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PubMed1979

Chemotactic factor-induced superoxide radical generation by human neutrophils: requirement for proteinase (esterase) activity.

The requirement for proteinase (esterase) activity in the generation of O2- by human peripheral neutrophils was investigated. Neutrophils were activated by exposure to the chemotactic peptide FMLP and superoxide generation was assessed by ferricytochrome C reduction. Inhibition of O2- generation was observed by pretreating cells with the chloromethyl ketone derivatives of tosyl-phenylalanine (TPCK, ID50 2.2 X 10(-5)M) and tosyl-lysine (TLCK, ID50 1.9 X 10(-4)M). Dose-dependent inhibition was also noted with synthetic proteinase substrates, especially those of chymotrypsin-like specificity, phenylalanine and tryptophan methyl esters (ID50S approximately 1.5 X 10(-4)M), as compared to derivatives of basic amino acids, arginine and acetyl-lysine methyl esters, which caused negligible inhibition at 10(-3M. The inhibition of O2- generation demonstrated by TryME was time- and temperature-dependent and reversible with washing of the cells, whereas the inhibition seen with TPCK was irreversible. DFP at high concentrations, 10(-3)M or greater, caused moderate inhibition of O2- generation that was unaffected by exogenous serine and almost completely reversible by washing the cells. TPCK and TryME, at concentrations that caused marked inhibition of O2- generation, had no effect on the fmlp-induced calcium-45 uptake by the cells. These studies suggest that intact proteinase function is required for O2- generation and that this step follows the calcium influx in the activation sequence induced by FMLP.

Calcium↗
PubMed1990

[Changes in the quantitative characteristics of the efferent activity of the scratching generator when the influence of the descending systems is removed].

The effects of signals coming from descending systems of the spinal cord on parameters of the activity of scratching generators were investigated on the decerebrated immobilized animals. It was shown that the removal of modulation influences of the cerebellum on the nuclei of descending systems (decerebellation) caused changes in the ratio of the activity of the generator half-centres, rose instability of the generator work, weakened the statistical dependence between changes in parameters of the activity of the generator half-centres and strengthened the generators sensitivity to afferent signals. The total removal of influences of descending systems (after decapitation) induced reduction of the intensity of the efferent activity and afferent sensitivity of the scratching generator. The functional role of descending signals in the work of the scratching generator is discussed.

Afferent Pathways↗
PubMed1989

In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma.

We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.

Blood Coagulation↗
PubMed1988

Activation of the neutrophil by calcium-mobilizing ligands. I. A chemotactic peptide and the lectin concanavalin A stimulate superoxide anion generation but elicit different calcium movements and phosphoinositide remodeling.

The relevance of phosphoinositide remodeling to calcium movements and to the physiological response of superoxide anion (O2-) generation was probed in neutrophils stimulated by the chemotactic peptide fMet-Leu-Phe and the lectin concanavalin A. fMet-Leu-Phe and concanavalin A triggered O2- generation but elicited different patterns of calcium mobilization and phosphoinositide remodeling. fMet-Leu-Phe (10(-7) M) triggered a rise in cytosolic calcium by mobilization of intracellular calcium (fura-2) and increased calcium permeability (45Ca uptake), while concanavalin A (100 micrograms/ml) elicited a rise in cytosolic calcium, primarily by uptake of extracellular calcium (45Ca uptake). fMet-Leu-Phe triggered rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate and phosphatidylinositol, and generation of inositol 1,4,5-trisphosphate (IP3). In contrast concanavalin A triggered breakdown of phosphatidylinositol, but not PIP2, nor was there a significant increase in IP3. However, both fMet-Leu-Phe and concanavalin A triggered a rapid biphasic increase in levels of labeled diacylglycerol (in [3H]arachidonate or [14C]glycerol prelabeled cells) and a 3-fold increase in [32P] phosphatidic acid. These results are concordant with a role for PIP2 breakdown and generation of IP3 specifically in intracellular calcium mobilization but not for other aspects of the signaling pathway for O2- generation. Calcium permeability changes were associated with elevated diacylglycerol and [32P]phosphatidic acid, although a cause and effect relationship is not apparent. Ligands such as concanavalin A enhance cytosolic calcium and trigger generation of O2- without significant PIP2 remodeling; elevated diacylglycerol and cytosolic calcium are the common events associated with ligand-induced O2- generation.

