Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability.

BACKGROUND AND PURPOSE: The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. EXPERIMENTAL APPROACH: In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor alpha (TNFalpha)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced endothelial permeability was evaluated. Flow cytometry was used to determine the effect of roflumilast on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced CD11b upregulation on human neutrophils. KEY RESULTS: In vivo, roflumilast, given 1 h before lipopolysaccharide (LPS), dose-dependently reduced leukocyte-endothelial cell interactions in rat mesenteric postcapillary venules. It also diminished histamine-induced microvascular permeability. Immunohistochemical analyses revealed that roflumilast prevented LPS-induced endothelial P- and E-selectin expression. In vitro, roflumilast N-oxide concentration-dependently suppressed neutrophil adhesion to TNFalpha-activated HUVEC and CD11b expression on fMLP-stimulated neutrophils. It also reduced TNFalpha-induced E-selectin expression on HUVEC, when PDE3 activity was blocked. HUVEC permeability elicited by thrombin was concentration-dependently suppressed by roflumilast N-oxide. While roflumilast N-oxide was as potent as roflumilast at inhibiting stimulated endothelial cell and neutrophil functions, both compounds were significantly more potent than the structurally unrelated PDE4 inhibitors, rolipram or cilomilast. CONCLUSIONS AND IMPLICATIONS: These findings further support earlier observations on the inhibition of inflammatory cell influx and protein extravasation by roflumilast in vivo.

Neutrophils migrate across intestinal epithelium using beta2 integrin (CD11b/CD18)-independent mechanisms.

Recruitment of polymorphonuclear leucocytes (PMN) across the intestinal epithelium is dependent on specific adhesion molecules and chemoattractants diffusing from the intestinal lumen. The present understanding is that in response to fMLP, PMN migration across a T84 colon carcinoma monolayer is dependent on the beta(2) integrin, Mac-1 (CD11b/CD18). To further understand PMN transepithelial migration, we sought to determine whether migration to C5a, IL-8 and LTB(4) was similarly Mac-1-, or even CD18-dependent. T84 epithelial cell monolayers growing on Transwell filters were used in combination with radiolabelled peripheral blood PMN. The number of migrated PMN was established by the amount of radioactivity recovered from the well after the migration period. Monoclonal antibodies were used to block integrin function. Whereas essentially all migration to fMLP across T84 monolayers was prevented by anti-CD18 antibody, significant migration to C5a, IL-8 or LTB(4) persisted despite anti-CD18 antibody, indicating PMN are capable of beta(2) integrin-independent transepithelial migration. An antibody to CD11b but not CD11a blocked migration to an extent similar as with anti-CD18. CD18-independent PMN migration to C5a occurred only in the basolateral-to-apical direction across epithelial cells. Co-stimulation of PMN with C5a and fMLP or IL-8 plus LTB(4) and fMLP still resulted in CD18-independent migration. Thus CD18 use during PMN migration across this model epithelium is a function of the chemoattractant inducing migration. The finding of CD18-independent migration mechanisms needs to be considered when developing antiadhesion molecule strategies to reduce or reverse intestinal inflammation.

Neutrophil migration into indomethacin induced rat small intestinal injury is CD11a/CD18 and CD11b/CD18 co-dependent.

BACKGROUND: Neutrophils may exacerbate intestinal inflammatory diseases through secretion of proteolytic enzymes and reactive oxygen and nitrogen intermediates. AIMS: To define the mechanisms involved in neutrophil infiltration into the non-steroidal anti-inflammatory disease inflamed intestine to develop strategies to regulate this process. METHODS: The small intestinal epithelium of (15 mg/kg) indomethacin treated rats was examined for cytokine mRNA. The kinetics of neutrophil accumulation into the gastrointestinal tract (including lumen contents) of inflamed rats was determined using radiolabelled (111In) neutrophils injected intravenously followed by a three hour migration period. To determine which adhesion molecules were critical for migration, rats were also injected with function blocking monoclonal antibodies to the beta2 (CD11/CD18) integrins. RESULTS: Interleukin 1beta, interleukin 1 receptor II, tumour necrosis factor alpha, and monocyte inflammatory peptide 2 but not monocyte chemoattractant protein 1 mRNA were detected in the epithelium within hours of indomethacin injection. Neutrophils were detectable in the small intestine and intestinal lumen by six hours and continued to accumulate until 48 hours post indomethacin injection. Neutrophil accumulation in the intestine was essentially blocked by anti-CD18, and partially blocked by either anti-CD11a or CD11b antibody treatment. Migration into the intestinal lumen was reduced by anti-CD11b. CONCLUSIONS: The small intestinal epithelium acts as one source of cytokines with properties important in the recruitment of neutrophils. In turn, neutrophil migration into the indomethacin inflamed small intestine is mediated by CD11a/CD18 and CD11b/CD18.

