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A C Issekutz

Publications and source records attributed to A C Issekutz.

At least 55 records · Page 3Linked to original sources
PubMed1996

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.

Allergens↗
PubMed1996

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.

Animals↗
PubMed1996

Mac-1 (CD11b/CD18) is the predominant beta 2 (CD18) integrin mediating human neutrophil migration through synovial and dermal fibroblast barriers.

Recently we reported that polymorphonuclear leucocyte (PMNL) migration in vitro through a barrier of human synovial fibroblasts (HSF) involves both beta 2 (CD18) and beta 1 (CD29) integrins on the PMNL. Here we investigated the role of the beta 2 integrin family members, lymphocyte function-associated (LFA)-1 (alpha L beta 2 or CD11a/CD18) and Mac-1 ( alpha M beta 2 or CD11b/CD18), in PMNL migration through HSF and human dermal fibroblast (HDF) monolayers. Treatment of PMNL with monoclonal antibody (mAb) to LFA-1 (anti-alpha L) did not inhibit PMNL migration through either monolayer in response to C5a. In contrast, mAb to Mac-1 (Cd11b) inhibited (by 30-40%) PMNL migration, and by virtually the same extent as mAb to the beta 1 integrin chain (CD18) (40% inhibition). Addition of mAb to LFA-1 to mAb to Mac-1 did not result in greater inhibition. This was in contrast to PMNL migration through human umbilical vein endothelium (HUVE) monolayers, where mAb to LFA-1 or to Mac-1 each partially inhibited PMNL transendothelial migration, and when these mAbs were combined, synergistic inhibition of migration was observed, reaching 90-95% inhibition. Intercellular adhesion molecule 1 (ICAM-1) was not required for Mac-1 mediated migration through HSF or HDF, because treatment of the fibroblasts with mAb R6.5 (F(ab)2) to ICAM-1, which blocks the Mac-1 binding site on ICAM-1, did not inhibit PMNL migration. An LFA-1-ICAM-1 mechanism of PMNL migration through HSF and HDF monolayers could be detected after treatment (4 hr) of the monolayers with TNF-alpha plus IFN-gamma, which upregulated ICAM-1 on the fibroblasts. The results demonstrate the beta 2 integrin dependent PMNL migration in connective tissue may involve primarily Mac-1, with little involvement of LFA-1 or ICAM-1, a situation in marked contrast to PMNL migration across endothelium. However, in inflammatory conditions in which TNF-alpha and/or IFN-gamma may be generated, a role for LFA-1-ICAM-1 may be induced.

CD18 Antigens↗
PubMed1996

Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen-4 integrins suppresses neutrophil and T-lymphocyte migration to the joints and improves clinical disease.

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the alpha 4 integrin, very late activation antigen-4 (VLA-4), and beta 2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti-alpha 4 (TA-2) or anti-beta 2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti-alpha 4 6.6; anti-beta 2 6.8; anti-alpha 4 + anti-beta 2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti-alpha 4 + anti-beta 2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti-alpha 4 + anti-beta 2 7.9). Treatment with anti-alpha 4 + anti-beta 2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66-79% and of spleen T lymphocytes by 56-75%, depending on the joint. In contrast, PMNL migration was abolished (> 98%) and T-cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to alpha 4 and beta 2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T-lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from alpha 4 (CD49d) and beta 2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.

Animals↗
PubMed1996

Expression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.

In chronic inflammatory conditions, mononuclear cells infiltrate the involved connective tissue and may persist, perhaps because of binding to adhesion molecules on connective tissue cells, as well as extracellular matrix. Here we investigated whether vascular cell adhesion molecule-1 (VCAM-1) may be induced on human dermal fibroblasts by proinflammatory cytokines. Expression of VCAM-1 was determined by Northern blotting, cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Only trace amounts of VCAM-1 mRNA or protein were constitutively expressed on dermal fibroblasts but both were rapidly (within 4 hr) upregulated by tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) and somewhat slower (20 hr) by interferon-gamma (IFN-gamma). The combination of TNF-alpha and IFN-gamma had at least an additive effect. The adhesion function of the expressed VCAM-1 was examined by studying the adhesion of the Jurkat T lymphocytes to fibroblasts. Adhesion of Jurkat cells to dermal fibroblasts was markedly increased by stimulation of fibroblasts with TNF-alpha and IFN-gamma (22% of cells adhered versus 9% on unstimulated fibroblasts). The increased adhesion was inhibited to that on unstimulated fibroblasts by monoclonal antibody (mAb) to domain 1 of VCAM-1, but not by mAb to domain 4. MAb to very late antigen-4 (VLA-4), the integrin counter-receptor on lymphocytes for VCAM-1, completely inhibited the increase in Jurkat cell adhesion to activated fibroblasts and partly also inhibited basal adhesion to unstimulated fibroblasts. These results suggest that VCAM-1 expression by dermal fibroblasts is inducible at the mRNA, protein and functional levels by proinflammatory cytokines. The VLA-4/VCAM-1 pathway maybe involved in adhesive interactions between T lymphocytes and activated fibroblasts in the skin during chronic dermal inflammatory conditions.

