The amyloidogenic peptide human amylin augments the inflammatory activities of eosinophils.

The amyloidogenic peptides, amyloid-beta (A beta) and human amylin, are the major constituents of amyloid deposits found in patients with the chronic degenerative disorders Alzheimer's disease (AD) and type 2 diabetes, respectively. Recent studies have shown that a variety of inflammatory proteins such as cytokines are associated with the amyloid deposits of AD brain tissues. Therefore, in the present study, we sought to determine whether A beta and/or human amylin could modulate the various inflammatory activities of eosinophils. We observed that human amylin but not A beta peptides inhibited the in vitro interleukin-5 (IL-5)-mediated survival of cord blood-derived eosinophils (CBEs) in a concentration-dependent manner. By contrast, rat amylin, a nonamyloidogenic peptide that is highly homologous to human amylin, failed to affect the IL-5-mediated survival of CBEs. Similar inhibitory effects of human amylin were observed for peripheral blood eosinophils. Human amylin also enhanced the release of the cytokine granulocyte-macrophage colony-stimulating factor by CBEs that were stimulated with the calcium ionophore A23187 but was incapable of directly stimulating CBEs to release cytokines. In addition, the A23187-induced release of the inflammatory lipid mediator leukotriene C4 by CBEs was augmented by human amylin. These results suggest that the amyloidogenic peptide human amylin is capable of amplifying the various inflammatory activities of eosinophils.

Retina-specifically expressed novel subtypes of bovine cyclophilin.

The Drosophila ninaA gene encodes photoreceptor-specific cyclophilin thought to play a critical role in rhodopsin folding or transport during its synthesis or maturation in the most abundant subclass of photoreceptors. Cyclophilins comprise a highly conserved family of proteins which are the primary targets of the potent immunosuppressive drug, cyclosporin A (CsA), and which display peptidyl prolyl cis-trans-isomerase (PPIase) activity. In an attempt to identify mammalian cyclophilins with properties similar to the NinaA protein, a probe derived from the ninaA cDNA was used to screen bovine retina cDNA libraries. The screen identified two major alternatively spliced forms of cDNA that would encode proteins containing a region of high homology to other cyclophilins and that are expressed specifically in the retina. These proteins represent a new class of cyclophilins with novel structural features and greatly reduced PPIase and CsA binding activities in comparison to other known cyclophilins. Tissue in situ hybridization and immunolocalization of the proteins showed that the RNA and protein products are expressed in photoreceptors as well s other retinal neurons. However, among photoreceptors, the proteins are found predominantly in cones. Thus, mammalian retinas do contain cyclophilins that are retina-specifically and photoreceptor class-preferentially expressed. The results suggest that, in cones, the main function of these proteins is, like the NinaA protein, to facilitate proper folding or intracellular transport of opsins.

Antigen-induced recruitment of eosinophils: importance of CD4+ T cells, IL5, and mast cells.

Eosinophils of sensitized mice readily recruit to the site of antigen challenge. In the present study, experiments were performed to determine the involvement of different cell types in the antigen-induced recruitment of eosinophils. We demonstrated that a single treatment with anti-L3T4 monoclonal antibody (mAb) on the day of allergen challenge significantly decreased antigen-induced recruitment of eosinophils. Treatments with anti-L3T4 mAb during the sensitization period also caused a substantial reduction in the migration of eosinophils into the site of challenge with antigen. Thus, it appears that both stages of eosinophil recruitment, sensitization and antigen-challenge, are dependent upon the presence of L3T4+ T cells. Moreover, while treatments with anti-IL5 mAb blocked eosinophil migration, anti-IL2 mAb failed to alter the antigen-induced recruitment of eosinophils. In addition, significant numbers of eosinophils from the mast-cell-deficient mice were found to migrate into the peritoneal cavities upon allergen challenge. Eosinophil migration was also observed in several mouse strains of different H-2 haplotypes. The present findings suggest that CD4+ T cells and IL5 but not IL2 may play important roles in modulating the recruitment of eosinophils. Moreover, the involvement of mast cells does not appear to be essential for eosinophil migration. Finally, the development of antigen-induced recruitment of eosinophils is probably not under the immunogenetic regulation by genes within the H-2 complex.

