Targeting Mac-1-mediated leukocyte–RBC interactions uncouples the benefits for acute vaso-occlusion and chronic organ damage
2016; Elsevier BV; Volume: 44; Issue: 10 Linguagem: Inglês
10.1016/j.exphem.2016.06.252
ISSN1873-2399
AutoresGrace Chen, Jungshan Chang, Dachuan Zhang, Sandra Pinho, Jung-Eun Jang, Paul S. Frenette,
Tópico(s)Erythrocyte Function and Pathophysiology
Resumo•SCD.Mac-1−/− mice are protected from inflammation-triggered acute vaso-occlusion.•Genetic deletion of Mac-1 integrin does not benefit chronic organ damage in SCD.Mac-1−/− mice.•Distinct mechanisms contribute to acute vaso-occlusion and chronic organ damage in SCD. Vaso-occlusive crisis (VOC) is one of the most common complications of sickle cell disease (SCD). Recurrent episodes of VOC may cause irreversible organ damage and early mortality in patients with SCD. Emerging evidence suggests that VOC arises from a complex cascade that involves interactions among multiple blood and endothelial cells in the vasculature. Previous studies have identified αMβ2 integrin (Mac-1) as a critical molecule that mediates heterotypic interactions between red blood cells (RBCs) and adherent leukocytes and promotes VOC in SCD mice. Here, we show that RBC–leukocyte interactions are significantly diminished in Mac-1-deficient SCD mice, leading to an improvement of blood flow rates and prolonged survival time in a tumor necrosis factor-alpha and surgical-trauma-induced VOC model. Mac-1-deletion, however, was not sufficient to reduce SCD-related chronic organ damage. Our results thus suggest uncoupled mechanisms between acute VOC benefits and the long-term complications of SCD that should be considered in future clinical trials. Vaso-occlusive crisis (VOC) is one of the most common complications of sickle cell disease (SCD). Recurrent episodes of VOC may cause irreversible organ damage and early mortality in patients with SCD. Emerging evidence suggests that VOC arises from a complex cascade that involves interactions among multiple blood and endothelial cells in the vasculature. Previous studies have identified αMβ2 integrin (Mac-1) as a critical molecule that mediates heterotypic interactions between red blood cells (RBCs) and adherent leukocytes and promotes VOC in SCD mice. Here, we show that RBC–leukocyte interactions are significantly diminished in Mac-1-deficient SCD mice, leading to an improvement of blood flow rates and prolonged survival time in a tumor necrosis factor-alpha and surgical-trauma-induced VOC model. Mac-1-deletion, however, was not sufficient to reduce SCD-related chronic organ damage. Our results thus suggest uncoupled mechanisms between acute VOC benefits and the long-term complications of SCD that should be considered in future clinical trials. Sickle cell disease (SCD), an inherited blood disorder, results from a single nucleotide substitution in the gene encoding β-chain of hemoglobin (Hbβs) [1Stuart M.J. Nagel R.L. Sickle-cell disease.Lancet. 2004; 364: 1343-1360Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar]. Hbβs polymerizes upon deoxygenation, which produces less deformable sickle red blood cells (sRBCs) with increased propensity to adhere to the endothelium. sRBCs can obstruct blood vessels and cause painful vaso-occlusive crisis (VOC) [2Frenette P.S. Atweh G.F. Sickle cell disease: old discoveries, new concepts, and future promise.J Clin Invest. 2007; 117: 850-858Crossref PubMed Scopus (266) Google Scholar]. Recurrent episodes of VOC are thought to lead to irreversible organ damage and to contribute to morbidity and mortality in patients with SCD [3Platt O.S. Brambilla D.J. Rosse W.F. et al.Mortality in sickle cell disease: life expectancy and risk factors for early death.N Engl J Med. 1994; 330: 1639-1644Crossref PubMed Scopus (2475) Google Scholar]. Emerging evidence has demonstrated that VOC is a complex, multicellular process involving sRBC adhesion [4Kaul D.K. Fabry M.E. Nagel R.L. Erythrocytic and vascular factors influencing the microcirculatory behavior of blood in sickle cell anemia.Ann N Y Acad Sci. 1989; 565: 316-326Crossref PubMed Scopus (47) Google Scholar, 5Kaul D.K. Fabry M.E. Nagel R.L. Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.Proc Natl Acad Sci U S A. 1989; 86: 3356-3360Crossref PubMed Scopus (270) Google Scholar], endothelial activation with upregulation of adhesion molecules [6Sultana C. Shen Y. Rattan V. Johnson C. Kalra V.K. Interaction of sickle erythrocytes with endothelial cells in the presence of endothelial cell conditioned medium induces oxidant stress leading to transendothelial migration of monocytes.Blood. 1998; 92: 3924-3935PubMed Google Scholar], platelet activation and aggregation [7Wun T. Paglieroni T. Tablin F. Welborn J. Nelson K. Cheung A. Platelet activation and platelet-erythrocyte aggregates in patients with sickle cell anemia.J Lab Clin Med. 1997; 129: 507-516Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 8Tomer A. Harker L.A. Kasey S. Eckman J.R. Thrombogenesis in sickle cell disease.J Lab Clin Med. 2001; 137: 398-407Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 9Wun T. Paglieroni T. Rangaswami A. et al.Platelet activation in patients with sickle cell disease.Br J Haematol. 1998; 100: 741-749Crossref PubMed Scopus (114) Google Scholar], and leukocyte activation and adhesion [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 11Chang J. Patton J.T. Sarkar A. Ernst B. Magnani J.L. Frenette P.S. GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice.Blood. 2010; 116: 1779-1786Crossref PubMed Scopus (179) Google Scholar, 12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar, 13Belcher J.D. Marker P.H. Weber J.P. Hebbel R.P. Vercellotti G.M. Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion.Blood. 2000; 96: 2451-2459PubMed Google Scholar]. We have demonstrated previously that intravascular accumulation and activation of neutrophils is a key pathogenic event in the development of VOC in SCD [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 11Chang J. Patton J.T. Sarkar A. Ernst B. Magnani J.L. Frenette P.S. GMI-1070, a novel pan-selectin antagonist, reverses acute vascular occlusions in sickle cell mice.Blood. 2010; 116: 1779-1786Crossref PubMed Scopus (179) Google Scholar, 12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar]. Notably, adherent neutrophils can capture circulating RBCs in inflamed venules, leading to critical reduction in blood flow and vaso-occlusion [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar]. Further, we identified that activated αMβ2 integrin (Mac-1) captured RBCs and mediated the RBC–leukocyte interaction in SCD [12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar]. Blocking Mac-1 using M1/70 antibody was sufficient to diminish the RBC–leukocyte interaction significantly, preventing VOC and prolonging survival in SCD mice challenged by tumor necrosis factor-alpha (TNF-α) and surgical trauma [12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar]. These studies highlighted the role of Mac-1 in the pathophysiology of acute VOC in SCD. Whether therapeutic strategies targeting acute VOC would benefit chronic organ damage remains unclear. SCD mice exhibit chronic organ damage reminiscent of human SCD characterized by multiorgan infarcts, progressive iron deposition in multiple organs, enlarged glomeruli in kidney, mononuclear cell infiltration in kidney and liver, extramedullary hematopoiesis, and hepatic sinusoidal and pulmonary capillary congestion [14Manci E.A. Hillery C.A. Bodian C.A. Zhang Z.G. Lutty G.A. Coller B.S. Pathology of Berkeley sickle cell mice: similarities and differences with human sickle cell disease.Blood. 