Benzofurans↗
PubMed1986

Killer-helper effect of L cells in the generation of anti-MM cytotoxic T lymphocytes: replacement by beta-interferon and mechanisms of action.

Cytotoxic T lymphocytes (CTL) for MM tumor cells were generated only when L-MM tumor hybrid (L-FM3A#2)-primed spleen cells were stimulated in vitro with a mixture of mitomycin C-treated parental FM3A/R tumor cells and L cells, but not when they were stimulated with FM3A/R or L cells alone. Killer-helper activity of L cells was replaced by a beta-interferon (IFN) preparation (L-cell IFN). Anti-(alpha + beta)-IFN antibodies completely inhibited the killer-helper activity of L-cell IFN. Several lines of evidence suggested that the killer-helper effect of L-cell IFN resulted from an action at an early step in the generation of anti-MM CTL, as follows. The killer-helper effect of L-cell IFN diminished as the addition was delayed during culture for 5 days. The presence of L-cell IFN during the first 24 hr was enough to generate a maximal CTL response. Anti-(alpha + beta)-IFN antibodies showed no inhibitory activity on the generation of anti-MM CTL when they were added to the culture on day 2 or 4. It was also demonstrated that the killer-helper effect of L-cell IFN resulted from an effect on primed spleen cells, but not from an effect on stimulator cells. Furthermore, it was observed that IFN acted on nylon wool-adherent cells in the generation of anti-MM CTL. Plastic-adherent cells preincubated with L-cell IFN also enhanced the generation of anti-MM CTL when they were added to a culture where primed spleen cells were stimulated with FM3A/R tumor cells. These results suggest that activation of macrophages by L-cell IFN at the initiation step is essential for the generation of anti-MM CTL.

Animals↗
PubMed1985

Characterization of three monoclonal antibodies which induce and modulate superoxide anion generation in guinea pig macrophages.

Three monoclonal antibodies (Ab), termed KY 12, KY 22, and KY 25 and raised against guinea pig macrophages, induced superoxide anion (O2-) generation in the cells. Although each monoclonal Ab bound to macrophages, each had a different pattern of binding to other cell types. In response to each of the Ab, the amount of O2- generated by 5 X 10(5) macrophages was between 0.5 and 0.7 nmol/min and was augmented threefold to fivefold by the addition of F(ab')2 fragments of rabbit Abs to mouse Ig. When macrophages were pretreated with soluble immune complexes (I.C.) prior to stimulation by the monoclonal Ab, the O2- generation stimulated by KY 12 or KY 22 was reduced by more than 70%. In contrast, pretreatment of macrophages with I.C. did not reduce O2- generation in response to KY 25. KY 12 and KY 22 stimulated adenyl cyclase activity in macrophages, but KY 25 did not. Pretreatment of the cells with soluble I.C. did not interfere with the enhancing effect of the monoclonal Ab on adenyl cyclase activity. Pretreatment of macrophages with KY 12 reduced by over 60% of subsequent generation of O2- in response to wheat germ agglutinin, I.C., formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, KY 22, or KY 25. KY 22 or KY 25 did not suppress the generation of O2- in response to other stimuli. These results suggest that KY 22 and KY 25 activate O2- generation in a manner that differs from that of KY 12. These monoclonal Ab should prove useful in examining the regulation of O2- production.

Adenylyl Cyclases↗
PubMed1985

Adenosine; a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A2 receptor on human neutrophils.

Adenosine specifically inhibits superoxide anion generation by N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils without affecting either degranulation or "aggregation." We present data that also supports the hypothesis that adenosine engages a specific cell surface receptor to mediate inhibition of stimulated neutrophils. Theophylline (10 and 100 mu M), a competitive antagonist at adenosine receptors, reversed the effects of adenosine (0.1 mu M) on superoxide anion generation by stimulated neutrophils. The adenosine analogue 5'N-ethylcarboxamidoadenosine (NECA) was a more potent inhibitor of superoxide anion generation than either N6-phenylisopropyladenosine (PIA) or adenosine, an order of potency consistent with that previously demonstrated for adenosine A2 receptors. 2-Chloroadenosine inhibited superoxide anion generation at concentrations similar to NECA. [3H]-NECA and [3H]-2-chloroadenosine bound to a single receptor on intact neutrophils. The characteristics of the receptors for [3H]-NECA and [3H]-2-chloroadenosine were similar (Kd = 0.22 and 0.23 mu M, respectively; number of binding sites = 9.31 and 11.1 X 10(3) sites/cell, respectively). NECA, 2-chloroadenosine, adenosine, and PIA inhibited binding of [3H]-NECA with a rank order similar to that for inhibition of superoxide anion generation (NECA = 2-chloroadenosine greater than adenosine greater than PIA). There was 50% inhibition of superoxide anion generation by NECA at approximately 20% receptor occupancy. Adenosine, derived from damaged tissues, may serve as a specific, endogenous modulator of superoxide anion generation by activated neutrophils through interaction at this newly described receptor on human neutrophils.