Blotting, Northern↗
PubMed1996

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.

Adult↗
PubMed1995

Domains 1 and 4 of vascular cell adhesion molecule-1 (CD106) both support very late activation antigen-4 (CD49d/CD29)-dependent monocyte transendothelial migration.

We investigated the role of the 6 domain (6D) and 7 domain (7D) forms of human VCAM-1 as counter-receptors for the alpha 4 beta 1 integrin (VLA-4) in monocyte migration induced by C5a. Across Chinese hamster ovary (CHO) cell monolayers transfected with VCAM-6D or VCAM-7D, monocyte migration was not inhibited by treatment of monocytes with mAb to CD18. Addition of mAb to alpha 4 to the CD18 mAb inhibited monocyte migration by 90% across CHO VCAM-6D and CHO VCAM-7D. mAbs to domain 1 (4B9) or domain 4 (GH12) of VCAM-1 each inhibited migration across CHO VCAM-7D partially, when monocytes were also treated with anti-CD18 mAb. When the VCAM-1 mAbs were combined, migration of these monocytes across CHO VCAM-7D was further inhibited to the same degree as with mAbs to alpha 4 plus CD18. IL-1-treated human umbilical vein endothelium (HUVE) supported CD18-independent, VLA-4-mediated monocyte migration to C5a. A mAb to domain 1 of VCAM-1 almost completely inhibited the CD18-independent migration across HUVE activated with IL-1 for 2 h or 20 h, but was less inhibitory when HUVE was treated with IL-1 for 5 h. However, when mAbs to domain 1 and domain 4 were combined, CD18-independent migration was inhibited completely under all conditions tested. These results suggest that either domain 1 or domain 4 of VCAM-1 can mediate VLA-4-dependent monocyte transendothelial migration, that VLA-4 interaction with these two domains can account for all of the VLA-4-mediated migration, and that the expression of VCAM-1 variants on HUVE depends partly on the duration of IL-1 activation.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Antibodies, Monoclonal↗
PubMed1995

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)

Cell Adhesion Molecules↗
PubMed1995

Migration of human polymorphonuclear leukocytes through a synovial fibroblast barrier is mediated by both beta 2 (CD11/CD18) integrins and the beta 1 (CD29) integrins VLA-5 and VLA-6.

Polymorphonuclear leukocytes (PMNL) accumulate in joint fluid in inflammatory arthritides. We investigated the molecular mechanisms required for PMNL migration through a barrier of human synovial fibroblasts (HSF) grown on microporous filters, as a model of PMNL migration through synovial connective tissue and compared this process with PMNL migration through human dermal fibroblast (HDF) barriers and through human umbilical vein endothelium (HUVE). A small amount of PMNL migration occurred spontaneously only through the synovial fibroblast/filter unit (6-10%). Migration markedly increased through all cell monolayers when the chemotactic factors C5a, IL-8, or zymosan-activated plasma (containing C5adesArg) were added to form a chemotactic gradient. The migration induced by C5a, IL-8, or C5adesArg across HSF was partially inhibited (25-76% depending on stimulus) by mAb to CD18 (beta 2 integrin). The CD18-independent migration induced by IL-8 or C5adesArg was almost completely inhibited by mAbs to beta 1 integrin, but with C5a, inhibition by mAb to beta 1 integrin was only partial (40-50%). Inhibition by mAb to beta 1 integrin required treatment of the PMNL, but not the HSF and was only observed when the function of CD11/CD18 on PMNL was also blocked by a mAb. Treatment of PMNL with mAb to alpha 5 (VLA-5) plus alpha 6 (VLA-6) in combination, was required to inhibit CD18-independent migration through HSF to the degree observed with mAb to beta 1 integrin. There was no qualitative difference in the mechanisms utilized by PMNL for migration through HSF or HDF in response to chemotactic factors. In contrast, PMNL migration across HUVE was almost completely CD18-dependent (85%) with no role for beta 1 integrins. The results suggest that (a) PMNL migration through HSF in response to chemotactic factors utilizes both CD11/CD18 and beta 1 (CD29) integrins; (b) the VLA-5 and VLA-6 members of beta 1 integrins are involved in mediating migration; and (c) PMNL utilize similar mechanisms for migration through HSF and HDF, which are distinct from migration through HUVE.