2006; 107: 1651-1658Crossref PubMed Scopus (100) Google Scholar]. Because Mac-1 blockade has been shown to improve acute VOC, we sought to evaluate whether the genetic deletion of Mac-1 would also lead to acute benefits and to investigate whether such a genetic deletion would translate into improved end-organ damage. Berkeley sickle cell mice (Tg[Hu-miniLCRα1GγAγδβS] Hba−/− Hbb−/−), referred to as SCD mice, have been described previously [15Ryan T.M. Ciavatta D.J. Townes T.M. Knockout-transgenic mouse model of sickle cell disease.Science. 1997; 278: 873-876Crossref PubMed Scopus (210) Google Scholar, 16Paszty C. Brion C.M. Manci E. et al.Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.Science. 1997; 278: 876-878Crossref PubMed Scopus (372) Google Scholar]. Itgam−/− (Itgamtm1Myd; Mac-1-deficient) mice and C57BL/6 mice were purchased from The Jackson Laboratory. SCD mice deficient (SCD.Mac-1−/−) or heterozygous (SCD.Mac-1+/−) in Mac-1 were generated in our laboratory by breeding SCD mice with Itgam–/– mice. All experimental procedures in this study were approved by the Animal Care and Use Committee of Albert Einstein College of Medicine. We generated SCD.Mac-1+/−, SCD.Mac-1−/−, and C57BL/6 mice by transplantation of bone marrow (BM) nucleated cells from SCD.Mac-1+/−, SCD.Mac-1−/−, and C57BL/6 mice into lethally irradiated C57BL/6 recipients as described previously [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 17Chang J. Shi P.A. Chiang E.Y. Frenette P.S. Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion.Blood. 2008; 111: 915-923Crossref PubMed Scopus (78) Google Scholar]. For intravital microscopy experiments, we used mice that were at least 3 months post-BM transplantation (BMT). Mice used in chronic tissue damage analysis were 6 months post-BMT. Blood was harvested through the retro-orbital sinus from SCD.Mac-1+/− and SCD.Mac-1−/− mice, collected in EDTA, and RBCs were lysed with lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). Single-cell suspensions from blood were stained with fluorophore-conjugated anti-mouse Gr-1 and Mac-1 antibodies (eBioscience) and DAPI (Sigma-Aldrich) and analyzed by flow cytometry using an LSRII (BD Biosciences). Data were analyzed with FlowJo software. Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of 2% α-chloralose and 10% urethane in phosphate-buffered saline (PBS) and were treated with 0.5 μg of TNF-α. A polyethylene tube was inserted into the trachea of anesthetized animals to facilitate spontaneous respiration. The cremaster muscle was gently exteriorized and then continuously superfused throughout the experiment with warmed (37°C) bicarbonate-buffered (pH 7.4) saline aerated with a mixture of 95% N2 and 5% CO2. Ninety minutes after TNF-α administration, 10 venules of each mouse were videotaped over a period of 60 minutes, with each venule recorded continuously for 2 minutes. Venules were visualized with a custom-designed intravital microscope (MM-40; Nikon), using a 60 × water-immersion objective (Nikon). Images were recorded with a charge-coupled device video camera (Hamamatsu) and video recorder (SVHS, SVO-9500; Sony). Venular diameter was measured with a video caliper. Centerline red cell velocity (VRBC) was measured for each venule in real time with an optical Doppler velocimeter (Texas A&M). Blood flow rate was calculated as Q = Vmeanπd2/4, where d is venule diameter and Vmean is estimated as VRBC/1.6. All analyses were made with playback assessment of videotapes as described previously [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar, 17Chang J. Shi P.A. Chiang E.Y. Frenette P.S. Intravenous immunoglobulins reverse acute vaso-occlusive crises in sickle cell mice through rapid inhibition of neutrophil adhesion.Blood. 