Platelets modulate ischemia/reperfusion-induced leukocyte recruitment in the mesenteric circulation.

P-selectin-dependent leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds, including the gut. Because platelet-endothelial (P/E) cell adhesion also occurs in postischemic venules, the possibility exists that the expression of P-selectin on the surface of platelets that are adherent to venular endothelial cells may mediate the leukocyte recruitment elicited by I/R. P-selectin expression [dual radiolabeled monoclonal antibody (MAb) technique] and neutrophil accumulation [myeloperoxidase (MPO) activity] were measured in the postischemic small intestine of untreated rats and rats treated with either antiplatelet serum (APS) or MAbs directed against either P-selectin, GPIIb/IIIa, or fibrinogen. The increases in P-selectin expression and tissue MPO normally elicited by I/R were significantly attenuated in the different treatment groups, suggesting that I/R-induced neutrophil recruitment is a platelet-dependent, P-selectin-mediated process. Intravital microscopy was then employed to examine this process relative to leukocyte-endothelial cell adhesion in postischemic rat mesenteric venules. The recruitment of adherent and emigrated leukocytes after I/R was attenuated by pretreatment with a MAb against, either P-selectin, GPIIb/IIIa, or fibrinogen, as well as an Arg-Gly-Asp peptide. Whereas thrombocytopenia greatly blunted leukocyte emigration, it did not alter the leukocyte adherence response to I/R. These findings suggest that platelet-associated P-selectin contributes to the accumulation of leukocytes in postischemic tissue via a mechanism that alters transendothelial leukocyte migration.

Recruitment of neutrophils across the blood-brain barrier: the role of E- and P-selectins.

The adult central nervous system parenchyma is resistant to inflammation, but in juvenile rats the injection of inflammatory mediators, interleukin-1 beta for example, gives rise to extensive neutrophil recruitment and neutrophil-dependent blood-brain barrier breakdown. The factors that confer this resistant phenotype are unknown. In this study, the authors demonstrate that E- and P-selectin expression is increased to a similar extent in adult and juvenile brain after the intracerebral injection of IL-1 beta. Thus, the refractory nature of the brain parenchyma cannot be attributed to an absence of selectin expression. However, in injuries where the resistant characteristic of the brain parenchyma is compromised, and neutrophil recruitment occurs, selectin blockade may be an advantage. The authors investigated the contribution that selectins make to neutrophil recruitment during acute inflammation in the brain. The authors examined neutrophil recruitment by immunohistochemistry on brain sections of juvenile rats killed four hours after the intracerebral injection of IL-1 beta and the intravenous injection of neutralizing anti-selectin monoclonal antibodies (mAb). The administration of the P-selectin blocking mAb inhibited neutrophil recruitment by 85% compared with controls. Surprisingly, E-selectin blockade had no effect on neutrophil recruitment to the brain parenchyma. Thus, P-selectin appears to play a pivotal role in mediating neutrophil recruitment to the brain parenchyma during acute inflammation.

Growth factor regulation of neutrophil-endothelial cell interactions.