Antibodies, Monoclonal↗
PubMed1995

Polymorphonuclear leucocyte migration through human dermal fibroblast monolayers is dependent on both beta 2-integrin (CD11/CD18) and beta 1-integrin (CD29) mechanisms.

Accumulation of leucocytes in inflammation involves their migration through vascular endothelium and then in the connective tissue. We investigated human polymorphonuclear leucocyte (PMNL) migration through a biological barrier of human dermal fibroblasts grown on microporous filters, as a model of PMNL migration in the connective tissue. PMNL did not migrate through a fibroblast monolayer unless a chemotactic factor, e.g. C5a, interleukin-8 (IL-8) or zymosan-activated plasma (ZAP; C5adesArg), was added. This migration was partially inhibited (35-70%, depending on the stimulus) by treatment of PMNL with monoclonal antibody (mAb) to CD18 (beta 2-integrins). Most of the CD18-independent migration was inhibited by mAb to beta 1-integrins (CD29). Inhibition by mAb to beta 1 was observed when the PMNL, but not the fibroblasts, were treated with mAb. The role of beta 1-integrins in PMNL transfibroblast migration was detectable only when the function of the CD11-CD18 complex was blocked, because mAb to beta 1-integrin alone had no significant effect on PMNL migration. Migration induced by C5a was more CD18-independent compared to IL-8 or C5adesArg. The CD18-independent migration was also inhibited by mAb to the beta 1-integrin subunits alpha 5 (of very late antigens-5; VLA-5) and alpha 6 (of VLA-6). Treatment of the fibroblasts (4 hr) with tumour necrosis factor-alpha (TNF-alpha) or IL-1 alpha enhanced C5a-induced PMNL transfibroblast migration and increased the proportion of migration utilizing the CD11-CD18 mechanism. However, TNF-alpha treatment had no effect on the degree of beta 1-integrin-dependent migration. These findings suggest that in response to the chemotactic factors C5a, IL-8 and C5adesArg, PMNL migration in the connective tissue is mediated by both CD11-CD18 (beta 2) and beta 1-integrins on the PMNL. The VLA-5 and VLA-6 members of beta 1-integrins are involved in this process. This is in contrast to PMNL migration across endothelium in this system, which is virtually all CD18 dependent with no significant role for beta 1-integrins.

CD18 Antigens↗
PubMed1994

Neutrophils migrate to delayed-type hypersensitivity reactions in joints, but not in skin. Mechanism is leukocyte function-associated antigen-1-/Mac-1-independent.

We have previously found that polymorphonuclear leukocyte (PMNL) migration to adjuvant arthritic joints of rats was only partially inhibited by mAbs to the adhesion molecules LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), suggesting that there is a CD11/CD18-independent mechanism for PMNL migration to inflamed joints. Adjuvant arthritis in rats is believed to be initiated by a T lymphocyte-dependent immune response and maintained by proinflammatory cytokines such as IL-1 and TNF-alpha. Here we studied two types of joint inflammation: that induced by a delayed-type hypersensitivity (DTH) reaction in the joint and that induced by intra-articular (i.a.) injection of cytokines, to explore PMNL migration to inflamed joints and examine the role of CD18. 51Cr-labeled blood PMNL were used to measure PMNL migration in rats to inflammatory reactions in joints and compared with reactions in skin. A large number of PMNL migrated to the carpal and talar joints after i.a. injection of Mycobacterium purified protein derivative in sensitized animals to induce DTH, but there was minimal PMNL migration to this DTH reaction in the skin. This migration to the joints was not inhibited by mAbs to LFA-1 alone or mAbs to LFA-1 plus Mac-1 that almost completely inhibited PMNL accumulation in dermal inflammatory reactions induced by zymosan-activated serum (C5adesArg), endotoxin, IL-1 alpha, or TNF-alpha in the same rats. Intra-articular injection of the cytokines IL-1 alpha and TNF-alpha, but not IFN-gamma, induced marked PMNL accumulation in the joints; this was strongly inhibited by the treatment of rats with anti-LFA-1 and anti-Mac-1. Thus, PMNL migrate to DTH induced in joints but not in skin, and this migration is CD18-independent, but migration to i.a. IL-1 alpha and TNF-alpha is largely CD18 dependent in both joints and skin. This suggests that both the joint microenvironment and the T cell dependence of the inflammatory reaction in the joint governs the mechanism of PMNL recruitment.