2008; 111: 915-923Crossref PubMed Scopus (78) Google Scholar]. Briefly, adherent leukocytes were defined as those remaining stationary for at least 30 seconds over a 100 μm venular segment. RBCs were identified by their size and shape (discoid and sickle-shaped cells). An interaction between RBCs and adherent leukocytes was defined as the arrest of an RBC on an adherent leukocyte for more than two video frames (>0.07 sec). Blood was collected in EDTA through the retro-orbital sinus. The RBC count, hemoglobin concentration (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and mean corpuscular hemoglobin concentration (MCHC) were determined using an ADVIA 120 Hematology System (Siemens). Mice were deprived of water for 16 hours before urine collection. Urine osmolality was measured with Model 3320 osmometer according to the manufacturer's protocol (Advanced Instruments). Organs were excised and fixed in 10% neutral buffered formalin for 36–72 hours and routinely processed for paraffin embedding. Samples for histopathology diagnostics were sectioned to a thickness of 5 μm and stained with hematoxylin and eosin (H&E), Prussian blue (for iron), and trichrome (for fibrosis). Samples collected from SCD.Mac-1+/−, SCD.Mac-1−/−, and C57BL/6 mice were evaluated histologically by board-certified veterinary pathologists blinded to the conditions and using a qualitative grading scheme. The grading scale was on a 0–5 range where 0 = no lesion, 1 = minimal finding, 2 = mild finding, 3 = moderate finding, 4 = marked finding, and 5 = severe finding. Data are represented either as mean ± SEM or median with interquartile range. Comparisons between two samples were done using the unpaired Student's t test or Mann–Whitney test. Statistical analyses were performed with GraphPad Prism 6 software (*p < 0.05, **p < 0.01). Previously, we have demonstrated that administration of an antibody to Mac-1 (M1/70) in SCD mice significantly decreased interactions between RBC and adherent leukocytes and prolonged survival of SCD mice compared with mice infused with isotype control antibody [12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar]. To confirm and extend studies on the role of Mac-1 integrin in SCD pathophysiology, we generated SCD.Mac-1−/− mice (Fig. 1A). F1 hybrids were generated by breeding SCD males with Mac-1-deficient females because SCD females exhibit difficulties in feeding their pups [16Paszty C. Brion C.M. Manci E. et al.Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.Science. 1997; 278: 876-878Crossref PubMed Scopus (372) Google Scholar]. Because the genes encoding murine β-globin (Hbb) and Mac-1 (Itgam) are closely linked on mouse chromosome 7 and are mapped in close proximity (∼15 cM apart; MGI database for the laboratory mouse), the F1 hybrids would carry Hbb(−) on paternal chromosome and Itgam(−) on maternal chromosome (trans-configuration) (Fig. 1A). We screened for F2 hybrids with Hbb(−) and Itgam(−) in cis configuration (termed cis mice) from the crossing between F1 hybrids and wild-type mice (Fig. 1A) and finally generated SCD.Mac-1−/− mice from intercrossing the F2 cis mice. We confirmed the absence of Mac-1 expression on Gr-1+ blood leukocytes of SCD.Mac-1−/− mice (Fig. 1B). Mac-1 deficiency did not significantly change systemic leukocyte counts in SCD mice (Fig. 1C). We next evaluated leukocyte behavior by intravital microscopy of cremasteric postcapillary venules. We found that the number of leukocytes recruited in venules of TNF-α-treated SCD.Mac-1+/− and SCD.Mac-1−/− mice was not affected by Mac-1 deficiency (Fig. 1D). However, Mac-1 deficiency significantly diminished the heterotypic interaction between RBCs and adherent leukocytes in SCD mice (Fig. 1E). The interaction between RBCs and adherent leukocytes can trigger VOC, which impedes blood flow and leads to early death in SCD mice [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar]. In parallel with the reduced RBC–leukocyte interactions, the microcirculatory blood flow rate of SCD.Mac-1−/− mice was significantly higher than that of SCD.Mac-1+/− mice (Fig. 1F). In addition, Mac-1 deficiency significantly prolonged the survival of SCD mice during and after the experimental procedure (Fig. 1G). These data indicate that Mac-1 integrin mediates the interaction between RBC and adherent leukocytes and promotes VOC in SCD. To investigate whether there is a beneficial effect of Mac-1 deficiency on chronic organ damage of SCD mice, we harvested blood, spleen, kidney, lung, and liver from SCD.Mac-1+/− and SCD.Mac-1−/− mice at 6 months after BMT. RBC counts and indices were comparable between SCD.Mac-1+/− and SCD.Mac-1−/− mice (Table 1). Both groups of mice exhibited severe anemia with reduced RBC count, decreased concentration of Hgb in the blood, and reduced Hct, MCV, and MCHC, similar to previously published data on SCD mice [15Ryan T.M. Ciavatta D.J. Townes T.M. Knockout-transgenic mouse model of sickle cell disease.Science. 1997; 278: 873-876Crossref PubMed Scopus (210) Google Scholar, 16Paszty C. Brion C.M. Manci E. et al.Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease.Science. 1997; 278: 876-878Crossref PubMed Scopus (372) Google Scholar].Table 1Hematologic values for SCD.Mac-1+/−, SCD.Mac-1−/−, and C57BL/6 miceRBCs (×106 cells/μL)Hgb (g/dL)Hct (%)MCV (fL)MCH (pg)MCHC (g/dL)SCD.Mac-1+/− mice (n = 4)6.5 ± 0.35.1 ± 0.326.3 ± 1.140.3 ± 0.17.8 ± 0.119.4 ± 0.3SCD.Mac-1−/− mice (n = 4)6.0 ± 0.15.2 ± 0.225.0 ± 0.842.0 ± 0.38.6 ± 0.120.5 ± 0.1C57BL/6 mice (n = 3)9.8 ± 0.113.2 ± 0.150.3 ± 1.251.7 ± 0.613.6 ± 0.126.4 ± 0.4Samples were obtained from animals 6 months after bone marrow transplantation. Values are shown as mean ± SEM. The means for each parameter were significantly different between C57BL/6 and SCD (Mac-1+/− or Mac-1−/−) mice (p < 0.0001). Open table in a new tab Samples were obtained from animals 6 months after bone marrow transplantation. Values are shown as mean ± SEM. The means for each parameter were significantly different between C57BL/6 and SCD (Mac-1+/− or Mac-1−/−) mice (p < 0.0001). Histological examination of the kidneys revealed membranous glomerulopathy characterized by thickened capillary basement membranes (Fig. 2A, upper panels). Histopathological changes, however, were similar between SCD.Mac-1+/− and SCD.Mac-1−/− mice. A similar amount of iron was present in the proximal tubular epithelial cells in SCD.Mac-1+/− and SCD.Mac-1−/− kidneys (Fig. 2A, middle panels, arrows), but was not present within the C57BL/6 kidneys (Fig. 2A, middle right panel). The trichrome staining confirmed increased collagen in glomerular tufts and demonstrated minimally increased interstitial collagen in both groups of mice (Fig. 2A, lower panels). Notably, interstitial and glomerular trichrome staining was slightly greater in SCD.Mac-1−/− mice than in SCD.Mac-1+/− mice (Fig. 2A, lower panels). To evaluate kidney function, we determined the ability to concentrate urine after 16 hours of water deprivation. We found that urine osmolality was significantly reduced in SCD, SCD.Mac-1+/− and SCD.Mac-1−/− mice compared with healthy C57BL/6 mice (Fig. 2B). Consistent with the similar alterations in renal histopathology, Mac-1 deficiency did not ameliorate the severity of renal dysfunction in SCD mice (Fig. 2B). Both SCD.Mac-1+/− and SCD.Mac-1−/− mice had similar splenomegaly (680 ± 41 and 658 ± 31 mg, respectively) compared with that of C57BL/6 mice (64 ± 3 mg). Histologically, spleens from both SCD.Mac-1+/− and SCD.Mac-1−/− mice had moderate lymphoid depletion in the white pulp and increased extramedullary hematopoiesis in the red pulp, with no statistically significant difference detected (Fig. 2C). Livers in both