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.).

Monocyte migration to arthritis in the rat utilizes both CD11/CD18 and very late activation antigen 4 integrin mechanisms.

In human and experimental models of arthritis, blood monocytes migrate into the inflamed synovium and joint space. The mechanisms required for monocyte migration across the vascular endothelium in joints is poorly defined. Radiolabeled rat blood monocytes were used to measure monocyte migration to the inflamed joints of rats with adjuvant arthritis, and the role of monocyte adhesion molecules was analyzed. Monocyte accumulation in the inflamed joints was maximal 14-21 d after immunization with adjuvant, when arthritis had fully developed. Blocking mAbs to lymphocyte function-associated antigen 1 (LFA-1), Mac-1, and very late activation antigen 4 (VLA-4) were used to evaluate the role of these integrins in the migration. Migration to the joints was not inhibited by treatment of the animals with mAb to LFA-1, Mac-1, or VLA-4 alone, and was partially (50%) inhibited in only the most arthritic joint, the talar joint, by the combination of mAb to LFA-1 plus Mac-1. In contrast, this combination inhibited migration to dermal inflammation induced by C5ades Arg, endotoxin, tumor necrosis factor alpha, and polyinosine-cytosine by 60-70%. When mAbs to LFA-1 and VLA-4 were combined, migration to all the inflamed joints was strongly inhibited (80-98%, depending on the joint). Treatment with the combination of the three mAbs to LFA-1, Mac-1, and VLA-4 completely eliminated monocyte migration to all joints and dermal inflammation. The results show that 51Cr blood monocytes can be used to quantify monocyte migration to arthritic joints in the rat. LFA-1 alone or VLA-4 alone is sufficient to mediate most of this migration, and either LFA-1 or VLA-4 is required for monocyte migration to joint inflammation. These results indicate that both the VLA-4 and LFA-1 integrins should be therapeutic targets for suppression of monocyte infiltration of joints in arthritis.

Rat blood neutrophils express very late antigen 4 and it mediates migration to arthritic joint and dermal inflammation.

Blood neutrophils contribute to joint injury in human and experimental models of arthritis. Neutrophil migration out of the blood in joint inflammation involves both the CD18 (beta2) integrins and a CD18 integrin-independent pathway. To investigate this migration, radiolabeled rat blood neutrophils were used to measure neutrophil accumulation in the inflamed joints of rats with adjuvant arthritis and the role of leukocyte integrins in migration to these joints and to dermal inflammation was determined. Neutrophils migrated rapidly (<2 h) to the inflamed joints 14-18 d after immunization with adjuvant. Blocking monoclonal antibodies (mAbs) to both LFA-1 and Mac-1 together, as well as a mAb to CD18, inhibited neutrophil accumulation in the inflamed joints by 50-75%. However, migration to dermal inflammation induced by C5a(des Arg)' tumor necrosis factor alpha, lipopolysaccharide, and poly-inosine:cytosine was inhibited by approximately 90%. Flow cytometry revealed the expression of low levels of very late antigen 4 (VLA-4) on nearly all rat blood neutrophils. Treatment with anti-VLA-4 plus anti-LFA-1 but neither mAb alone, strongly (60-75%) inhibited neutrophil accumulation in arthritic joints. This mAb combination also inhibited neutrophil migration to dermal inflammatory reactions by 30-70%. Blocking VLA-4 together with the CD18 integrins inhibited neutrophil accumulation by 95-99%, virtually abolishing neutrophil accumulation in cutaneous inflammation. A similar blockade of VLA-4 and CD18 decreased neutrophil accumulation in the inflamed joints by 70-83%, but a significant portion of the neutrophil accumulation to these joints still remained. In conclusion, rat blood neutrophils express functional VLA-4 that can mediate neutrophil migration to both inflamed joints and dermal inflammatory sites. VLA-4 appears to be able to substitute for LFA-1 in this migration and is particularly important for accumulation in inflamed joints. However, there exists an additional CD18- and VLA-4-independent pathway of neutrophil migration to arthritic joints that is not involved in acute dermal inflammation.