The effects of the angiogenic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on human polymorphonuclear leukocyte (PMNL)-endothelial cell adhesion and transendothelial migration (TEM) were investigated. Stimulation of human umbilical vein endothelial cells by VEGF or bFGF for 18 h up-regulated intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 expression and significantly increased PMNL adhesion and TEM in response to complement fragment 5a (C5a) or interleukin (IL)-8. In contrast, continued exposure to bFGF (24 h-6 days) down-regulated basal and IL-1- or tumor necrosis factor (TNF)-induced intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin expression as well as PMNL adhesion and TEM. These effects could be reversed by introduction of high concentrations of TNF-alpha, C5a, or IL-8. None of these inhibitory effects was observed with VEGF. The acute effects of bFGF and VEGF may facilitate PMNL emigration during acute inflammation, but continued bFGF production may have anti-inflammatory actions during chronic inflammation, angiogenesis, and tumor defense by inhibition of endothelial activation for leukocyte recruitment.

E-selectin, but not P-selectin, is required for development of adjuvant-induced arthritis in the rat.

OBJECTIVE: To determine the role of the endothelial cell adhesion molecules E- and P-selectin in the development and severity of adjuvant-induced arthritis (AIA) in the rat. METHODS: Lewis rats were immunized subcutaneously with Mycobacterium butyricum (Mb), and blocking monoclonal antibodies (mAb) to rat E- and P-selectin were administered. Clinical score, radiolabeled (51Cr and 111In) blood polymorphonuclear leukocyte (PMN) and monocyte migration to joints, and histologic features were monitored. RESULTS: When mAb treatment was started on day 5 postimmunization with Mb (preclinical stage), development of AIA was significantly (P < 0.01) inhibited by mAb to E- but not to P-selectin (mean score on day 14 control 10.2, anti-E 2.8, anti-P 9.1). This was associated with markedly decreased migration (by 66-94%) of PMN and monocytes to arthritic joints and diminished cartilage degradation. When treatment was delayed until animals showed signs of arthritis (day 10 postimmunization), only a marginal and variable effect was observed as compared with blockade during the preclinical (day 5) stage. E-selectin blockade on day 5 and day 7 postimmunization resulted in inhibition of antigen-dependent T cell-mediated inflammation, since it decreased T cell migration to sites of dermal-delayed hypersensitivity induced by Mb without affecting migration to concanavalin A or cytokines. The proliferative response of T cells to Mb in vitro was not altered. CONCLUSION: E-selectin plays an important role early in the development of AIA. This adhesion molecule may contribute to the migration of antigen-reactive T cells to peripheral tissues, including the joints where T cells initiate the arthritis.

The beta2, alpha4, alpha5 integrins and selectins mediate chemotactic factor and endotoxin-enhanced neutrophil sequestration in the lung.

Intravascular chemotactic factor activation of neutrophils (polymorphonuclear leukocytes; PMNLs), associated with actin polymerization resulting in PMNL stiffening, induces rapid and transient sequestration in the pulmonary vasculature and lung dysfunction. Recent studies have proposed that this sequestration is mediated by physical lodging of PMNLs because of loss of deformability. To examine the contribution of cell adhesion molecules in this process, we used blocking monoclonal antibodies (mAbs) to rat selectins and integrins in a model of PMNL margination (reflected by acute blood neutropenia) induced by N-formyl-met-leu-phe (FMLP) chemotactic factor infusion in normal or lipopolysaccharide (LPS)-primed rats. Blood PMNL levels dropped by 70% within 1 minute and for the duration of FMLP infusion (20 minutes) in normal or by 90% in LPS-primed rats. Pretreatment with mAbs to beta2(WT.3), VLA-4(TA-2 F(ab)(2)), and VLA-5 (HMalpha5 F(ab)(2)) in combination inhibited the decrease by 50% and to a greater degree than beta2 blockade alone (35% inhibition). F(ab)(2) mAbs to L-(HRL-3), P-(RMP-1), plus E-(RME-1) selectins had no effect but they potentiated inhibition by anti-beta2 + anti-VLA-4 + anti-VLA5 mAb treatment (69% inhibition, P < 0.05). Similar results were observed in the first 6 minutes in LPS-primed rats with complete inhibition of sequestration thereafter by combined selectin and integrin blockade. These results indicate that besides PMNL stiffening because of actin polymerization, both selectins and integrins substantially contribute to activated PMNL sequestration in the lung.

The alpha 4 beta 1 (very late antigen (VLA)-4, CD49d/CD29) and alpha 5 beta 1 (VLA-5, CD49e/CD29) integrins mediate beta 2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation.