genotypes exhibited degenerative and inflammatory histological changes with increased hepatocellular and macrophage accumulations of iron and increased sinusoidal and perivascular collagen compared with wild-type mice. Similar histological findings were identified in SCD.Mac-1+/− and SCD.Mac-1−/− livers, including sinusoidal congestion (Fig. 2D, upper panels, black arrows), leukocyte infiltration (Fig. 2D, upper panels, white arrow heads), siderosis (Fig. 2D, middle panels, black arrows), and extensive areas of chronic hepatocellular degeneration, necrosis, fibrosis, and mixed inflammation. Increased collagen, aside from that associated with areas of visible fibrosis, was evident along sinusoids and around vessels in both SCD.Mac-1+/− and SCD.Mac-1−/− mouse livers (Fig. 2D, lower panels), but the extent of fibrosis was similar between the two groups. In the lungs, both groups of mice had evidence of patchy alveolar leukocyte accumulation (Fig. 2E, upper panels, yellow arrowheads) and intravascular sickle cell congestion (Fig. 2E, upper panels, black arrows). In addition, both groups of mice had minimally increased trichrome staining present in the alveolar septa, with no difference observed between groups (Fig. 2E, lower panels). Collectively, both SCD.Mac-1+/− and SCD.Mac-1−/− mice revealed end-organ damage characteristic of SCD that includes loss of urine-concentrating ability, splenomegaly, liver damage characterized by chronic inflammation and iron deposition, and pulmonary congestion. Our data thus indicate that Mac-1 deficiency does not alleviate the chronic organ damage induced by SCD. Leukocytes play an important role in promoting sickle cell vaso-occlusion. Early clinical studies revealed a positive correlation between high leukocyte counts and poor disease outcome in patients with SCD [3Platt O.S. Brambilla D.J. Rosse W.F. et al.Mortality in sickle cell disease: life expectancy and risk factors for early death.N Engl J Med. 1994; 330: 1639-1644Crossref PubMed Scopus (2475) Google Scholar, 18Anyaegbu C.C. Okpala I.E. Akren'Ova Y.A. Salimonu L.S. Peripheral blood neutrophil count and candidacidal activity correlate with the clinical severity of sickle cell anaemia (SCA).Eur J Haematol. 1998; 60: 267-268Crossref PubMed Scopus (65) Google Scholar, 19Kinney T.R. Sleeper L.A. Wang W.C. et al.Silent cerebral infarcts in sickle cell anemia: a risk factor analysis: the Cooperative Study of Sickle Cell Disease.Pediatrics. 1999; 103: 640-645Crossref PubMed Scopus (246) Google Scholar, 20Castro O. Brambilla D.J. Thorington B. et al.The acute chest syndrome in sickle cell disease: incidence and risk factors: the Cooperative Study of Sickle Cell Disease.Blood. 1994; 84: 643-649PubMed Google Scholar, 21Ohene-Frempong K. Weiner S.J. Sleeper L.A. et al.Cerebrovascular accidents in sickle cell disease: rates and risk factors.Blood. 1998; 91: 288-294PubMed Google Scholar]. Intravital microscopy studies using SCD mouse models have identified adherent neutrophils as the major population that interacts with sRBCs and promotes vaso-occlusion [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar, 22Chiang E.Y. Hidalgo A. Chang J. Frenette P.S. Imaging receptor microdomains on leukocyte subsets in live mice.Nat Methods. 2007; 4: 219-222Crossref PubMed Scopus (70) Google Scholar]. Blockade of leukocyte adhesion or the RBC–neutrophil interaction effectively improves the blood flow rates and prolongs survival during VOC in SCD mouse models [10Turhan A. Weiss L.A. Mohandas N. Coller B.S. Frenette P.S. Primary role for adherent leukocytes in sickle cell vascular occlusion: a new paradigm.Proc Natl Acad Sci U S A. 2002; 99: 3047-3051Crossref PubMed Scopus (355) Google Scholar, 12Hidalgo A. Chang J. Jang J.E. Peired A.J. Chiang E.Y. Frenette P.S. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury.Nat Med. 2009; 15: 384-391Crossref PubMed Scopus (263) Google Scholar]. These studies have led to exciting clinical trials of anti-adhesion therapies, including reagents that target selectins (e.g., rivipansel, SelG1) or neutrophil activation (e.g., intravenous immunoglobulin, PF04447943) [23Zhang D. Xu C. Manwani D. Frenette P.S. Neutrophils, platelets, and inflammatory pathways at the nexus of sickle cell disease pathophysiology.Blood. 2016; 127: 801-809Crossref Scopus (244) Google Scholar]. However, whether anti-adhesion therapies can benefit chronic tissue damage in SCD remains unclear. Here, we show that whereas RBC–leukocyte interactions are significantly diminished in SCD.Mac-1−/− mice, leading to an improvement of blood flow rate and prolonged survival in acute VOC, Mac-1 deletion is insufficient to alleviate chronic organ damage, as evidenced by the lack of improvement in multi-organ histopathology of SCD.Mac-1−/− mice compared with SCD.Mac-1+/− mice. These results therefore suggest distinct mechanisms for acute vaso-occlusion and chronic tissue damage in SCD and argue that Mac-1 can be a useful therapeutic target for managing acute VOC, but not for preventing chronic organ damage in SCD. The development of end-organ damage in SCD is likely multifactorial. Chronic hemolysis, oxidative stress, and recurrent vaso-occlusive episodes may emerge as major determinants of SCD-related organ damage [24Kato G.J. Gladwin M.T. Steinberg M.H. Deconstructing sickle cell disease: reappraisal of the role of hemolysis in the development of clinical subphenotypes.Blood Rev. 2007; 21: 37-47Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, 25Nur E. Biemond B.J. Otten H.M. Brandjes D.P. Schnog J.J. Group C.S. Oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management.Am J Hematol. 2011; 86: 484-489Crossref PubMed Scopus (142) Google Scholar]. Intravascular hemolysis releases cell-free hemoglobin into the circulation, which can deplete nitric oxide, generate oxidative stress, and increase the heme concentration in the blood [26Schaer D.J. Buehler P.W. Alayash A.I. Belcher J.D. Vercellotti G.M. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins.Blood. 2013; 121: 1276-1284Crossref PubMed Scopus (473) Google Scholar]. A large fraction of extracellular heme is carried by the erythrocyte-derived microparticles and can be transferred to the endothelium, which further promote inflammation and vascular injury [27Camus S.M. De Moraes J.A. Bonnin P. et al.Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease.Blood. 2015; 125: 3805-3814Crossref PubMed Scopus (177) Google Scholar, 28Wagener F.A. Eggert A. Boerman O.C. et al.Heme is a potent inducer of inflammation in mice and is counteracted by heme oxygenase.Blood. 2001; 98: 1802-1811Crossref PubMed Scopus (353) Google Scholar]. Excessive reactive oxygen species, heme, and inflammatory cytokines produced by several leukocyte populations, including monocytes and invariant natural killer T cells, together produces a hypercoagulable, pro-inflammatory, and pro-adhesive state in SCD that leads to progressive tissue damage [13Belcher J.D. Marker P.H. Weber J.P. Hebbel R.P. Vercellotti G.M. Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion.Blood. 2000; 96: 2451-2459PubMed Google Scholar, 23Zhang D. Xu C. Manwani D. Frenette P.S. Neutrophils, platelets, and inflammatory pathways at the nexus of sickle cell disease pathophysiology.Blood. 2016; 127: 801-809Crossref Scopus (244) Google Scholar, 25Nur E. Biemond B.J. Otten H.M. Brandjes D.P. Schnog J.J. Group C.S. Oxidative stress in sickle cell disease; pathophysiology and potential implications for disease management.Am J Hematol. 2011; 86: 484-489Crossref PubMed Scopus (142) Google Scholar, 29Wallace K.L. Linden J. Adenosine A2A receptors induced on iNKT and NK cells reduce pulmonary inflammation and injury in mice with sickle cell disease.Blood. 