VLA-4 integrin can mediate CD11/CD18-independent transendothelial migration of human monocytes.

The migration of human monocytes across unactivated and activated human umbilical vein endothelium (HUVE) in response to chemotactic factors was studied, and the adhesion molecules involved were characterized. Migration of blood monocytes or U937 cell line-derived monocytes across unactivated HUVE induced by C5a, was partially inhibited (by 75%) by mAbs (R15.7 or 60.3) to CD18 of the CD11/CD18 complex on the monocyte. However, when the HUVE was pretreated for 5 h with IL-1 alpha (0.1 ng/ml), TNF-alpha (100 U/ml), or LPS (1 ng/ml), migration induced by C5a was no longer inhibited; i.e., migration became CD18 independent. The monocyte CD18-independent migration was completely blocked by mAbs against alpha 4 or beta 1 integrin chains of VLA-4. This migration was also partially inhibited by mAbs against vascular cell adhesion molecule-1 (VCAM-1), a major counter-receptor on HUVE for VLA-4, but not by mAbs to E-selectin or intercellular adhesion molecule-1. The significant CD18-independent migration across "unactivated" HUVE was also inhibited by mAbs against alpha 4 or beta 1 chains of VLA-4, although mAbs against VCAM-1 did not inhibit under these conditions. Finally, considerable VLA-4-dependent transendothelial migration to C5a was also observed with monocytes from a patient with CD18 deficiency (leukocyte adhesion deficiency). These results suggest that (a) there is a major CD18-independent component in monocyte chemotactic factor-dependent migration across activated and unactivated endothelium; (b) that VLA-4 integrin on the monocyte has a major role in this migration; and (c) that VCAM-1 on activated endothelium functions as a counter-receptor in this process, but other ligands for VLA-4, especially on unactivated endothelium, may also be involved.

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.

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.

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.

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.

CD11/CD18-independent transendothelial migration of human polymorphonuclear leukocytes and monocytes: involvement of distinct and unique mechanisms.

Monocytes and polymorphonuclear leukocytes (PMNLs) migrate across cytokine (interleukin-1, tumor necrosis factor) activated endothelium or unstimulated endothelium in response to chemotactic factors in vitro and in vivo utilizing the CD11/CD18 (i.e., beta 2 integrin) adhesion molecule complex. However, in vivo studies have suggested that under some conditions and/or in certain tissues, leukocyte migration can also proceed via CD11/CD18-independent mechanisms. Here we compared adhesion mechanisms involved in the migration of 51Cr-labeled blood monocytes and PMNLs across human umbilical vein endothelium (HUVE) monolayers. We observed that monocyte transendothelial migration was not inhibited by monoclonal antibody (mAb) to CD18, when the HUVE was activated with IL-1 and the chemotactic factor C5a induced the migration. This CD18-independent monocyte migration was blocked by treatment of the monocyte with mAb to beta 1 or alpha 4 integrin, suggesting that very late activation antigen 4 (VLA-4) on the monocyte served as the alternative migration mechanism. In contrast to monocytes, mAb to CD18 inhibited PMNL migration to C5a across IL-1-activated HUVE, but only by 66%, significantly less than with C5a alone (84%) or IL-1-activated HUVE alone (95%). The migration of anti-CD18 mAb-treated PMNLs was not inhibited by function-blocking mAbs to sialyl Lewisx, L-selectin, beta 1 or alpha 4 integrin, the beta 3-related leukocyte response integrin, IL-8, or platelet-activating factor (PAF) antagonists, alone or in combination. Antibody-blocking studies of the ligands on HUVE indicated that E-selectin may be partially involved in this CD18-independent PMNL migration but that ICAM-1, VCAM-1, PECAM-1, and P-selectin are not involved. Of several chemotactic factors tested, C5a and C5adesArg in activated plasma were the most active in inducing CD18-independent migration of PMNLs across IL-1-activated HUVE. These results demonstrate that (1) monocytes can utilize VLA-4 for optimal transendothelial migration and (2) PMNLs may have a novel CD18-independent migration mechanism that is activated by C5a in conjunction with one or more ligands on cytokine-activated endothelium. This may involve, in part, E-selectin interacting with a yet to be identified counterreceptor on PMNLs.

Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration.

We evaluated the relative contribution of ICAM-1 and ICAM-2, known ligands on endothelium for LFA-1 and Mac-1, in spontaneous neutrophil (PMN) transendothelial migration (TEM) across IL-1-activated HUVEC monolayers or TEM induced by C5a or IL-8 across unstimulated HUVEC grown on polycarbonate filters. Adhesion blocking mAb to ICAM-1 [R6.5 F(ab)2] or ICAM-2 [CBR IC2/2 F(ab)2] tended to inhibit TEM under each condition but, in general, inhibition was significant only with both ICAM-1 and ICAM-2 blockade. mAb to LFA-1 partially inhibited migration to C5a or IL-8 across unstimulated HUVEC and inhibition was not altered by additional treatment of HUVEC with mAbs to ICAM-1 and -2. In contrast, with IL-1 HUVEC, mAb to ICAM-1 significantly inhibited this LFA-1-independent TEM. mAb to Mac-1 alone partially inhibited TEM and, when combined with mAb to LFA-1, migration was almost completely blocked with all TEM conditions tested. The contribution of alternate ligands for Mac-1 in mediating Mac-1-dependent but ICAM-1/-2-independent C5a-induced TEM was examined using anti-LFA-1-treated PMN and anti-ICAM-treated resting HUVEC. Addition of RGD peptides, fibronectin, fibrinogen, heparins, collagens alone or in combination, even to heparinase-treated HUVEC, did not inhibit this Mac-1-mediated PMN TEM. The results indicate that: (1) LFA-1 mediates PMN TEM primarily by interaction with ICAM-1 and ICAM-2; (2) ICAM-2 may function in concert with ICAM-1 in this role, especially on unstimulated endothelium, and (3) Mac-1 on PMN also plays a major role in TEM and can utilize yet to be identified ligands distinct from ICAM-1 or -2, especially on unstimulated endothelium.

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.

An endotoxin-induced factor distinct from interleukin-1 and tumour necrosis factor alpha produced by the THP-1 human macrophage line stimulates polymorphonuclear leukocyte infiltration in vivo.

Endotoxin and gram-negative bacteria induce vigorous inflammatory reactions. Our previous work showed that rabbit macrophages (M phi) incubated with endotoxin produce a 45,000 dalton protein that recruited polymorphonuclear leukocytes (PMNL) into the skin of rabbits. This factor was separated from interleukin-1 (IL-1) but could not be unequivocally distinguished from rabbit tumour necrosis factor (TNF alpha). Here we have examined the human M phi cell line, THP-1, for the production of an analogous protein. After exposure to phorbol diester the THP-1 cells assumed the characteristic M phi phenotype and function. During 6 hours of culture with LPS these M phi released a factor(s) that caused PMNL recruitment into the skin of rabbits when injected intradermally, measured using 51Cr-labelled blood leukocytes. This activity, referred to as PMNL recruiting activity (PRA), was heat labile, and its production was blocked by cycloheximide, suggesting that this is most likely a de novo synthesized protein. Sephadex-G 100 and Superose-12 FPLC chromatography indicated a molecular weight in the 45,000-65,000 dalton range. The active fractions were free of IL-1 activity (less than 0.2 U/ml), and Superose-12 chromatography separated the peak of PRA, which eluted around 45,000 daltons, from TNF alpha eluting at 20,000 daltons. The peak PRA was not neutralized by antiserum to IL-1 alpha, IL-1 beta TNF alpha, IL-6, and granulocyte-macrophage colony-stimulating factor (GMCSF), indicating that it was distinct immunologically from these cytokines. The major PRA did not induce migration of rabbit or human PMNLs in vitro in a Boyden chamber chemotaxis assay, although peaks of chemotactic activity and weak PMNL recruitment in vivo were detected in fractions eluting around 15,000 daltons and 800 daltons. The generation of PRA by a human M phi cell line is analogous to that reported previously with rabbit M phi. Here we extend these observations to a human M phi system and confirm that this molecule is distinct from several other M phi cytokines and M phi chemotactic factors with inflammatory properties.