The beta(2) integrin cell adhesion molecules (CAM) mediate polymorphonuclear leukocyte (PMNL) emigration in most inflamed tissues, but, in the lung, other yet to be identified CAMs appear to be involved. In Lewis rats, the intratracheal injection of Escherichia coli-LPS induced acute (6-h) PMNL accumulation in the lung parenchyma (280 x 10(6) by myeloperoxidase assay; PBS control = 35 x 10(6)) and bronchoalveolar lavage fluid (BALF = 27 x 10(6); PBS = 0.1 x 10(6)). Parenchymal accumulation was not inhibited by a blocking Ab to beta(2) integrins and only minimally inhibited (20.5%; p < 0.05) in BALF. We examined the role of alpha(4)beta(1) and alpha(5)beta(1) integrins and of selectins in this PMNL recruitment. Treatment with mAbs to alpha(4)beta(1) or alpha(5)beta(1), even in combination, had no effect on PMNL accumulation induced by intratracheal LPS. However, anti-alpha(4) combined with anti-beta(2) mAbs inhibited PMNL recruitment to the parenchyma by 56% (p < 0.001) and to BALF by 58% (p < 0.01). The addition of anti-alpha(5) mAb to beta(2) plus alpha(4) blockade inhibited PMNL accumulation further (by 79%; p < 0.05). In contrast, blockade of L-, P-, and E-selectins in combination or together with beta(2), alpha(4), and alpha(5) integrins had no effect. LPS-induced BALF protein accumulation was not inhibited by treatment with anti-beta(2) plus alpha(4) mAbs, but was prevented when alpha(5)beta(1) was also blocked. Thus, while selectins appear to play no role, alpha(4)beta(1) and alpha(5)beta(1) function as major alternate CAMs to the beta(2) integrins in mediating PMNL migration to lung and to pulmonary vascular and epithelial permeability.

Neutrophil migration stimulates rat intestinal epithelial cell cytokine expression during helminth infection.

We are interested in understanding the role of epithelial cells during inflammation, and we previously reported that rat small intestinal epithelial cells express interleukin-1beta (IL-1beta) during infection by Trichinella spiralis. We now report that the epithelium also produces the potent neutrophil chemotactic factor, macrophage inflammatory protein-2 (MIP-2), and an IL-1 antagonist: the type II IL-1 receptor. Consequently we investigated the pattern of neutrophil infiltration into the infected intestine, which closely paralleled the epithelial cytokine expression. Speculating that neutrophil infiltration may provoke epithelial cytokine expression, neutrophil migration into the infected gut was reduced by depleting circulating cells through the use of a specific antibody, or by preventing migration through the use of a function-blocking anti-CD18 monoclonal antibody. Either treatment reduced the number of neutrophils recoverable from the small intestinal epithelium and was paralleled by reduced mRNA levels for epithelial cytokines. These results demonstrate that neutrophil infiltration of the small intestinal epithelium contributes to the stimulation of epithelial cell cytokines.

Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT(1) and AT(2) receptor-mediated P-selectin upregulation.

BACKGROUND: Angiotensin II (Ang II) plays a critical role in the development of vascular lesions in hypertension, atherosclerosis, and several renal diseases. Because Ang II may contribute to the leukocyte recruitment associated with these pathological states, the aim of the present study was to assess the role of Ang II in leukocyte-endothelial cell interactions in vivo. METHODS AND RESULTS: Intravital microscopy of the rat mesenteric postcapillary venules was used. Sixty minutes of superfusion with 1 nmol/L Ang II induced a significant increase in leukocyte rolling flux (83.8+/-20. 7 versus 16.4+/-3.1 cells/min), adhesion (11.4+/-1.0 versus 0.8+/-0. 5 cells/100 microm), and emigration (4.0+/-0.7 versus 0.2+/-0.2 cells/field) without any vasoconstrictor activity. These effects were not mediated by mast cell activation. Intravenous pretreatment with AT(1) (losartan) or AT(2) (PD123,319) receptor antagonists significantly reduced Ang II-induced responses. A combination of both receptor antagonists inhibited the leukocyte rolling flux, adhesion, and extravasation elicited by Ang II at 60 minutes. Pretreatment of animals with fucoidin or an adhesion-blocking anti-rat P-selectin monoclonal antibody abolished Ang II-induced leukocyte responses. Furthermore, rat platelet P-selectin expression was not affected by Ang II stimulation. CONCLUSIONS: -Ang II induces significant leukocyte rolling, adhesion, and emigration, which may contribute not only to hypertension but also to the onset and progression of the vascular damage associated with disease states in which plasma levels of this peptide are elevated.

