Disparate cleavage of poly-(ADP-ribose)-polymerase (PARP) and a synthetic tetrapeptide, DEVD, by apoptotic cells.

In the present investigations, we have shown differential cleavage of cellular PARP and a caspase 3-selective synthetic tetrapeptide substrate, Z-DEVD-AFC or Ac-DEVD-AMC using a T lymphoblastoid cell line Jurkat, and its variant clone E6.1(J-E6). Anti-Fas antibody-mediated apoptosis resulted in DNA fragmentation and PARP cleavage in both Jurkat and J-E6 cells. However, unlike Jurkat, J-E6 cells did not cleave a synthetic tetrapeptide substrate efficiently. The failure to cleave the DEVD tetrapeptide by apoptotic J-E6 cells was not due to insufficient expression or processing of caspase 3 in J-E6 cells. Interestingly, when the J-E6 cells were transiently transfected with a cDNA encoding caspase 3, efficient cleavage of Z-DEVD-AFC was achieved. The observations that apoptotic J-E6 cells barely cleaved a synthetic DEVD tetrapeptide, but efficiently cleaved endogenous PARP, potentially at the most preferred DEVD site, suggest that active caspases may have disparate characteristics to recognize substrates presented in different context.

Receptor-mediated gene delivery to human peripheral blood mononuclear cells using anti-CD3 antibody coupled to polyethylenimine.

Gene transfer to primary cells, especially to lymphoid cells, using a nonviral delivery system has been very challenging. In the present studies, we have used a cationic polymer, polyethylenimine (PEI) coupled to an anti-CD3 antibody for achieving receptor-mediated gene delivery to human peripheral blood mononuclear cells (PBMC). Naive, unstimulated PBMC did not express transfected genes, whereas the transgenes were expressed efficiently in PHA activated PBMC. Transiently expressed gene products were detected maximally at 24 and 48 h following transfection. Gene expression was detected until 96 h with a gradual diminution in the signal after 48 h. Receptor-mediated gene delivery was successfully used for freshly isolated, as well as previously frozen lymphocyte samples. The transfections performed using ligands other than anti-CD3 were not as efficient as anti-CD3-PEI. These results suggest that in addition to receptor-mediated endocytosis, signaling subsequent to engagement of the CD3 receptor with anti-CD3-PEI appears to be important for the efficacy of anti-CD3-PEI mediated gene delivery.

Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition.

The IkappaB kinase (IKK) signaling complex is responsible for activating NF-kappaB-dependent gene expression programs. Even though NF-kappaB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-kappaB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cells versus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKgamma/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-kappaB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118, Pea-15/MAT1, CD82, CD40L, Wnt10a, Notch 1, R-ras, Rgs-16, PAC-1, ISG15, and CD36) and five repressed (CCR2, VpreB, lambda5, SLPI, and CMAP/Cystatin7) genes, respectively, were bona fide NF-kappaB targets by virtue of their response to a transdominant IkappaBalphaSR (super repressor). MyD118 and ISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-kappaB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

Effect of deoxycoformycin and Val-boroPro on the associated catalytic activities of lymphocyte CD26 and ecto-adenosine deaminase.

CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.

Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms.

Solution structures of active and inactive forms of the DP IV (CD26) inhibitor Pro-boroPro determined by NMR spectroscopy.

Synthesis of the boronic acid analog of the dipeptide Pro-Pro yields a mixture of diastereomers Pro-L-boroPro and Pro-D-boroPro, one of which is a potent inhibitor [Ki = 16 pM; Gutheil, W. G., & Bachovchin, W. W. (1993) Biochemistry 32, 8723-8731] of dipeptidyl amino peptidase type IV (DP IV), also known as CD26. The structures of both diasteremers are determined here in aqueous solution by means of 1D and 2D NMR of 1H, 13C, and 11B, and force-field calculations, and the inhibitor is proven to have the L-L configuration. At low pH values (approximately 2), both diastereomers are trans with respect to the peptide bond. Populations of proline ring conformers are determined by pseudorotation analysis, using vicinal proton spin-coupling constants obtained by computer analysis of 1D1H NMR spectral fine structure. At neutral pH values, the Pro-boroPro inhibitor of DP IV undergoes slow, reversible inactivation (Gutheil & Bachovchin, 1993). By structural determination of the decomposition products of both diasteromers, the process is shown here to involve formation of a six-membered ring between the residues by means of trans-cis conversion and formation of a B-N bond, producing chiral nitrogen atoms in both cases having the S configuration. Analogy to cyclic dipeptides suggests the new compounds be named cyclo(Pro-L-boroPro) and cyclo(Pro-D-boroPro).

