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1.
J Immunol ; 163(5): 2844-9, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10453030

ABSTRACT

P-selectin plays an important role in leukocyte adherence to microvascular endothelium and is expressed in synovial tissue from patients with rheumatoid arthritis (RA). However, the contribution of P-selectin to the initiation and chronicity of joint inflammation is not well understood. In these studies, collagen-induced arthritis (CIA) was induced in P-selectin mutant (-/-) mice to explore the role of P-selectin in the development of joint inflammation. Surprisingly, CIA onset was accelerated and severity was increased in P-selectin mutant mice, compared with wild-type mice (+/+). Increased levels of anti-type II collagen IgG were detected in both nonarthritic and arthritic P-selectin mutant mice from days 14-91. In addition, splenocytes isolated from immunized and nonimmunized P-selectin mutant mice produced significantly less IL-2 and IL-4, but significantly higher levels of IL-10 and IL-5 than splenocytes from wild-type mice. These observations show that P-selectin-mediated leukocyte rolling is not required for the development of murine CIA and that P-selectin expression exerts a controlling effect on the development of Ag-driven inflammatory joint disease, possibly by mediating the recruitment and/or trafficking of specific leukocyte subtypes into lymphoid tissue or inflammatory foci.


Subject(s)
Arthritis, Experimental/etiology , Arthritis, Experimental/genetics , Collagen/immunology , P-Selectin/genetics , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Autoantibodies/biosynthesis , Autoantibodies/blood , Cytokines/biosynthesis , Disease Progression , Female , Forelimb , Hindlimb , Incidence , Male , Mice , Mice, Knockout , Severity of Illness Index , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Wrist Joint/pathology
2.
Am J Respir Cell Mol Biol ; 18(2): 158-67, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9476902

ABSTRACT

We investigated the effects of in vivo intraperitoneal treatment with the rat monoclonal antibody (mAb), YN1.7.4 (YN1) against intercellular adhesion molecule-1 (ICAM-1) on the ovalbumin (OA)-inhalation-induced infiltration of leukocytes into the airways of OA-sensitized mice. YN1 (100 to 400 microg) given over a period of 72 h dose-dependently reduced the influx of lymphocytes and eosinophils into the bronchial lumen by > 60% and > or = 70%, respectively, when compared with saline or purified rat IgG-treated controls. Alveolar macrophages (AM) in the bronchoalveolar lavage fluid (BALF) were also decreased by > 50%. Lung tissue inflammation as determined by histopathologic examination was reduced. The number of neutrophils in the blood of OA-sensitized mice 3 days after challenge was significantly increased by treatment with YN1. However, at 24 h and 72 h after OA-challenge, the numbers of eosinophils and mononuclear cells in the bone marrow were reduced by YN1 treatment. Additionally, at 72 h after OA-challenge, the numbers of bone-marrow neutrophils were depressed. BALF levels of interleukin-5 (IL-5) and of IgA were lower for YN1-treated mice than for controls. With increasing doses of YN1, the levels of anti-ICAM-1 mAb in the plasma were proportionally increased. To correlate these results with YN1 treatment, blood and BALF T cells and BALF eosinophils were examined with flow cytometry. Blood T cells from YN1-treated mice were unable to bind phycoerythrin (PE)-labeled anti-ICAM- mAb ex vivo. These results implied that ICAM-1 on these cells was bound (occupied) by YN1 administered in vivo. Dose-related decreases were observed in the percentage and mean channel fluorescence (MCF) values of ICAM-1+ BALF T cells and eosinophils. The percentages of CD11a+ or CD49d+ eosinophils were also suppressed. Our data suggest that ICAM-1 is an important molecule involved in the recruitment of leukocytes into the airways of sensitized mice after pulmonary challenge.


Subject(s)
Bronchoalveolar Lavage Fluid/immunology , Eosinophils/immunology , Intercellular Adhesion Molecule-1/immunology , Lung/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/metabolism , Antigens/immunology , Bone Marrow Cells , Chemotaxis, Leukocyte/immunology , Disease Models, Animal , Eosinophils/metabolism , Female , Immunoglobulin A/analysis , Interleukin-5/analysis , Leukocytes/immunology , Macrophages, Alveolar/immunology , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Pulmonary Eosinophilia/immunology , Rats , T-Lymphocytes/metabolism
3.
Bioorg Med Chem Lett ; 8(9): 1093-6, 1998 May 05.
Article in English | MEDLINE | ID: mdl-9871714