Cytokine mRNA in the joints and draining lymph nodes of rats with adjuvant arthritis and effects of cyclosporin A.

TNF-alpha and IL-1beta promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-alpha, IL-1beta, IL-6, IFN-gamma, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF-alpha and IFN-gamma mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1beta and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1beta and TNF-alpha throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-alpha, IL-1beta, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF-alpha levels were reached on d20, while IL-1beta peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN-gamma mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1beta or TNF-alpha was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN-gamma mRNA in synovium being analogous to human rheumatoid arthritis.

Differential mechanisms of neutrophil and monocyte adhesion on neuroblastoma cells: CD18 and VLA-4 integrins mediate adhesion to SK-N-SH, but not to SK-N-MC cell line.

We examined the adhesion of monocytes and polymorphonuclear leukocytes (PMNLs) to the neuroblastoma (NB) cell lines SK-N-SH and SK-N-MC, which have some distinct differentiation characteristics. Monocytes adhered to SK-N-SH and SK-N-MC to the same extent (20 +/- 1.4% and 24 +/- 0.8% of monocytes added). Monocyte adhesion to SK-N-SH but not SK-N-MC was partially inhibited by treating monocytes with a mAb to the CD18 (beta2) integrin chain. The adhesion was further inhibited when monocytes were treated with a combination of mAb to CD18 and VLA-4. Treatment of both NB cell lines with interleukin-1alpha (0.5 ng/ml), tumor necrosis factor alpha (100 U/ml), interferon gamma (200 U/ml), or their combinations increased monocyte adhesion to SK-N-SH and SK-N-MC. With each condition, monocyte adhesion to SK-N-SH was partially blocked by mAb to CD18. The inhibition of adhesion to IL-1alpha- or TNFalpha-treated SK-N-SH cells was greater when the monocytes were treated with mAb to both CD18 and VLA-4. In contrast, monocyte adhesion to IL-1alpha or IFNgamma treated SK-N-MC was only slightly inhibited with a combination of mAb to CD18 + VLA-4 and there was no inhibition at all to TNFalpha-treated SK-N-MC. Spontaneous PMNL adhesion to SK-N-SH was almost negligible but increased by treating the cell line with IL-1alpha, TNFalpha, IFNgamma or their combinations. A mAb to CD18 blocked this increase in each case. The pattern of adhesion of PMNLs to SK-N-MC was totally different. PMNL adhesion to unstimulated SK-N-MC was very high (24 +/- 1.3%), was not inhibited by mAb to CD18, and did not increase by stimulating the cell line with IL-1alpha, TNFalpha, IFNgamma or their combinations. Overall, these results suggest two distinct patterns of monocyte and PMNL interaction with neural cells, such as the SK-N-SH and MC cell lines. While monocyte and PMNL adhesion to SK-N-SH is mainly via CD18/VLA-4 or the CD18 mechanisms, respectively, leukocyte adhesion to SK-N-MC is CD18- and VLA-4-independent. Thus, leukocyte-neural cell interactions share some mechanisms common also to leukocyte-endothelium interaction, but there are also unique mechanisms which may be neural cell and differentiation specific.

Heme oxygenase modulates selectin expression in different regional vascular beds.