2-Chloroadenosine↗
PubMed1987

Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fc gamma receptor-mediated superoxide generation in macrophages.

A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Eosinophil granule major basic protein regulates generation of classical and alternative-amplification pathway C3 convertases in vitro.

Eosinophil major basic protein (MBP), a highly charged polycation, forms the core of the eosinophil granule and mediates tissue damage in allergic disease. Purified MBP was studied for capacity to regulate the generation of classical and alternative-amplification pathway C3 convertases because previous studies have shown that other polycations (protamine, poly-L-lysine) and polyanions (heparin) may play important roles in regulating C activation. MBP inhibited the generation of EAC1,4b,2a and EAC4b,3b,Bb,P but appeared to inhibit the generation of classical pathway convertase more than the alternative amplification pathway convertase at a given dose. Dose-response curves with MBP were steeper than curves seen with polyanion (heparin). MBP did not lyse cellular intermediates at concentrations that caused almost total inhibition of convertase generation. One mechanism of inhibition of convertase generation may have been through an action on C3b, because preincubation of MBP with an EAC4b,3b cellular intermediate interfered with the ability of this cellular intermediate to be lysed. Furthermore, MBP prevented consumption of B in a reaction mixture that contained factors B, D, and C3b, also suggesting an action on C3b. Reduced and alkylated MBP (A-MBP) was compared with native MBP, which possesses two reactive sulfhydryl groups, to determine whether charge alone is responsible for blocking convertase generation; native MBP rapidly associates and is relatively insoluble at neutral and alkaline pH whereas A-MBP remains soluble. A-MBP impaired convertase generation, did not appear to remain bound to cellular intermediates and did not suppress B consumption in the fluid phase assay. This suggests that the ability of MBP to regulate C activation is complex and not entirely through its net charge. Finally, although heparin or MBP alone may prevent C activation, when these substances were present at the same time there was no effect on C activation suggesting that charge neutralization may abrogate the effects of these charged substances on C activation. Taken together, these studies suggest that MBP at physiologic concentrations may regulate in vivo C activation at the tissue level.

Alkylation↗
PubMed1987

Dynamics of generation of antigen loss variants from L1210 murine leukemia clones detected by a tumor-specific T-cell clone.

Originally T-cell clone K7L-sensitive L1210 murine leukemia clones were tested for their capacity to generate K7L-insensitive variants at various times after cloning. All of the L1210 clones (L1210/1, -2, -4, and -7) maintained in vitro for 1 month were severely inhibited in their growth in the culture in which K7L was added and in mice given injections of K7L at the initial stage. This indicated that any L1210 clone tested was not a mixture of K7L-sensitive and K7L-insensitive clones at the time of cloning. By both in vivo and in vitro K7L-mediated tumor suppression assays, K7L-insensitive antigen loss variants were then found to be generated from some (L1210/4, L1210/7) but not other (L1210/1, L1210/2) originally K7L-sensitive L1210 clones during 1 month of maintenance. Ratios of variant cells to total clone cells 1 month after cloning were estimated around 0.1% for L1210/7, 0.01% for L1210/4, and less than 0.001% (undetectable) for L1210/1 and L1210/2. Neither L1210/1 nor L1210/2 generated detectable K7L-insensitive variant cells during long-term (14-month) maintenance. All of the ten subclones of L1210/7 which were obtained 7 or 11 months after the initial cloning of L1210/7 were K7L sensitive, and not all the subclones generated K7L-insensitive variants in 1-2 months of maintenance after recloning. However, all of the subclones of L1210/7 which were maintained for 7 months generated antigen loss variants. All eight clone cells obtained from original L1210 and K7L-insensitive L1210 expressed H-2Kd and H-2Dd antigens detected by H-2Kd or Dd-specific cytotoxic T-lymphocyte clones or monoclonal antibodies. These results suggest that the antigen loss variants arise in originally K7L-sensitive L1210 clones at different times after cloning, and the probability of generation of the variants is clonally determined. The antigen loss variants seem to be generated by rare (once per 1 to 2 months or less frequent) chance with unproportionally rapid growth rather than by more frequent development for simple accumulation. The ratio of K7L-insensitive variant cells to total L1210/7 cells did not increase progressively during long-term (13 months or more) maintenance in vivo or in vitro and was always below 0.1%. It was suggested that the population size of antigen loss variants was controlled biphasically.