ABSTRACT

Bisphosphonate ester 2 is an inhibitor of inflammation, but is devoid of antiarthritic effects. SAR studies on a series of related bisphosphonate esters resulted in compounds 6e, 6i, 6j, and 6m, which exhibited excellent inhibition of an arthritis model, in addition to potent anti-inflammatory effects.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Arthritis, Experimental/drug therapy , Diphosphonates/chemical synthesis , Inflammation/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Diphosphonates/chemistry , Diphosphonates/therapeutic use , Drug Design , Granuloma/drug therapy , Granuloma/immunology , Hypersensitivity, Delayed , Indicators and Reagents , Mice , Molecular Structure , Structure-Activity Relationship
4.
Expert Opin Investig Drugs ; 6(7): 805-9, 1997 Jul.
Article in English | MEDLINE | ID: mdl-15989642

ABSTRACT

Drugs used to treat arthritis can be broadly classified into either anti-inflammatory or immunosuppressive/immunomodulatory agents. Non-steroidal anti-inflammatory drugs (NSAIDs) are used for both osteoarthritis (OA) and rheumatoid arthritis (RA), while drugs with an immunological mechanism of action are only applicable to RA. The total market value for anti-arthritic drugs is estimated to be 8.4 bn dollars, with the OA segment worth 6.9 bn dollars. In contrast, the major RA markets are valued at approximately 1.5 bn dollars. Drugs currently in late phase development reflect the accepted approaches towards treating the symptoms of arthritis, or suppressing immunological mechanisms considered to be the driving force for the underlying disease process in RA. The size of both the RA and OA markets, however, creates an incentive to develop new therapeutics which do not conform to these two approaches, but target new molecular mechanisms. Compounds currently in preclinical development demonstrate that it may be possible to combine anti-inflammatory and slow-acting antirheumatic activity into a single therapeutic. This new generation of anti-arthritic compounds has the potential to redefine the way in which common forms of arthritis, mixed connective tissue diseases and musculoskeletal diseases are treated in the future.

5.
Am J Physiol ; 272(2 Pt 1): L219-29, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9124372

ABSTRACT

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.


Subject(s)
Antigens, CD/physiology , Leukocytes/physiology , Lung/physiopathology , Vascular Cell Adhesion Molecule-1/physiology , Animals , Antibodies, Monoclonal , Bronchi/pathology , Cell Movement , Female , Immunization , Immunohistochemistry/methods , Integrin alpha4 , Leukocytes/pathology , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Pneumonia/immunology , Pneumonia/pathology , Rats , Staining and Labeling
6.
Blood ; 88(8): 2973-9, 1996 Oct 15.
Article in English | MEDLINE | ID: mdl-8874194

ABSTRACT

Leukocyte rolling and emigration in response to inflammatory stimuli appears to involve both E-selectin- and P-selectin-dependent adhesion, which suggests that these molecules have overlapping functions. To clarify their relative contributions in chronic inflammation, we examined delayed-type contact hypersensitivity (DTH) responses in P-selectin, E-selectin, and E-/P-selectin-deficient mice. Oxazolone-induced increases in ear thickness and ear weight were equivalent in wild-type mice and in P-selectin and E-selectin mutants, but were significantly reduced in E-/P-selectin mutants. The number and area of microabscesses on the ears of E-/P-deficient mice were decreased by 72% and 93%, and the number of leukocytes invading the subdermal ear tissue was reduced. T cells from E-/P-deficient mice transferred oxazolone reactivity into naive wild-type mice. However, when donor T cells from wild-type mice were transferred into E-/P-selectin-deficient mice, the DTH response was significantly impaired. These results show that leukocyte recruitment into a subacute inflammatory reaction can occur when either P-selectin or E-selectin is present, but is significantly reduced when both selectins are absent. Both P- and E-selectin are likely to play important roles in the development and maintenance of inflammatory diseases.