Human cortical neuronal (HCN) cell lines: a model for amyloid beta neurotoxicity.

Human cortical neuronal cell lines HCN-1A and HCN-2 are killed for following exposure of the differentiated cells to amyloid beta-peptide(1-40), a component of senile plaques and other amyloid deposits in brains from Alzheimer's patients. We present a model of A beta toxicity uncomplicated by the presence of other cell types that can be used to address the mechanism of A beta neurotoxicity. This model will be useful in the evaluation of neuroprotective compounds which may attenuate cortical neuronal loss in Alzheimer's disease.

FK506 and rapamycin modulate the functional activities of human peripheral blood eosinophils.

Numerous studies suggest that eosinophils may play a role in the inflammatory process of various diseases. Eosinophils are potent effectors of a variety of end-stage inflammatory activities. In addition, eosinophils are highly capable of synthesizing several cytokines which can stimulate the inflammatory responses of other cell types. Eosinophils are also responsive to a number of cytokines in that their in vitro survival as well as their effector functions can be enhanced by various cytokines. Finally, it is well established that the immunosuppressive drugs, FK506 and rapamycin, can inhibit the activation of T cells and mast cells. In the present study, the effects of FK506 and rapamycin on the activities of human peripheral blood eosinophils were investigated. It was first shown that human peripheral blood eosinophils upon stimulation with the calcium ionophore A23187 produced significant amounts of the cytokines, GM-CSF and IL3. FK506 was found to inhibit the synthesis of IL3 and GM-CSF, whereas rapamycin failed to suppress the cytokine production of eosinophils. Since FK506 and rapamycin are structurally related, the ability of rapamycin to reverse the FK506-mediated inhibition on cytokine production of eosinophils was next examined. It was observed that a 250-fold excess of rapamycin was required to antagonize the suppressive effects of FK506 on the cytokine secretion mediated by eosinophils. The capacity of rapamycin to alter the in vitro IL5-mediated survival of eosinophils was also studied. The results of this study demonstrated that rapamycin was effective in reducing the ability of IL5 to maintain the survival of eosinophils in culture; by contrast, FK506 had minimal effects on the IL5-mediated survival of eosinophils. In conclusion, the effects of FK506 and rapamycin on the activities of eosinophils appear to parallel those of mast cells and T cells. Hence, it is possible that FK506 and rapamycin might be acting on signal pathways or molecules that are shared by eosinophils, mast cells as well as T cells. Moreover, these findings suggest that both FK506 and rapamycin can potentially interfere with the capacity of eosinophils to engage in interactive activities that may contribute to the chronicity of inflammatory diseases such as asthma.

Effects of various anti-T cell receptor antibodies on the development of type II collagen-induced arthritis in mice.

Recent genetic studies of David and coworkers suggest that subsets of T cells utilizing specific V beta TcR genes may play important roles in the susceptibility to collagen-induced arthritis (CIA). Hence, in vivo depletion of such T cell subsets may significantly affect the development of CIA. To address this possibility, we first examined the effects of in vivo treatments with various monoclonal antibodies (mAbs) that are specific for particular TcR V beta families on the induction of CIA. Results presented in this study demonstrated that treatments with either anti-V beta 6, anti-V beta 8 or anti-V beta 11 did not suppress the development of arthritis in collagen-immunized mice. While combined treatments with these V beta specific mAbs which resulted in the in vivo elimination of V beta 6+, V beta 8+ and V beta 11+ T cells were not very effective in preventing the onset of CIA, the severity of the arthritic disease was somewhat reduced in animals that had received the triad of anti-V beta mAbs. By contrast, depletion of T cells expressing the alpha beta receptors by in vivo treatments with a pan anti-alpha beta mAb significantly decreased the incidence of CIA. Therefore, although an effect on the development of CIA was achieved by in vivo treatments with a mAb that detects all alpha beta + T cells, the elimination of only a few subsets of T cells which included the V beta 6+, V beta 8+, and V beta 11+ cells did not profoundly alter the incidence of CIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-1 enhances the development of type II collagen-induced arthritis only in susceptible and not in resistant mice.