Animals↗
PubMed1987

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines.

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.

Animals↗
PubMed1984

Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential.

Hematopoietic stem cells of high proliferative potential such as the giant macrophage colony-forming cell HPP-CFC, were present in the marrow of mice treated with high dose 5-fluorouracil (5Fu) (150 mg/kg i.v.), whereas most committed granulocyte-macrophage progenitors, GM-CFU-C, were depleted. Enrichment of primitive stem cells in post 5-Fu bone marrow (5FuBM) was reflected in an enhanced capacity to proliferate in suspension cultures stimulated by the mixture of lymphokines present in Con A spleen-conditioned medium supernatant (Con A CM) when compared to normal bone marrow. The population of blast-like cells harvested at 5 days from suspension cultures of 5FuBM with Con A CM showed marked increases in stem cells GM-CFU-C and HPP-CFC. For this reason, 5FuBM was utilized to study the cell surface characteristics of putative pluripotential stem cells capable of giving rise to committed stem cells in suspension cultures. Treatment of 5FuBM (BDF1 mice) before suspension culture with a high concentration of either of two cytotoxic monoclonal antibodies directed against the Thy-1.2 surface antigen in the presence of rabbit complement reduced or abrogated the generation of stem cells HPP-CFC and GM-CFU-C in suspension cultures, even though the input content of HPP-CFC and GM-CFU-C in treated 5FuBM compared with control 5FuBM showed little reduction by the antibody plus complement treatment. The Thy-1+ cell required for generation of stem cells was not a T cell, because reconstitution of Thy-1.2-depleted 5FuBM with spleen nylon nonadherent (T) cells did not reconstitute the generation of stem cells, even though T cells did grow in the suspension cultures. In addition, depletion from 5FuBM of cells expressing Lyt-1 and Lyt-2 antigens, unambiguous markers of T cell-thymocyte differentiation, did not ablate the generation of HPP-CFC and GM-CFU-C. Rather, performance of Thy-1 cell depletion at lower efficiency, which still abrogated T cell function, ablated generation of HPP-CFC but did not affect the generation of GM-CFU-C. It was concluded that 5FuBM contains distinct Thy-1+ primitive stem cells expressing different amounts of Thy-1 antigen correlating with their respective generation potentials. Some of these Thy-1+ progenitor cells may be pluripotential.

Animals↗
PubMed1981

Prostaglandin-generating factor of anaphylaxis. Identification and isolation.

Anaphylaxis of human lung in vitro is accompanied by prostaglandin (PG) formation. To search for factors capable of generating PG, supernatants prepared by reversed anaphylaxis of human lung tissue were added to guinea pig lung and PG formation examined. The PG-generating activity fractionated between Mr = 500 and 5000 by ultramembrane filtration and was designated prostaglandin-generating factor of anaphylaxis or PGF-A. Partially purified PGF-A was soluble in 80% ethanol and insoluble in chloroform or ethyl acetate. Paper chromatography of anti-IgE-treated human lung preparations revealed a ninhydrin-positive spot at RF 0.27 to 0.41 with PG-generating activity. When subjected to gel filtration on Sephadex G-50 or G-25, PGF-A demonstrated peak PG-generating capacity at Mr = 1200 to 1800. Peak fractions were pooled and chromatographed on CM52 cellulose with PGF-A activity eluting at 0.16 to 0.3 M NH4COOH. The pooled peak of PGF-A activity from Sephadex G-25 filtration and CM52 chromatography eluted as a single peak at 0.024 M NH4HCO3 on DE52. Application of the peak off DE52 to a paper chromatogram revealed a single ninhydrin-positive spot with an RF 0.27 to 0.41 with the capacity to generate PGs. Amino acid analysis of three purifications revealed the following: Glu/Asp/Gly/Ser/Thr (6:3:2:1:1). Based on these analyses, a Mr = 1450 was calculated for the PGF-A. Therefore, PGF-A represents a novel mediator of lung anaphylaxis which may be partly responsible for PG generation accompanying allergic reactions.