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.

IL-1 activation of endothelium supports VLA-4 (CD49d/CD29)-mediated monocyte transendothelial migration to C5a, MIP-1 alpha, RANTES, and PAF but inhibits migration to MCP-1: a regulatory role for endothelium-derived MCP-1.

We investigated the effect of interleukin-1 (IL-1) activation of human umbilical vein endothelium (HUVE) on human monocyte transendothelial migration induced by chemotactic factors. Monocyte migration across unactivated endothelium in response to macrophage inflammatory protein-1 alpha (MIP-1 alpha), RANTES, platelet-activating factor (PAF), or monocyte chemoattractant protein-1 (MCP-1) was completely inhibited (90%) by monoclonal antibodies (mAbs; 60.3) to CD18 of the CD11/CD18 complex on the monocyte and partially inhibited (by 75%) in response to C5a. When the HUVE was stimulated with IL-1 alpha (5 h, 0.1 ng/ml), monocyte migration in response to C5a, MIP-1 alpha, RANTES, or PAF was no longer inhibited by mAb to CD18. However, migration was blocked by the combination of mAb to the alpha 4-integrin (CD49d) chain of very late antigen-4 (CD49d/CD29) with the mAb to CD18. In contrast to the above stimuli, activation of the HUVE with IL-1 alpha inhibited the transendothelial migration of monocytes in response to MCP-1. mAbs to the adhesion molecules up-regulated on HUVE by IL-1, i.e., E-selectin (CD62E), intercellular adhesion molecule-1 (CD54) or vascular cell adhesion molecule-1 (CD106), did not reverse the inhibitory effect. Transendothelial migration in response to MCP-1 but not to C5a was inhibited by the treatment of monocytes with culture supernatant from IL-1 alpha-stimulated (but not from unstimulated) HUVE. Such supernatant contained chemotactic activity for monocytes, and a mAb to MCP-1 blocked the migration inhibitory effect of IL-1 activation of the HUVE monolayer, as well as the chemotactic activity in the supernatant from IL-1-stimulated HUVE. The inhibitory effect on migration of IL-1-stimulated HUVE was specific for monocytes because polymorphonuclear leukocyte transendothelial migration in response to IL-8 (a related chemokine) was not inhibited by IL-1 activation of HUVE.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Deficient tumor necrosis factor secretion by cord blood mononuclear cells upon in vitro stimulation with Listeria monocytogenes.

We examined the production of tumor necrosis factor (TNF) by mononuclear cells (MNC) after incubating adult or cord blood MNC with Listeria monocytogenes in vitro. With adult MNC cultures, we found that TNF activity reached a peak at 6 h (606 +/- 120 x 10(3) units/liter) and declined to the baseline by day 3. In contrast, using cord blood MNC, we found that TNF activity increased gradually reaching a peak at 24 h. In addition, the peak TNF activity using newborn MNC (189 +/- 26 x 10(3) U/liter) at 24 h was still lower than the peak using adult MNC at 6 h (p < 0.0002). In seeking an explanation for the decreased TNF secretion from newborn MNC, we examined the possibility that newborn cells produce TNF but failed to secrete it. However, lysates of newborn cells contained functionally and antigenically less TNF than adult cells. Based on these observations, we conclude that the overall TNF production by newborn cells incubated with L monocytogenes is decreased compared with similarly stimulated adult cells.

Removal of gram-negative endotoxin from solutions by affinity chromatography.

Endotoxins liberated by gram-negative bacteria are frequent contaminants of aqueous and physiological solutions. Because of their potent biological effects in vivo and in vitro, it is often necessary to eliminate even minute quantities of endotoxin from such solutions. A process is described which exploits the high binding affinity of polymyxin B for the lipid A moiety of most endotoxins in order to remove endotoxins from solutions by chromatography on polymyxin B Sepharose 4B. This method was simple, very effective, resulting in essentially complete removal of several endotoxins from heavily contaminated solutions (1-10 micrograms/ml by Limulus amoebocyte lysate assay) and employed mild physiological conditions.