Heme oxygenase (HO) catalyzes the degradation of heme to biliverdin, iron, and CO. The inducible isoform (HO-1) has been implicated as a modulator of the inflammatory response. HO-1 activity can be induced by hemin and inhibited with zinc protoporphyrin IX (ZnPP). Using these reagents, we assessed the possibility that HO-1 modulates the inflammatory response by altering the expression of endothelial cell adhesion molecules. Endotoxin (lipopolysaccharide, LPS)-induced expression of P- and E-selectin expression was quantified in different vascular beds of the rat using the dual radiolabeled monoclonal antibody technique. Pretreatment with hemin attenuated, whereas ZnPP treatment exacerbated, the increased selectin expression normally elicited by LPS. Biliverdin, at an equimolar dosage, was as effective as hemin in attenuating LPS-induced selectin expression in the lung, kidneys, liver, and intestines. These findings indicate that the anti-inflammatory properties of HO-1 may be related to an inhibitory action of P- and E-selectin expression in the vasculature. Biliverdin (or its metabolite, bilirubin), rather than CO, may account for this action of HO-1 on endothelial cell adhesion molecule expression.

The role of alpha(4) and LFA-1 integrins in selectin-independent monocyte and neutrophil migration to joints of rats with adjuvant arthritis.

Monocytes and neutrophils are chronically recruited to joints in rheumatoid arthritis. In the joints of rats with adjuvant arthritis, this is mediated, in part, by selectin-dependent and selectin-independent mechanisms. To define the selectin-independent mechanisms, (51)Cr-labeled blood monocytes, (111)In-labeled neutrophils and function blocking mAb to the selectins and integrins were utilized. Integrins contributed to the selectin-independent monocyte migration to arthritic joints with 58-70% inhibition of this recruitment by anti-alpha(4) or anti-LFA-1 mAb, relative to selectin blockade alone. alpha(4) plus P-selectin blockade was as effective as combined blockade of alpha(4), P-, E- and L-selectin, mediating approximately 83% of the overall monocyte migration to the joints. In contrast, LFA-1 was the predominant selectin-independent mechanism for neutrophil recruitment to the joints. LFA-1 together with P-selectin had essential roles in the talar joint. In dermal inflammation in the arthritic rats, LFA-1 accounted for most (69%) of the selectin-independent monocyte migration to the chemoattractant C5a(desArg) (zymosan-activated serum), whereas LFA-1 and Mac-1 both contributed to selectin-independent neutrophil recruitment to C5a(desArg). alpha(4) integrin and P-selectin in concert mediated monocyte recruitment to lipopolysaccharide and IFN-gamma lesions (81%). Thus: (1) either alpha(4) or LFA-1 can mediate monocyte migration to arthritic joints in the absence of selectin function and alpha(4) together with P-selectin is particularly important; (2) LFA-1 is the predominant mechanism of selectin-independent migration of neutrophils to inflamed joints; and (3) in arthritic rats, selectin-independent migration of monocytes and neutrophils to dermal inflammation is mediated by alpha(4) or LFA-1 or both LFA-1 and Mac-1, depending on the leukocyte type, and inflammatory stimulus.

Liposome encapsulation of a soluble recombinant fragment of the respiratory syncytial virus (RSV) G protein enhances immune protection and reduces lung eosinophilia associated with virus challenge.

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children and infants. Previous animal studies have shown that immunizing intramuscularly or intraperitoneally with the RSV G protein has elicited protective as well as harmful immune responses upon RSV challenge. In an RSV immunization strategy designed to target the respiratory tract directly (the site of RSV replication), we immunized BALB/c mice intranasally with a liposome-encapsulated, prokaryotically expressed thioredoxin fusion protein consisting of amino acids 128-229 of the RSV G protein (Trx-G(128-229)). Upon intranasal challenge with RSV, a 100 to 500-fold reduction in lung RSV replication was observed in mice immunized with liposome-encapsulated Trx-G(128-229) compared to a sham-immunized control group. Analysis of bronchoalveolar lavage fluids revealed an influx of eosinophils (18% of total cells) in mice immunized with Trx-G(128-229) alone. Such eosinophilic infiltration was diminished (to 4.5% of total cells), however, in mice immunized with liposome-encapsulated Trx-G(128-229). Histological analysis of lung tissue revealed an accumulation of cells around the bronchioles and vessels in mice immunized with Trx-G(128-229) alone followed by RSV challenge which was not increased further in mice immunized with liposome-encapsulated Trx-G(128-229). These results show that intranasal immunization of BALB/c mice with Trx-G(128-229), when encapsulated in liposomes, can reduce the level of RSV replication in the lung as well as specifically reduce the degree of eosinophilic infiltration compared to mice immunized with Trx-G(128-229) alone. This demonstrates the potential of liposomes and particular recombinant fragments of the RSV G protein as an effective combination in RSV vaccine studies.

