lane 8:</i> whole platelet lysate; <i>lane 9:</i> whole platelet lysate.</p

LAIR-2/Fc inhibits collagen but not TRAP-induced platelet aggregation.

<p>Aggregation of platelet rich plasma (PRP) in response to collagen was measured using an optical aggregometer. <i>Panel</i> A: Platelet aggregation in response to 50 µM TRAP alone (PBS) or in the presence of 100 µg/ml LAIR-1/Fc, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> B: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 100 µg/ml LAIR-1/F, LAIR-2/Fc or SIRL-1/Fc. <i>Panel</i> C: Platelet aggregation in response to collagen (1 µg/ml) alone (PBS) or in the presence of 0.01 µg/ml, 0.1 µg/ml or 1.0 µg/ml LAIR-2/Fc. <i>Panel</i> D: Platelet aggregation in response to 0.5 µg/ml, 1 µg/ml, 2 µg/ml or 4 µg/ml collagen in the presence of 1.0 µg/ml LAIR-2/Fc.</p