Quantitation of eosinophil and neutrophil infiltration into rat lung by specific assays for eosinophil peroxidase and myeloperoxidase. Application in a Brown Norway rat model of allergic pulmonary inflammation.

Conditions for measuring selectively eosinophil peroxidase (EPO) and the neutrophil myeloperoxidase (MPO) in inflamed rat lung were determined. EPO could be specifically measured with o-phenylene diamine as chromogen at pH 8.0 in the presence of 3 mM bromide and MPO with tetramethylbenzidine as chromogen at pH 5.0 in the absence of bromide but with the EPO inhibitor, resorcinol. Aeroallergen challenge of sensitized Brown Norway rats with ovalbumin, but not with saline, resulted in a pronounced eosinophilic lung inflammation with some focal hemorrhages and an increase in lung wet weights. Quantitation of the eosinophil and neutrophil accumulation required lyophilization of lung samples, a hypotonic wash to remove contaminating hemoglobin, which interfered with the MPO assay, followed by extraction with the detergent cetyltrimethylammonium chloride. Based on lung EPO and MPO activities and standardization of enzyme activity with purified eosinophils and neutrophils, the total number of eosinophils and neutrophils in the lungs was calculated at 24 h (n = 19), 48 h (n = 9) and 72 h (n = 4) after challenge, as 56 +/- 6.4 x 10(6), 119 +/- 28 x 10(6) and 108 +/- 33 x 10(6) for eosinophils, respectively, and 94 +/- 6.8 x 10(6), 49 +/- 5.0 x 10(6) and 32 +/- 5.5 x 10(6) for neutrophils, respectively. We conclude that, with the assay conditions outlined here, EPO and MPO can be used to quantitate the tissue infiltration of eosinophils and neutrophils in the rat even in mixed inflammatory reactions.

Interleukin-1 and tumour necrosis factor alpha induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: the effect of apical versus basal monolayer stimulation.

The cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) enhance polymorphonuclear leukocyte (PMNL) adhesion to vascular endothelium by an endothelial cell dependent mechanism in vitro and induce PMNL infiltration in vivo In this study, we employed human umbilical vein endothelium (HUVE) cultured on microporous membrane filters to form a monolayer, a system in which PMNL adherence and PMNL transendothelial migration could be measured using 51Cr-labelled human PMNL. In this system, it was found that PMNL adhesion and migration were dependent on prior treatment of the HUVE monolayer with IL-1 or TNF alpha for at least 2 h and that cytokine could be removed prior to the addition of PMNL without any effect on the response. PMNL adherence to the HUVE was maximal by 30 min and was followed by progressive migration of PMNL across the monolayer and the membrane filter into the lower chamber. The effect of apical surface versus basal surface exposure of the HUVE monolayer to IL-1 alpha and TNF alpha on subsequent PMNL interaction with the HUVE monolayer in the absence of cytokine was examined. Apical or basal stimulation induced comparable PMNL adherence at 30 min following addition of PMNL (35.5% and 43.1%). However, basal (i.e., abluminal) exposure to IL-1 or TNF alpha of the HUVE induced significantly greater PMNL transendothelial migration (e.g., 27.8% vs. 15.4%; P less than 0.01). The expression of endothelial-leukocyte adhesion molecules ELAM-1 and ICAM-1 following apical versus basal stimulation was determined by ELISA on viable cells. These adhesion molecules were upregulated to a similar extent under both conditions. These observations suggest that spacial localization or orientation of adhesion molecules may be influenced by basal versus apical cytokine stimulation or that other mechanisms are responsible for the preferential PMNL migration with basal stimulation. These findings may have implications for the in vivo interactions of PMNL with vascular endothelium, depending on whether the endothelium is exposed to IL-1 of TNF alpha via the blood on the luminal (apical) surface or via the extravascular space on the abluminal (basal) surface.

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma.

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.