The development of type II collagen-induced arthritis (CIA) in DBA/1 mice is readily accelerated by treatments with interleukin-1 beta (IL-1 beta). In an attempt to further characterize this IL-1 beta-mediated enhancement of CIA, we first examined the effects of IL-1 beta treatments in other "CIA-susceptible" strains and "CIA-resistant" mice. It was observed that treatments with IL-1 beta also enhanced the onset of arthritis in two B10 recombinant CIA-susceptible strains, B10.T (6R) and B10.DA, and in the SJL mice which develop CIA with a relatively low and variable incidence. On the other hand, IL-1 beta failed to augment the expression of arthritic disease in several CIA-resistant strains. We also investigated the potentiating effects of IL-1 beta in mice that were depleted of L3T4+ T cells. It was found that the ability of IL-1 beta to accelerate the development of CIA was significantly reduced in DBA/1 mice pretreated with the monoclonal anti-L3T4 antibody. In further studies, we demonstrated that the induction of CIA upon transfer with collagen-primed spleen cells was also augmented by IL-1 beta, and this enhancing effect by IL-1 beta on the adoptive transfer of CIA was associated with a significant increase in the levels of serum anti-collagen antibodies. Moreover, IL-1 beta treatments did not potentiate the induction of CIA in mice that were transferred with either collagen-immune splenic cells that were depleted of L3T4+ T cells or only T cells obtained from collagen-immunized animals. However, IL-1 beta enhanced the development of arthritis in animals that had been transferred with two subpopulations of collagen-immune cells: (i) enriched T cells and (ii) splenic cells that were depleted of L3T4+ T cells. Thus, IL-1 beta potentiated the inflammatory responses in animals that were genetically predisposed to developing arthritis. In contrast, IL-1 beta was incapable of accelerating the development of arthritis in various mouse strains that were genetically resistant to CIA. The administration of IL-1 beta also failed to potentiate the development of CIA in L3T4-deficient mice or in animals transferred with collagen-primed spleen cells that were depleted of L3T4+ T cells. These results indicate that IL-1 beta readily accelerates the induction of arthritis when the disease is present, but that IL-1 beta is incapable of promoting the expression of the arthritis in the absence of underlying disease.

Anti-inflammatory activity of BF389, a Di-T-butylphenol, in animal models of arthritis.

Biofor 389 (BF389), dihydro-4-[[3,5-bis(1,1-dimethyl)-4-hydroxyphenyl] methylene]-2-methyl-2H-1,2-oxazin-3(4H)-one, was tested for anti-inflammatory activity in various animal models of arthritis. Initial evaluation in the lipoidalamine (LA) arthritis model in rats (5-day dosing protocol) resulted in an oral ED50 of 4.9 mg/kg for inhibition of paw swelling. No effects on splenomegaly were observed, suggesting that the compound was efficacious as a result of anti-inflammatory rather than immunomodulatory effects. BF389 was efficacious in interleukin 1 (IL-1)-enhanced type II collagen arthritis in rats (oral ED50 less than 1.0 mg/kg) as assessed by paw volume measurement and histologic evaluation of joints. Mice with IL-1-enhanced type II collagen arthritis given 30 mg/kg of BF 389 had significantly lower histological scores for joint damage than did untreated controls. Normal rats given single oral doses of BF389 had significant suppression of arachidonate-stimulated whole blood prostaglandin E2 and thromboxane B2 production 2 hr postdosing (ED50 = 0.1 mg/kg). Leukotriene B4 production in these animals was not decreased. After it became apparent that the compound was a potent inhibitor of prostaglandin production in vivo, a study was done to compare the efficacy and toxicity of BF389 with several currently marketed nonsteroidal anti-inflammatory drugs, piroxicam, naproxen and diclofenac. Lipoidalamine-injected rats were given daily oral doses of BF389 or the comparators for 21 days. Quantitation of effects on arthritis on day 21 resulted in ED50 values of 0.9 mg/kg (BF389), 3.9 mg/kg (naproxen), 4.9 mg/kg (diclofenac) and 0.6 mg/kg (piroxicam).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1 mediated acceleration of type II collagen-induced arthritis: effects of anti-inflammatory or anti-arthritic drugs.