Subject(s)
Chemotaxis, Leukocyte/physiology , E-Selectin/physiology , Hypersensitivity, Delayed/physiopathology , P-Selectin/physiology , Abscess/etiology , Adoptive Transfer , Animals , E-Selectin/genetics , Edema/etiology , Hypersensitivity, Delayed/complications , Hypersensitivity, Delayed/pathology , Leukocyte Count , Mice , Mice, Knockout , Oxazolone/toxicity , P-Selectin/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/transplantation
7.
J Immunol ; 157(7): 3153-8, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8816427

ABSTRACT

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the firm adhesion of leukocytes to venular endothelium and facilitates leukocyte extravasation from the vasculature into inflamed tissue. In addition, ICAM-1 is an important costimulatory molecule during Ag presentation to lymphocytes. Using mice deficient in ICAM-1, we have investigated the role of this molecule in the development of collagen-induced arthritis. After immunization with type II collagen, 71% of wild-type mice developed arthritis compared with 50% of ICAM-1 heterozygote mutants and 18% of ICAM-1 homozygous mutants. In those ICAM-1 mutants that developed arthritis, the mean day of onset, the mean number of involved paws, and the severity of paw inflammation were not significantly different from those in wild-type mice. The reduced incidence of arthritis in the ICAM-1 homozygous mutant mice was not due to lack of immunity to type II collagen, since these mice developed similar levels of anti-type II collagen IgG compared with wild-type mice and had a positive delayed-type hypersensitivity reaction to type II collagen. The reduction of arthritis in heterozygous as well as homozygous deficient mice indicates that expression of ICAM-1 can be a pivotal variable in the pathogenesis of collagen-induced arthritis in mice. The results suggest that naturally occurring genetic variation in the expression of ICAM-1 or related inflammatory cell adhesion molecules might influence susceptibility to the complex disease of rheumatoid arthritis in humans and support the concept that pharmacologic approaches to chronic reduction in the expression or the function of ICAM-1 may be of therapeutic value.


Subject(s)
Antigen Presentation , Arthritis/immunology , Collagen/immunology , Intercellular Adhesion Molecule-1/physiology , Animals , Arthritis/etiology , Arthritis/pathology , Arthritis, Rheumatoid/genetics , Cell Adhesion , Disease Models, Animal , Disease Susceptibility , Hypersensitivity, Delayed/immunology , Immunocompromised Host , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Intercellular Adhesion Molecule-1/genetics , Leukocyte Count , Mice , Mice, Inbred DBA , Mice, Knockout
8.
Am J Physiol ; 271(2 Pt 1): L267-76, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8770066

ABSTRACT

We investigated the involvement of intercellular adhesion molecule-1 (ICAM-1; CD54) in ovalbumin (OA) antigen-induced lung inflammation in sensitized Brown Norway (BN) rats by using flow cytometry and in vivo treatment with a murine monoclonal antibody (MAb), 1A29, directed against rat ICAM-1. OA-challenge induced an eosinophil and lymphocyte-rich accumulation of leukocytes into the airway lumen. Between 75 and 90% of the T cells in bronchoalveolar lavage (BAL) fluid after challenge expressed CD54 and CD11a and were of the memory phenotype. 1A29 treatment produced dose-related increases in circulating 1A29 and blood neutrophils. In the BAL fluid of 1A29-treated animals, significant (P < 0.05) reductions in the numbers of eosinophils and lymphocytes, but not neutrophils or alveolar macrophages, were observed in association with a reduced inflammatory pathology in lung tissue. 1A29 administration reduced the number of detectable ICAM-1 binding sites on T cells in peripheral blood and BAL fluid examined ex vivo by flow cytometry. We conclude that ICAM-1 is critically important for the antigen-specific recruitment of eosinophils and lymphocytes into the lungs.


Subject(s)
Antigens/immunology , Intercellular Adhesion Molecule-1/physiology , Pneumonia/immunology , Animals , Antibodies, Monoclonal , Blood Cells/physiology , Bronchoalveolar Lavage Fluid/cytology , Immunization , Lung/immunology , Lung/pathology , Lymphoid Tissue/pathology , Ovalbumin/immunology , Phenotype , Pneumonia/pathology , Rats , Rats, Inbred BN , T-Lymphocytes/physiology
9.
Am J Respir Cell Mol Biol ; 15(2): 172-83, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8703473