Animals↗
PubMed1994

The effect of ebselen on T-lymphocyte migration to arthritic joints and dermal inflammatory reactions in the rat.

We have previously observed that ebselen (PZ 51, 2-Phenyl-1,2-Bensoisoselenazol-3-(2H)-one) can inhibit human polymorphonuclear leukocyte (PMNL) transendothelial migration in vitro and PMNL migration to arthritic joints and dermal inflammatory reactions in rats. In this study, we investigated the effect of ebselen on T-lymphocyte migration to the inflamed joints in rats with adjuvant arthritis (AA) and to dermal inflammation induced by cytokines (IFN gamma, mTNF alpha), cytokine inducing stimuli (poly I:C and LPS), or a delayed type hypersensitivity (DTH) reaction. Treatment of rats with AA with ebselen (100 mg/kg/day) p.o. for three days significantly reduced accumulation of 111In-labelled spleen T-cells (SPLT) in the arthritic joints, including forepaws, carpal joints, hindpaws and talar joints, and in all the above dermal inflammatory reactions. The inhibitory effect of ebselen on SPLT cell accumulation was greater than with indomethacin (2 mg/kg/day) and was observed within 3 h of initiation of ebselen treatment. Ebselen also inhibited SPLT migration to mandibular, axillary and mesenteric lymph nodes, and to the spleen. The results suggest that not only does ebselen inhibit SPLT migration to inflamed joints and to dermal inflammation but it also may inhibit lymphocyte homing and recirculation. Whether these effects of ebselen are related to its reported inhibition of cellular activation and intracellular signalling requires further investigation. However, the inhibition of T-lymphocyte migration reported here and of PMNL migration reported previously may both be beneficial in the treatment of human arthritis.

Animals↗
PubMed1994

The role of tumour necrosis factor-alpha and IL-1 in polymorphonuclear leucocyte and T lymphocyte recruitment to joint inflammation in adjuvant arthritis.

The mediators involved in leucocyte recruitment to joints during arthritis are not fully defined, but two important proinflammatory cytokines, IL-1 and tumour necrosis factor-alpha (TNF-alpha), are produced in joints in rheumatoid arthritis (RA). We investigated in the rat adjuvant arthritis model whether endogenous IL-1 and TNF-alpha contribute to joint inflammation and polymorphonuclear leucocyte (PMNL) and T lymphocyte infiltration. The migration of 51Cr-labelled rat blood PMNL and 111In-labelled T lymphocytes to the joints of rats with adjuvant arthritis was measured along with plasma protein extravasation, which was quantified using 125I-labelled human albumin. Rats with active arthritis of 5 days' duration received i.p. non-immune serum, polyclonal neutralizing anti-serum to rat TNF-alpha, antiserum to IL-1 alpha and IL-1 beta, or both anti-TNF plus anti-IL-1 for 5 days. Treatment with anti-IL-1 alpha and IL-1 beta did not affect plasma protein extravasation, or PMNL or T lymphocyte accumulation in the joints (i.e. talar joint, hind paws, and tail) despite the fact that this treatment inhibited 80-90% of the PMNL migration into dermal sites injected with IL-1 alpha or IL-1 beta. In contrast, anti-TNF-alpha treatment significantly improved clinical scores, decreased plasma protein extravasation by 60-80%, inhibited PMNL accumulation by 40-50% and decreased T lymphocyte accumulation by 30-50%. Treatment with anti-IL-1, together with anti-TNF-alpha, significantly potentiated the inhibition of T lymphocyte accumulation observed with anti-TNF-alpha alone. These results indicate that endogenous TNF-alpha production may play an important role in the inflammatory changes and leucocyte recruitment in this experimental model of human arthritis, while IL-1 may have a less important role in leucocyte recruitment to these joints.