Amino Acids↗
PubMed1983

Immunologic and ionophore-induced generation of leukotriene B4 from mouse bone marrow-derived mast cells.

Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation-secretion with the generation of leukotriene B4 (LTB4), a highly potent chemotactic factor. The antigen-initiated net percent release of the secretory granule marker beta-hexosaminidase and the generation of immunoreactive LTB4 and of immunoreactive leukotriene C4 (LTC4) were not diminished by washing the cells before challenge, indicating that interaction of the antigen occurred with the IgE fixed on cell membranes and was not due to phagocytosis of immune complexes formed in the fluid phase. The parallel dose-response relationship for the antigen-induced release of the performed mediator and the generation of both leukotrienes along with the superimposable time courses of their extracellular appearance indicate the origin of these mediators from a common cell type with IgE receptors. Resolution by reverse phase-high performance liquid chromatography (RP-HPLC) of the leukotrienes released from unsensitized cells by ionophore A23187 and from sensitized cells by the specific antigen revealed that the generation of LTB4 was accompanied by the production of the two diastereoisomers of 6-trans-LTB4, which were not immunoreactive. The immunoreactive LTB4 eluted from RP-HPLC at the same retention time as synthetic LTB4 was present in similar nanogram quantities when measured by either radioimmunoassay or integrated absorbance at 269 nm and exhibited a chemotactic activity for human neutrophils on a weight basis comparable to that of synthetic LTB4. The overall recoveries after RP-HPLC of immunoreactive LTB4 released from ionophore A23187-activated, bone marrow-derived mast cells and of added tritiated synthetic LTB4 in separate experiments with ionophore-activated cells were comparable and greater than 78%. The maximum generation of LTB4 by ionophore A23187-activated cells of 7.9 +/- 2.5 ng/10(6) cells (mean +/- SD), occurring at 40 min, was greater than the maximum generation of 4.5 +/- 0.8 ng/10(6) cells, with immunologic stimulation occurring 5 min after antigen challenge of sensitized cells. The initial rate of LTB4 generation after perturbation of the IgE-Fc receptors, however, exceeded that following ionophore A23187 stimulation and may represent one important variable in establishing a significant chemotactic gradient.

Animals↗
PubMedDate not supplied

The physical and chemical features of Cannabis plants grown in the United Kingdom of Great Britain and Northern Ireland from seeds of known origin--Part II: second generation studies.

A second generation of Cannabis plants has been grown from seeds produced by first generation plants which were raised in the United Kingdom of Great Britain and Northern Ireland from seeds taken from imported cannabis of known geographical origin and chemistry. The gross physical appearance of the second generation plants, in general, resembled their parents and the cannabis produced therefrom was not usually distinguishable from that derived from first generation plants. Although cannabinoid patterns were, in some cases, similar to those of both parents and original seedstock cannabis, there were many exceptions. The yields of cannabis were significantly higher than from first generation plants, but the tetrahydrocannabinol contents were lower. Although higher than in the original imported cannabis, the tetrahydrocannabinolic acid to tetrahydrocannabinol ratios in cannabis from second generation plants were lower than in cannabis produced from first generation plants.

Cannabidiol↗
PubMed1983

Generation of biologically active, complement-(C5) derived peptides by cathepsin H.