ABSTRACT

We used flow cytometry and treatment in vivo with a monoclonal antibody (mAb), TA-2, to the alpha 4 integrin to investigate the role of alpha 4 beta 1, CD49d/CD29 (VLA-4) in antigen-induced lung inflammation in Brown Norway (BN) rats. Ovalbumin (OVA) inhalation induced an accumulation of eosinophils and lymphocytes in the lungs and bronchoalveolar lavage (BAL) fluid of sensitized BN rats at 24 h after challenge. Phenotypic analyses demonstrated that the percentages of T cells expressing detectable alpha 4 and CD25 in the bronchial lumen after antigen challenge were dramatically increased compared with blood and lymph node T cells. The mean channel fluorescence values of alpha 4 expression were also increased on BAL T cells compared with blood or lymph node T cells. Treatment of OVA-sensitized rats in vivo with total cumulative doses of 0.75 to 6 mg/kg TA-2 mAb intraperitoneally produced dose-related increases in circulating TA-2 and a peripheral blood lymphocytosis, basophilia, and eosinophilia. Flow cytometric analysis of the peripheral blood T cells after in vivo TA-2 mAb administration showed decreases in detectable alpha 4 when these cells were examined ex vivo. Treatment with TA-2, but not an isotype-matched control mouse immunoglobulin G1 mAb, markedly inhibited the OVA-induced recruitment of lymphocytes and eosinophils into the airway lumen. Very few CD3+CD49d+ cells migrated into BAL fluid following anti-alpha 4 mAb treatment in vivo. Treatment with TA-2 also significantly attenuated OVA-induced inflammatory histopathology. We conclude that VLA-4 is a critically important adhesion molecule involved in antigen-specific lung inflammation in sensitized BN rats.


Subject(s)
Anti-Allergic Agents/immunology , Eosinophils/immunology , Integrins/physiology , Lung/immunology , Receptors, Lymphocyte Homing/physiology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/pharmacology , Bronchoalveolar Lavage Fluid/cytology , Eosinophils/cytology , Flow Cytometry , Immunophenotyping , Integrin alpha4beta1 , Leukocyte Count , Lung/cytology , Lymphocyte Subsets/immunology , Lymphoid Tissue/cytology , Male , Mice , Ovalbumin/immunology , Pneumonia/immunology , Pneumonia/pathology , Rats , Rats, Inbred BN , Respiratory Hypersensitivity/immunology , T-Lymphocytes/cytology
10.
J Med Chem ; 37(26): 4449-54, 1994 Dec 23.
Article in English | MEDLINE | ID: mdl-7818726

ABSTRACT

A study of the decomposition of the pyrazoline bisphosphonate ester 2 identified 3 as the sole bisphosphonate component. Evaluation in a delayed-type hypersensitivity granuloma model of chronic inflammation in mice (DTH-GRA) showed 3 to be a potent inhibitor of granuloma formation (sc, 10 mg/kg, 45%), but in a murine model of antigen-induced arthritis (AIA), no significant inhibition was observed. As a result, new ketonic bisphosphonate tetraethyl esters were synthesized from vinylidenebisphosphonic acid tetraethyl ester 4 and activated carbonyl compounds in 13-84% yield. 6 significantly inhibited the pathology of both the DTH-GRA (sc, 25 mg/kg, 45%) and AIA models (sc, 25 mg/kg, 55%). Other compounds in the series were not as potent. Our results show that bisphosphonate ester 6 can inhibit the chronic inflammatory response associated with cutaneous granuloma formation and erosive arthritis.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Arthritis/drug therapy , Diphosphonates/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Diphosphonates/pharmacology , Dose-Response Relationship, Drug , Female , Mice , Mice, Inbred C57BL , Structure-Activity Relationship
11.
Arthritis Rheum ; 36(9): 1305-14, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8216424

ABSTRACT

OBJECTIVE: To investigate the anti-arthritic effect of recombinant human interleukin-1 receptor antagonist protein (IRAP) in two experimental models of arthritis. METHODS: Recombinant IRAP was administered daily to mice with type II collagen-induced arthritis (CIA) or with antigen-induced arthritis (AIA) provoked by methylated bovine serum albumin (mBSA). Disease incidence and severity were assessed by a clinical index and histologic features. Serum antibody to type II collagen, spleen cell proliferation to mBSA, and anti-IRAP antibodies were measured as indices of immune function. RESULTS: IRAP reduced the incidence and delayed the onset of CIA and suppressed the antibody response to type II collagen. In contrast, IRAP did not affect the pathogenesis of AIA and had no effect on either humoral or cellular immune responses to mBSA in arthritic mice. CONCLUSION: These observations suggest that interleukin-1 may play a prominent role in the development of some, but not all, forms of arthritis.