Generation and characterization of an anti-idiotypic antibody specific for intercellular adhesion molecule-1.

Intercellular adhesion molecule-1 (ICAM-1) is an inducible ligand for the LFA-1/MAC-1 family of leukocyte adhesion molecules. The results reported herein show that a mouse monoclonal anti-idiotypic antibody, CA3, specific for R6.5 anti-ICAM-1 mAb, shares conformational homology with the original epitope bound by R6.5. The CA3 bound specifically to R6.5 F(ab) fragments and blocked the binding of R6.5 to its ICAM-1 epitope; no CA3 binding was detected to a second anti-ICAM-1 mAb F(ab) fragment nor to control mouse IgG F(ab) fragments. Similarly, the interaction of CA3 with R6.5 was inhibited by sICAM-1. However, CA3 was ineffective in inhibiting CD18-dependent cell aggregation. A rabbit anti-CA3 response (Ab3) further indicated that CA3 is of the Ab2 beta type. Significant binding of anti-CA3 to sICAM-1 was demonstrated and anti-CA3 competed with R6.5 for binding to sICAM-1. Anti-CA3 bound to both soluble and cell-surface-associated ICAM-1. However, unlike R6.5, significant binding both to reduced sICAM-1 and to native sICAM-1 was exhibited by anti-CA3, whereas the binding of R6.5 to reduced sICAM-1 was undetectable.

Interactions of leukocyte integrins with intercellular adhesion molecule 1 in the production of inflammatory vascular injury in vivo. The Shwartzman reaction revisited.

We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.

The Shwartzman response: a model of ICAM-1 dependent vasculitis.

The local Shwartzman response was produced in rabbits by the intradermal injection of endotoxin, followed 24 h later by intravenous zymosan. Hemorrhagic lesions developed in the prepared skin sites. We quantitated the Shwartzman-induced hemorrhage with autologous 99mTc-erythrocytes. We show that the development of the Shwartzman response depends on both leukocyte membrane CD18 glycoprotein activity as well as the participation of intercellular adhesion molecule-1 (ICAM-1). We discuss the possibility that the common property shared by the agents capable of preparing the skin for the Shwartzman response is the ability to induce ICAM-1 expression.

The effects of an anti-CD18 antibody (R15.7) in antigen-induced airway hyperresponsiveness (AH) and cell influx in guinea pigs.

Aerosol ovalbumin challenge (OA) of sensitized guinea pigs induced airway hyperreactivity (AH) to i.v. acetylcholine (Ach) and serotonin (5-HT) 24 hr post OA. Bronchoalveolar lavage fluid 24 hrs after OA showed increased leukocytes compared to unsensitized unchallenged animals. Treatment with monoclonal antibody R15.7 (3 mg/kg i.v.,) 1 hr prior and 4 hours after OA prevented the induction of AH to Ach but not to 5-HT and reduced influx of leukocytes. We conclude: 1) antigen inhalation induces an increase in AH with an increase in proinflammatory cell influx and 2) treatment with anti-CD18 antibody inhibits cell influx and airway hyperreactivity.

Binding of the T cell activation monoclonal antibody Ta1 to dipeptidyl peptidase IV.

The monoclonal antibodies, Ta1 and IOT15, define T cell activation cell surface markers and have been assigned to the CD26 leukocyte differentiation antigen cluster. Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) is an exoaminopeptidase that, among leukocytes, is expressed almost exclusively on activated T cells. Comparative binding studies showed that the Ta1 mAb binds to DPP IV purified from human placenta as well as in extracts of the human YT lymphoid cell line and of CD3 stimulated normal human peripheral blood mononuclear cells. The mAb IOT15 did not bind to DPP IV from any source even upon repeated incubations. Western blot analysis of YT cell extracts revealed that Ta1 and IOT15 bound to distinctly different molecular weight molecules. Immunofluorescent cell surface capping experiments showed that capping of the IOT15 did not alter the surface distribution of the Ta1 fluorescence. The capping results combined with the DPP IV binding results indicate that IOT15 and Ta1 mAb's bind to different, apparently unassociated, molecules on the surface of T cells and that only Ta1 binds the T cell surface enzyme DPP IV.