The thiol proteinase cathepsin H, isolated and purified from rat liver lysosomes, provokes acute inflammation characterized by the accumulation of polymorphonuclear leukocytes (PMN) when injected intracutaneously into newborn rats. We have examined the possibility that the accumulation of PMN at skin sites injected with cathepsin H is due, in part, to generation locally of C-derived chemotactic factors. We have found that cathepsin H acts in a concentration- and time-dependent fashion in whole human (and rat) EDTA-plasma to generate C5-derived peptides with chemotactic activity for PMN. Chemotactic activity was not generated in EDTA-plasma by either heat-inactivated cathepsin H or by a combination of active enzyme and a thiol proteinase inhibitor isolated from rat epidermis. Cathepsin H also acted in a concentration- and time-dependent fashion on isolated (functionally pure) human C5 to yield chemotactic activity for PMN as well as PMN lysosomal enzyme-releasing activity. Whereas 10 ng/ml cathepsin H generated significant chemotactic activity from isolated C5 (1000 CH50 U/ml), 7 to 10 micrograms/ml were required to generate chemotactic activity in whole EDTA-plasma. Cathepsin H not only was capable of generating biologically active, C5-derived peptides, but also was capable of degrading these peptides. Incubation of either whole EDTA-plasma or isolated C5 with high concentrations of cathepsin H (e.g., 25 micrograms/ml and 100 ng/ml, respectively) caused the rapid appearance of chemotactic activity followed by an equally rapid disappearance. PMN accumulated more rapidly in the skin of newborn rats injected with cathepsin H-treated C5 than in the skin of animals injected with cathepsin H alone. These data suggest that generation by cathepsin H of C-derived chemotactic activity contributes to the ability of this enzyme to induce dermal inflammation.

Animals↗
PubMed1983

Generation of alloreactive cytotoxic T lymphocytes: evidence for a differentiation factor distinct from IL 2.

In this report we provide evidence that the generation of cytotoxic T lymphocytes (CTL) involves a differentiation signal in addition to a proliferative signal, and that the differentiation signal is mediated by a factor distinct from interleukin 2 (IL 2). Generation of CTL from nylon wool-nonadherent splenocytes stimulated by heat-treated thymocytes did not correlate with the addition of IL 2-containing supernatants to this factor-dependent bioassay. IL 2 from two different sources and prepared by different methods (purified IL 2 from the LBRM33 cell line and partially purified IL 2 from the EL4 cell line) was not sufficient to generate a cytotoxic response in the bioassay. The inability of IL 2 to generate a cytotoxic response was not due to the use of heat-treated stimulator cells because similar results were obtained with UV-treated cells. As we previously reported, supernatant fluids containing a differentiation/activation factor did generate CTL from nylon wool-nonadherent splenocytes. Supernatant fluid from co-cultures of activated T cells and M phi as well as from cultures of activated M phi contained this CTL helper factor; however, only the former supernatant contained IL 2 activity. These results suggest a factor distinct from IL 2 is providing a differentiation/activation signal that is necessary for CTL development. Although IL 2 was not effective at generating CTL from nylon wool-nonadherent splenocytes, it was capable in some, but not all, experiments of generating a cytotoxic response from unseparated splenocytes. The ability of IL 2 to induce a cytotoxic response from unseparated splenocytes appeared to correlate with the presence of a weak background response seen when unseparated cells were used as responder cells.

Animals↗
PubMed1982

Prostaglandin D2 generation after activation of rat and human mast cells with anti-IgE.

Anti-IgE-dependent activation of rat and human mast cells resulted in the preferential generation of the cyclooxygenase products prostaglandin D2 (PGD2) and prostaglandin I2 (PGI2) in the rat and PGD2 in the human. The average net generation of PGD2, determined by gas chromatography-mass spectrometry, was 13.1 ng/10(6) purified rat mast cells and 39.5 ng/10(6) dispersed, enriched human mast cells. After IgE-dependent activation, there was a linear relationship between the net quantities of PGD2 generated and of histamine secreted from dispersed human pulmonary cells when the number of mast cells was varied but the total number of cells was held constant, indicating that it is the number of mast cells participating in IgE-dependent activation, rather than total mast cell number, that determines PGD2 generation. A linear relationship was also shown between PGD2 generation, determined by radioimmunoassay, and the release of the granule marker beta-hexosaminidase from purified rat mast cells on the dose-response portion of the plot of their response to anti-IgE challenge. With higher concentrations of anti-IgE, PGD2 generation from rat mast cells plateaued, whereas net percent beta-hexosaminidase release increased further. In kinetic studies of rat mast cells activated with anti-IgE, the onset (1 to 2 min) and time of maximum generation (5 to 10 min) for PGD2 were delayed relative to the onset (15 to 30 sec) and completion (1 to 2 min) of beta-hexosaminidase release. Thus, the extracellular appearance of PGD2 during IgE-dependent mast cell activation represents a response additional to the secretion of granule-associated mediators.

Animals↗