We previously demonstrated that treatments with rIL-1 beta accelerated the onset and progression of CIA in mice. In the present study, it was observed that IL-1 also enhanced the development of CIA in rats. Like the mouse model, maximal incidence (80-100%) of arthritis occurred within 7 days after the first treatment with IL-1 in rats. Thus, the acceleration of CIA by IL-1 (IL-1 CIA) may be an improved model for the rapid screening of anti-inflammatory and/or anti-arthritic drugs. As a first step to determining the utility of the IL-1 CIA model as a drug screen, we examined the ability of various known anti-inflammatory and anti-arthritic drugs to modify the IL-1 mediated enhancement of CIA in both rats and mice. The results of these studies showed that when analyzed in the IL-1 CIA model, rats and mice exhibited differences in their responses to several of these drugs. For example, dexamethasone, cyclophosphamide, azathioprine, various non-steroidal anti-inflammatory drugs (NSAIDs) as well as methotrexate were found active in the IL-1 CIA of rats. By contrast, the NSAIDs were found to be less effective in suppressing the IL-1 accelerated disease in mice. In both rats and mice, cyclosporine A and several disease modifying anti-arthritic drugs failed to the prevent the development of CIA that was potentiated by IL-1. Thus, in the IL-1 CIA model NSAIDs appeared to be less active in mice than rats. In conclusion, because of the shorter latent period required for the development of arthritis in the IL-1 treated animals, the IL-1 accelerated CIA model in both mice and rats may be useful for screening anti-inflammatory or anti-arthritic compounds.

Interleukin 1 enhances the development of spontaneous arthritis in MRL/lpr mice.

We previously reported that treatments with human recombinant interleukin-1 beta (rIL-1 beta) in DBA/1 mice which were suboptimally immunized with native chick type II collagen (NcII) markedly accelerated the onset of collagen-induced arthritis (CIA). In the present study, we further characterized this IL-1-mediated enhancement of murine arthritis by examining the in vivo effects of rIL-1 beta in another arthritis model, namely, the spontaneous arthritis of the MRL/lpr mouse strain. The results of these studies demonstrated that IL-1 treatments also enhanced the onset and progression of the spontaneous arthritic disease in MRL/lpr mice. A substantial proportion of the IL-1-treated MRL/lpr mice that were between 3 and 3.5 months of age exhibited swelling in either the hind or front paws. Moreover, histopathologic studies demonstrated the presence of striking alterations within the various joints of these IL-1-treated MRL/lpr mice. Such abnormalities were not detected in the non-IL-1-treated, age-matched MRL/lpr mice. Therefore, as in the experimentally induced disease of CIA, IL-1 may also be capable of contributing to the pathogenesis of the spontaneous arthritis of MRL/lpr mice.

Two subsets of cloned T helper cells exhibit different activation requirements and characteristics.

An anti-clonotypic monoclonal antibody (mAb 211G7H) was generated against a T helper cell clone which specifically recognized type II collagen. Besides inhibiting the proliferative response of the immunizing T cell clone to type II collagen, mAb 211G7H (soluble form) also suppressed the antigen-induced proliferation of several other T cell clones which shared similar specificity for antigen and major histocompatibility complex with the immunizing T cell clone. On the other hand, mAb 211G7H did not inhibit the responses of clones exhibiting different antigenic specificities from the clonotype-positive T cell clones. It was also demonstrated that these clonotype-positive T cell clones responded differently to mAb 211G7H when it was immobilized. Based upon their proliferative responses to immobilized anti-clonotype 211G7H mAb, two subpopulations of T cell clones were defined. Collagen-specific T cell clones of the first group proliferated poorly when stimulated with immobilized anti-clonotype mAb, by contrast, T cell clones belonging to the second group proliferated well to immobilized mAb. Furthermore, upon stimulation with immobilized 211G7H mAb, the second subpopulation of cloned T cells produced both interleukin (IL) 2 and IL4, while the first group secreted IL2 but not IL4. The cloned T cells from the group which responded weakly to immobilized anti-clonotype mAb also mediated reduced proliferative responses to IL2 in the presence of immobilized 211G7H mAb. Finally, these two subsets of T cell clones were found to respond differently to other non-antigen stimuli such as IL4 and phorbol 12-myristate 13-acetate. Thus, from a panel of collagen-specific T cell helper clones which had similar receptor fine specificities, we have isolated two subsets of cloned T helper cells that displayed different activation requirements and lymphokine production.