Enhancement of monocyte transendothelial migration by granulocyte-macrophage colony-stimulating factor: requirement for chemoattractant and CD11a/CD18 mechanisms.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances and primes monocyte functions, but its role in monocyte migration is poorly understood. We examined monocyte migration across human umbilical vein endothelial cells (HUVEC) grown on filters. GM-CSF had no chemotactic or chemokinetic effect. However, GM-CSF enhanced monocyte transendothelial migration (TEM) through unstimulated and IL-1-activated (5 h) HUVEC in response to C5a or monocyte chemoattractant protein-1 in a dose-dependent fashion, increasing the migration from 28.7 +/- 5.3% to 41.8 +/- 6.2% (n = 8, p < 0.05) and from 34.8 +/- 6% to 50.3 +/- 3.1%, p < 0.05), respectively. The enhanced TEM was inhibited by monoclonal antibodies (mAb) to LFA-1, but not by mAb to Mac-1 or to VLA-4. Furthermore, GM-CSF up-regulated and activated LFA-1, as assessed by NKI-L16 neoepitope expression. The results indicate that: (1) GM-CSF can prime monocytes for increased TEM, (2) GM-CSF enhances LFA-1-mediated monocyte TEM and (3) this effect is in part mediated by increasing LFA-1 expression and activation. Thus, increased GM-CSF production may promote monocyte accumulation in inflammation not only by inducing monocytosis, but also enhancing migration.

alpha9beta1 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers.

Accumulation of leukocytes in inflamed tissue involves their migration through vascular endothelium and then in the connective tissue. Recently we utilized a barrier of human synovial, dermal, and lung fibroblasts (HSF, HDF, and HLF) grown on polycarbonate filters as a model of human polymorphonuclear leukocyte (PMN) migration through connective tissue. The beta2 integrins (CD 11/ CD18) and alpha4, alpha5, and alpha6beta1 (VLA-4, -5, and -6) integrins each contributed to this PMN migration. Here we report that on human blood leukocytes, alpha9beta1 (VLA-9) is expressed only on PMNs and that it is up-regulated after PMN activation. Based on monoclonal antibody (mAb) blocking studies, alpha9beta1 integrin contributed to C5a-induced PMN migration through fibroblast (HLF and HSF) barriers. This role was apparent only when alternate mechanisms such as CD18, alpha4, alpha5, and alpha6beta1 integrins were blocked and then mAb to alpha9beta1 integrin inhibited the residual PMN migration (by 40-50%) through the HLF or HSF barrier, resulting in > or = 75% inhibition overall. In contrast, PMN migration across interleukin-1-activated endothelium (HUVEC) in response to a C5a gradient, which is partly (30-40%) via CD11/CD18-independent mechanisms, was not inhibited by adhesion blocking by mAbs to alpha4, alpha5, alpha6, and alpha9beta1 even in combination. These results indicate that alpha9beta1 integrin on PMN may have a special role, in conjunction with other beta1 integrins, in mediating PMN migration in the extravascular space, and may contribute to differential neutrophil function within tissues.

Pressure is proinflammatory in lung venular capillaries.

Endothelial responses may contribute importantly to the pathology of high vascular pressure. In lung venular capillaries, we determined endothelial [Ca(2+)](i) by the fura-2 ratioing method and fusion pore formation by quantifying the fluorescence of FM1-43. Pressure elevation increased endothelial [Ca(2+)](i). Concomitantly evoked exocytotic events were evident in a novel spatial-temporal pattern of fusion pore formation. Fusion pores formed predominantly at vascular branch points and colocalized with the expression of P-selectin. Blockade of mechanogated Ca(2+) channels inhibited these responses, identifying entry of external Ca(2+) as the critical triggering mechanism. These endothelial responses point to a proinflammatory effect of high vascular pressure that may be relevant in the pathogenesis of pressure-induced lung disease.