Animals↗
PubMed1993

Quantitation and kinetics of blood monocyte migration to acute inflammatory reactions, and IL-1 alpha, tumor necrosis factor-alpha, and IFN-gamma.

Monocytes migrate from the blood into acute inflammatory reactions, where they differentiate into macrophages, and after 12 to 24 h become the predominant histologic feature of the inflammatory infiltrate. The quantitation of monocyte migration into these reactions has been difficult. This report employs a novel combination of techniques to isolate highly purified monocytes from the blood of rats, and shows that these cells have a normal t1/2 of 26 h and migrate efficiently after i.v. injection into cutaneous acute inflammatory sites. Monocytes labeled with 51Cr accumulated in delayed-type hypersensitivity reactions, and sites injected with killed Escherichia coli, LPS, poly-inosine: cytosine, zymosan-activated serum (ZAS), a source of C5adesArg, and the cytokines IL-1 alpha, TNF-alpha, and IFN-gamma. Both radiolabeled monocytes and neutrophils migrated rapidly to E. coli, LPS, ZAS, IL-1 alpha, and TNF-alpha with a large increase in cell accumulation by 2 h. Neutrophil migration declined rapidly to undetectable levels by 3 to 4 h to all five stimuli, and monocyte migration to ZAS and IL-1 alpha also declined by this time. In contrast, E. coli, LPS, and TNF-alpha caused a sustained migration of monocytes for 5 to 6 h, long after neutrophils had stopped accumulating. Intradermal IFN-gamma did not recruit neutrophils but stimulated a prolonged monocyte migration from 1 to 6 h. Combinations of LPS, TNF-alpha, and IFN-gamma synergistically enhanced the late (> 5 h) but not the early phase of monocyte recruitment. In conclusion, purified monocytes isolated from rat blood can be used to quantify monocyte migration in vivo, and these cells migrate rapidly to cutaneous inflammation and in response to chemotactic factors, IL-1 alpha, TNF-alpha, and IFN-gamma, with initial kinetics similar to those of neutrophils. However, monocyte-selective mechanisms are induced by IFN-gamma and also appear to be involved in prolonged monocyte migration to TNF-alpha, LPS, and E. coli.

Acute Disease↗
PubMed1993

A major portion of polymorphonuclear leukocyte and T lymphocyte migration to arthritic joints in the rat is via LFA-1/MAC-1-independent mechanisms.

The importance of the leukocyte CD11/CD18 beta 2 integrins for leukocyte adhesion and for leukocyte migration to inflamed tissues is well recognized. However, the role of CD11/CD18 molecules and their subtypes, such as LFA-1 and MAC-1, in leukocyte migration to arthritis is not known. Here we report the effects of a new monoclonal antibody (mAb) TA-3, which recognizes and blocks rat LFA-1 (CD11a/CD18), on the migration of 51Cr-labeled rat blood polymorphonuclear leukocytes (PMNL) and 111In-labeled spleen T lymphocytes into the inflamed joints of rats with adjuvant arthritis and to dermal inflammation. The mAb MRC OX-42, which reacts with and blocks rat MAC-1 (CD11b/CD18), was also evaluated. In established arthritis, iv anti-LFA-1 (TA-3) inhibited, up to 50% (P < 0.006) of PMNL accumulation in the talar and metatarsal joints but had no effect on T lymphocyte migration to the joints. In the same animals, anti-LFA-1 partially (35-50%) inhibited PMNL accumulation in acute dermal inflammation induced by zymosan-activated serum (ZAS, C5ades Arg), IL-1 or endotoxin (P < 0.01) and suppressed T lymphocyte migration to dermal DTH, IFN-gamma, LPS, and poly-I:C-induced inflammation by 60-80%. Anti-MAC-1 (OX-42) by itself had no effect on PMNL migration. Anti-MAC-1 combined with anti-LFA-1 did not inhibit PMNL or T lymphocyte migration to joints any more than anti-LFA-1 alone. However, the addition of anti-MAC-1 to anti-LFA-1 further inhibited PMNL migration to acute dermal inflammation by up to 90%. These findings demonstrate that LFA-1 on PMNL plays a major role in PMNL migration but not in T cell migration to arthritic joints. MAC-1 on PMNL appears less important. Furthermore, while LFA-1 and MAC-1 on PMNL and LFA-1 on T lymphocytes mediate much of the migration to dermal inflammation, the migration of these cells to arthritis has a major LFA-1/MAC-1-independent component.

Animals↗