Subject(s)
Arthritis/chemically induced , Arthritis/immunology , Collagen , Proteins/pharmacology , Receptors, Interleukin-1/antagonists & inhibitors , Serum Albumin, Bovine , Animals , Antibodies/immunology , Antibody Formation , Arthritis/physiopathology , Collagen/immunology , Female , Mice , Mice, Inbred DBA , Proteins/immunology , Recombinant Proteins , Serum Albumin, Bovine/immunology
12.
J Pharmacol Exp Ther ; 266(3): 1691-8, 1993 Sep.
Article in English | MEDLINE | ID: mdl-8371167

ABSTRACT

Diphosphonates (DP) are synthetic pyrophosphates with a P-C-P backbone and are predominantly used for the treatment of bone diseases. Several DP have also been shown to exert significant antiarthritic effects in the rat adjuvant-induced polyarthritis model; however, there is no direct evidence for the anti-inflammatory effects of these compounds. We therefore tested the effects of dichloromethylene diphosphonate on delayed-type hypersensitivity granuloma elicited by s.c. implantation of antigen-soaked hydroxyapatite disks in antigen-sensitized mice. Dichloromethylene diphosphonate induced a dose-related inhibition of the delayed-type hypersensitivity granuloma response (38-64% at 25-100 mg/kg/day s.c. or p.o.); novel DP analogs, U-81581, U-82579 and U-84849 were also effective in the same dose range. In contrast, all DP failed to suppress 24-hr delayed-type hypersensitivity paw edema in mice. In addition to rat adjuvant-induced polyarthritis, mouse antigen-induced erosive arthritis was also significantly suppressed by s.c. administration of all four DP. Toxicity was minimal for each DP (> 600 mg/kg p.o. or s.c.). We conclude that DP represent a novel class of anti-inflammatory agents with excellent therapeutic potential for chronic inflammatory diseases including rheumatoid arthritis.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arthritis, Experimental/drug therapy , Diphosphonates/pharmacology , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Edema/drug therapy , Female , Granuloma, Foreign-Body/drug therapy , Hypersensitivity, Delayed/drug therapy , Inflammation/drug therapy , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Rats , Rats, Wistar
13.
Agents Actions ; 39 Spec No: C86-8, 1993.
Article in English | MEDLINE | ID: mdl-8273596

ABSTRACT

The angiogenesis inhibitor AGM-1470 has recently been reported to inhibit collagen-induced arthritis in rats. To determine if the anti-arthritic effects of AGM-1470 might be due to T cell inhibition, we have studied its effects on T cell responses in vitro. Responses of human cells to tetanus toxoid (TT), and those of murine splenocytes to staphylococcal enterotoxin (SE), mitogens or a mls difference were inhibited by AGM-1470. Responses of human cells to SE, OKT3 and PHA were all partially inhibited on day 2 (d2) but not d3, and in fact were augmented on d6-8. The amount of IL-2 in SEA cultures was augmented on d4 and d5. There were no differences in the expression of CD3, CD4, CD8, CD25, CD45RA, CD45RO, LFA-1, VLA-4 or VLA-6 in inhibited cultures, except for slight decreases in CD25 and CD45RO in TT cultures. These results indicated that the angiogenesis inhibitor AGM-1470 also modulates human and murine lymphocyte function.


Subject(s)
Antibiotics, Antineoplastic/pharmacology , Interleukin-2/metabolism , Lymphocyte Activation/drug effects , Sesquiterpenes/pharmacology , T-Lymphocytes/drug effects , Animals , Cells, Cultured , Cyclohexanes , Humans , Mice , O-(Chloroacetylcarbamoyl)fumagillol , Staphylococcal Toxoid/toxicity , T-Lymphocytes/immunology , Tetanus Toxin/toxicity
14.
Agents Actions ; 39 Spec No: C93-5, 1993.
Article in English | MEDLINE | ID: mdl-8273598

ABSTRACT

Six diphosphonates were examined for their ability to alter proliferative responses of mouse bone marrow cells to recombinant human M-CSF and recombinant murine GM-CSF. Risedronate ([2-(3-pyridinyl)-ethylidene] hydroxy bisphosphonic acid) added to in vitro cultures at 10 microM, suppressed the response to M-CSF by 58%, but had no significant effect on GM-CSF-induced proliferation. Ethane-1-hydroxy-1,1-bisphosphonic acid (EHDP), dichloromethylene bisphosphonic acid (Cl2MBP), 3-amino-1-hydroxy-propylidene-1,1-bisphosphonic acid (APD), (4-chlorophenyl)-thiomethylene bisphosphonic acid (tiludronate) and (cycloheptylamino)-methylene bisphosphonic acid (YM-175) had no significant effect. Treatment of mice for 5 or 14 days with 200 mg/kg/day p.o., Cl2MBP or 3 mg/kg/day p.o. of risedronate failed to inhibit M-CSF- or GM-CSF-induced proliferation by recovered bone marrow cells. Addition of Cl2MBP or risedronate in vitro to these cells did not reveal any change in sensitivity to CSFs as a result of exposure to diphosphonate in vivo.