2010; 116: 5010-5020Crossref PubMed Scopus (107) Google Scholar]. In addition, sRBCs are reported to exhibit an increased propensity for adhesion to vascular endothelium [5Kaul D.K. Fabry M.E. Nagel R.L. Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications.Proc Natl Acad Sci U S A. 1989; 86: 3356-3360Crossref PubMed Scopus (270) Google Scholar], which would remain unaffected by Mac-1 deletion. The cumulative effects of sRBC interactions with the endothelium and the intermittent transient microvascular occlusions could also contribute to chronic organ damage in SCD. Neutrophils also promote sickle cell pathophysiology by releasing neutrophil extracellular traps (NETs), which contribute significantly to SCD-related pulmonary dysfunction [30Chen G. Zhang D. Fuchs T.A. Manwani D. Wagner D.D. Frenette P.S. Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.Blood. 2014; 123: 3818-3827Crossref PubMed Scopus (226) Google Scholar]. Cell-free heme has been identified as a potent inducer of NET formation upon inflammatory challenges in a SCD mouse model. DNase I and hemopexin treatment targeting NETs significantly alleviates acute pulmonary tissue damage in these mice [30Chen G. Zhang D. Fuchs T.A. Manwani D. Wagner D.D. Frenette P.S. Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease.Blood. 2014; 123: 3818-3827Crossref PubMed Scopus (226) Google Scholar]. These results therefore raise a possibility that NETs may contribute to the progressive tissue damage associated with SCD. More recently, an aged subset of neutrophils has been identified to exhibit enhanced Mac-1 activation and NET formation in response to inflammatory challenges and this subset expands in both the SCD mouse model and patients [31Zhang D. Chen G. Manwani D. et al.Neutrophil ageing is regulated by the microbiome.Nature. 2015; 525: 528-532Crossref PubMed Scopus (480) Google Scholar]. Microbiota-derived signals drive the generation of aged neutrophils and depletion of the microbiota with antibiotics administered through drinking water leads to a significant reduction of aged neutrophils and dramatic improvement of both acute vaso-occlusion and chronic organ damage [31Zhang D. Chen G. Manwani D. et al.Neutrophil ageing is regulated by the microbiome.Nature. 2015; 525: 528-532Crossref PubMed Scopus (480) Google Scholar]. Although these data suggest a role for neutrophils in chronic tissue damage, the present studies—inasmuch as the model reflects human disease—indicate that the inhibition of RBC–leukocyte interactions will not be sufficient to prevent these chronic complications. In addition, long-term interference of Mac-1-mediated leukocyte migration may lead to an increased number of aged and total neutrophils in the human circulation, which may enhance the risk of end-organ damage in SCD patients and should be carefully evaluated in future clinical studies. We thank Drs. Rani Sellers and Amanda Beck of the histology core for blind histopathological analyses and the New York State Department of Health (NYSTEM Program) for shared facility support (C029154). This work was supported by the National Institutes of Health (NIH Grants R01 HL069438, HL097700, and DK056638 to P.S.F.) and by the Albert Einstein Cancer Center (NIH Support Grant P30CA013330). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. G.C. designed the research, performed the experiments, analyzed the data, prepared the figures, and wrote the manuscript; J.C. performed the experiments and analyzed the data; S.P. and J.-E. J. performed the experiments; D.Z. analyzed the data and wrote the manuscript; and P.S.F. supervised the research, analyzed the data, and wrote the manuscript. The authors declare no competing financial interests.
Referência(s)