The effect of anti-intercellular adhesion molecule-1 on phorbol-ester-induced rabbit lung inflammation.

The role of the CD18 complex of leukocyte glycoproteins in adhesion-dependent functions of human leukocytes in vitro has been well documented. A ligand, intercellular adhesion molecule-1 (ICAM-1), for at least one member of the CD18 complex has been identified. This molecule is inducible on many cell types including vascular endothelium and keratinocytes by inflammatory mediators such as IL-1, TNF, and IFN-gamma. ICAM-1 has been shown to mediate, in part, the in vitro adhesion of lymphocytes and neutrophils to endothelial cells expressing ICAM-1. In the present study we have shown that mAb's to the human CD18 complex and to human ICAM-1 cross react with rabbit cells and that both anti-CD18 and anti-CD11b but neither anti-CD11a nor anti-ICAM-1 mAb's inhibit neutrophil migration, an adhesion-dependent function, in vitro. Pretreatment of rabbits with anti-CD18 and anti-ICAM-1 but not anti-CD11a mAb inhibited by greater than 60% neutrophil migration into PMA-induced inflamed rabbit lungs. This effect of anti-ICAM-1 mAb on pulmonary neutrophil influx after PMA injection has important implications. Specifically, that ICAM-1 can function as a ligand for CD18 and can mediate, at least in part, the migration of neutrophils to inflammatory sites.

A human urine-derived interleukin 1 inhibitor. Homology with deoxyribonuclease I.

We have previously reported that the urine of febrile humans contained large quantities of an inhibitor of IL-1-induced murine thymocyte proliferation that was a glycoprotein between 30 and 40 kD in size. In the present study this factor has been purified to homogeneity using a sequence of eight purification steps (ammonium sulfate precipitation, ion exchange chromatography, molecular sieve chromatography, hydrophobic affinity chromatography, hydroxylapatite chromatography, fast protein liquid chromatography, and two HPLC steps). SDS-PAGE analysis indicates that the purified material is a 38-kD molecule. Evidence based on a partial amino acid sequence analysis as well as enzyme studies indicates that this inhibitor is a type of human DNase I.

A model of pulmonary granulomata induced by beryllium sulfate in the rat.

In humans beryllium is known to cause pulmonary granulomata which are histologically indistinguishable from sarcoidosis. There is some evidence in man that beryllium-induced pulmonary granulomata are immunologically mediated. We set out to develop an animal model to study the immunopathogenesis of beryllium-induced granulomatous lung disease. Beryllium sulfate (BeSO4) was injected intratracheally (i.t.) into F344 rats previously immunized to BeSO4. This results in well-formed, sarcoid-like lung granulomata at 6 weeks post BeSO4. There was a conspicuous presence at 4 weeks post BeSO4 of numerous, perivascularly located Langhans' giant cells which preceded the development of well-formed granulomas at 6 weeks. Rats were sacrificed at 4, 6, 8 and 12 weeks after i.t. BeSO4. At the time of sacrifice bronchoalveolar lavage (BAL) was performed; B and T (W3/25+ helper 0X8+ suppressor/cytotoxic) lymphocyte populations were quantitated and compared to lymphocyte populations obtained from lung tissue. Both B and T cells were significantly elevated in lung tissue post BeSO4. At 4 weeks when granulomata were just developing, a W3/25+ to 0X8+ ratio of 20:1 in lavage and 2:1 in lung tissue was seen. At 6 weeks when granulomata were well-formed there was a predominance of W3/25+ cells in lavage but not in lung tissue. At 8 and 12 weeks, when the granulomata were regressing, lavage fluid still contained a W3/25+ predominance in contrast to lung tissue which contained a predominance of 0X8+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A rat model of purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the number of thymus cells (P less than 0.001) and in lymph node and spleen lymphocytes (P less than 0.01). These data showed that the administration of 8-aminoguanosine to rats provided an animal model of NP deficiency that will allow studies of the specific regulation of T-cell function.