Subject(s)
Bone Marrow/drug effects , Diphosphonates/pharmacology , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Macrophage Colony-Stimulating Factor/pharmacology , Animals , Bone Marrow Cells , Cell Division/drug effects , Cells, Cultured , Female , Mice , Mice, Inbred C57BL , Recombinant Proteins/pharmacology
15.
J Med Chem ; 36(1): 134-9, 1993 Jan 08.
Article in English | MEDLINE | ID: mdl-8421279

ABSTRACT

Vinylidenebisphosphonic acid tetraethyl ester (1) and diazo ketones 7a-1 in ether at 22 degrees C yield pyrazoline bisphosphonate tetraethyl esters 8a-1 in moderate to good yield. These compounds were evaluated in animal models of arthritis: rat adjuvant-induced polyarthritis (AIP) and murine antigen-induced arthritis (AIA) and a murine model of chronic inflammation, the delayed type hypersensitivity granuloma reaction (DTH-GRA). (5-Benzoyl-2,4-dihydro-3H-pyrazol-3-ylidene)-bisphosphonic acid tetraethyl ester (8a), and [5-(3-fluorobenzoyl)-2,4-dihydro-3H-pyraxol-3-ylidene]- bisphosphonic acid tetraethyl ester (8d) significantly inhibited the arthritis models, AIP (15 mg/kg) and AIA (25 mg/kg), as well as the DTH-GRA (25 mg/kg). Conversion of 8a to the corresponding bisphosphonic acid, 10a, resulted in loss of activity. Compounds with alkyl substituents on the pyrazoline nitrogen, 9a-d, were inactive in the DTH-GRA. These results show that 8a and 8d have novel antiinflammatory activity and are capable of inhibiting chronic arthritis and inflammation in animals. Such compounds might be useful in man for treating chronic tissue injury associated with arthropathies such as inflammatory joint disease as well as other chronic inflammatory diseases.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Diphosphonates/chemical synthesis , Pyrazoles/chemical synthesis , Animals , Arthritis/drug therapy , Diphosphonates/chemistry , Diphosphonates/pharmacology , Female , Male , Mice , Mice, Inbred C57BL , Pyrazoles/chemistry , Pyrazoles/pharmacology , Rats , Rats, Wistar , Structure-Activity Relationship
16.
J Protein Chem ; 11(6): 709-22, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1466764

ABSTRACT

We have modified recombinant human Interleukin-1 beta using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide at pH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI = 6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1 beta. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 beta and may be important for the binding of IL-1 to its receptor.


Subject(s)
Cross-Linking Reagents/chemistry , Ethyldimethylaminopropyl Carbodiimide/chemistry , Interleukin-1/chemistry , Amino Acid Sequence , Animals , Cell Division , Chromatography, Gel , Chromatography, Ion Exchange , Circular Dichroism , Interleukin-1/pharmacology , Isoelectric Point , Metalloendopeptidases/metabolism , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Mapping , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , Spectrometry, Mass, Fast Atom Bombardment , T-Lymphocytes/cytology
17.
J Biol Chem ; 267(5): 3122-8, 1992 Feb 15.
Article in English | MEDLINE | ID: mdl-1531337

ABSTRACT

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.


Subject(s)
2-Naphthylamine/analogs & derivatives , Cysteine , Interleukin-1/pharmacology , Lysine , 2-Naphthylamine/pharmacology , Amino Acid Sequence , Animals , Cells, Cultured , Chromatography, Ion Exchange , Interleukin-1/isolation & purification , Interleukin-1/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C3H , Models, Structural , Molecular Sequence Data , Peptide Mapping , Protein Conformation , Receptors, Immunologic/metabolism , Receptors, Interleukin-1 , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Spectrometry, Mass, Fast Atom Bombardment , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
18.
Arthritis Rheum ; 33(2): 253-60, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2306293

ABSTRACT

We examined the effect of interleukin-1 (IL-1) administration on a mild and transient inflammatory response in the knees of mice injected intraarticularly with methylated bovine serum albumin (mBSA). Injection of mBSA on day 0 into nonsensitized mice caused a weak inflammatory response confined to the infrapatellar fat pads and involved infiltration by mononuclear cells, neutrophils, and eosinophils. The response developed between days 4 and 7 and resolved by day 28. No erosion of cartilage or subchondral bone was seen. In contrast, mBSA-treated mice injected with recombinant human IL-1 beta subcutaneously in the ipsilateral footpad on days 0-3 developed a severe monarticular arthritis in the antigen-injected knee. Pannus developed, extending over the articular surfaces, and extensive erosion of cartilage and subchondral bone occurred. Multinucleated giant cells, together with fibrin-like material, were observed at sites of active bone erosion and debris, and large numbers of neutrophils were seen in the joint space. These pathologic features represent a new arthritis model in which IL-1 profoundly augments a weak inflammatory response and induces acute erosive joint destruction, supporting the hypothesis that IL-1 is an important cytokine in the pathogenesis of arthritis.


Subject(s)
Arthritis, Experimental/etiology , Arthritis/etiology , Interleukin-1 , Serum Albumin, Bovine , Acute Disease , Animals , Arthritis, Experimental/pathology , Cartilage, Articular/pathology , Female , Injections, Intra-Articular , Interleukin-1/administration & dosage , Knee Joint/pathology , Mice , Mice, Inbred C57BL , Neutrophils , Serum Albumin, Bovine/administration & dosage , Time Factors
19.
J Biol Chem ; 264(30): 17691-7, 1989 Oct 25.
Article in English | MEDLINE | ID: mdl-2808341

ABSTRACT

Recombinant human interleukin-1 beta (rIL-1 beta) was chemically modified by a 10-fold molar excess (reagent:protein) of sulfosuccinimidyl 6-(biotinamido) hexanoate (sulfo-NHS-LC-biotin) or sulfosuccinimidobiotin (sulfo-NHS-biotin) under mild conditions. The primary product was purified in each case by cation exchange high performance liquid chromatography (HPLC) and digested with endoproteinase Lys C. Peptide mapping by C18 reverse phase HPLC permitted identification of three sites of biotinylation using both reagents; N-terminal alanine, lysine 93, and lysine 94. Few additional singly modified rIL-1 beta products were obtained under these conditions, despite the presence of 15 lysine residues in this protein. These data support the view that the N terminus as well as the trilysine sequence (residues 92-94) are readily susceptible to chemical modification and are exposed on the surface of the protein. Chromatography of intact biotinylated rIL-1 beta by C4 reverse phase HPLC resolved a protein modified exclusively at the N-terminal alanine from two proteins modified singly at either lysine 93 or lysine 94. In addition, a protein product modified at lysine 103 was also obtained when rIL-1 beta was similarly modified with sulfo-NHS-biotin. Since the only difference between the two biotinylation reagents relates to spacer length and its associated hydrophobicity, these data suggest that lysine 103 is not as accessible to surface modification reagents as are lysine 93, lysine 94, or alanine 1. Initial experiments indicate that none of the modifications described above decrease thymocyte proliferation by more than one order of magnitude. Therefore, these amino acid residues are not crucial for bioactivity, and we anticipate the use of these monobiotinylated proteins in structure/function analysis of IL-1 beta.


Subject(s)
Biotin/analogs & derivatives , Interleukin-1/metabolism , Succinimides , Amino Acid Sequence , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Humans , Interleukin-1/isolation & purification , Molecular Sequence Data , Peptide Mapping , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
20.
Agents Actions ; 27(3-4): 290-3, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2508441

ABSTRACT

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1 alpha, IL-1 beta, TNF alpha, IL-2 and Ifn gamma. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1 alpha greater than rIL-1 beta greater than rTNF alpha greater than rIfn gamma = BSA (control) following a single sc. injection. However, only rIL-1 beta and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 beta lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These "in vivo" observations reinforce the roles of both IL-1 beta and IL-2 as potent mediators of chronic immunoinflammatory disease.


Subject(s)
Inflammation/chemically induced , Interferon-gamma/toxicity , Interleukin-1/toxicity , Interleukin-2/toxicity , Tumor Necrosis Factor-alpha/toxicity , Animals , Female , Inflammation/pathology , Injections, Subcutaneous , Mice , Recombinant Proteins/toxicity , Skin/pathology
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