A new murine CD4+ T cell subset with an unrestricted cytokine profile.

CD4+ T cell clones were derived from mice immunized to keyhole limpet hemocyanin to characterize the cytokine profiles of newly isolated clones. Surprisingly, several of the clones had an unrestricted profile, producing IL-2, IL-3, IL-4, IFN-gamma, and TNF after either Con A or Ag stimulation. The coproduction of IL-2 and IL-4 was confirmed at the mRNA level. Subclones were derived which contained RNA transcripts for, as well as secreted, both IL-2 and IL-4 thus confirming the clonality of the original T cell clones. CD4+ T cell clones that expressed an unrestricted cytokine profile upon Con A stimulation were also isolated from mice immunized to other Ag (hen egg lysozyme, OVA, or type II collagen). These data indicate that CD4+ T cell clones newly isolated from immunized mice do not necessarily segregate into the Th1 and Th2 subsets. We propose this new murine CD4+ cell subset with an unrestricted pattern of cytokine production be called Th0.

In vivo administration with IL-1 accelerates the development of collagen-induced arthritis in mice.

In an attempt to examine the in vivo proinflammatory properties of IL-1, the effects of rIL-1 beta on the development of collagen-induced arthritis in mice were investigated. The results presented in this paper demonstrated that the administration of rIL-1 beta via mini-osmotic pumps into DBA/1 mice which were suboptimally immunized with native chick type II collagen (NcII) markedly accelerated the onset as well as the progression of the arthritic disease. When IL-1-containing osmotic pumps were s.c. implanted onto mice 18 days post-collagen immunization, clinical signs of arthritis appeared within 3 to 4 days after the implant with the pumps. Maximal incidence of arthritis which was usually 80 to 100% occurred between the 6th and 7th day after the administration of rIL-1 beta. Histologic analyses revealed that the knee and ankle joints from mice which were treated with rIL-1 beta for 7 days were most severely and consistently affected. Furthermore, these IL-1-treated mice exhibited granulocytic hyperplasia within the marrow as well as marked peripheral blood neutrophilia. By contrast, arthritis was not observed during the 7-day course of the IL-1 study in the following control groups: 1) mice that were only immunized with NcII, and 2) collagen-immunized mice which received osmotic pumps containing PBS. A substantial number of these collagen-immunized mice which were not treated with IL-1 eventually developed arthritis but at later times after the incidence of arthritis had peaked in the IL-1-treated group. In addition, unimmunized mice failed to develop arthritis upon treatments with IL-1 beta. Moreover, the humoral responses to NcII were not altered in the IL-1-treated mice. Thus, these in vivo studies suggest that IL-1 is potentially capable of triggering the various inflammatory events of collagen-induced arthritis, and thereby, contribute to the pathogenesis of murine arthritis.

The progression of the inflammation in established collagen-induced arthritis can be altered by treatments with immunological or pharmacological agents which inhibit T cell activities.

Alterations in the progression of the inflammatory disease in mice with established collagen-induced arthritis (CIA) by in vivo treatments with either anti-T cell antibodies or an immunosuppressive drug, whose main targets of action are T cells, were studied. It was demonstrated that the in vivo administration of either monoclonal anti-L3T4, anti-Ly-2 or the combination of anti-L3T4 and anti-Ly-2 failed to modify the inflammatory disease after the arthritis had been initiated. Nevertheless, the progression of disease was decreased by treatments with rabbit anti-mouse lymphocyte sera (ALS) in mice with established CIA. While there was a substantial reduction in the proliferative responses to the T cell mitogen concanavalin A (Con A) in these ALS-treated mice, proliferation to a B cell mitogen, lipopolysaccharide, was unaffected. Furthermore, treatments with anti-Thy-1.2 monoclonal antibody (mAb) by itself did not alter the progression of the ongoing inflammatory disease, but combined treatments with both anti-Thy-1.2 and anti-L3T4 mAb prevented the further advancement of the arthritic disease. Although mice receiving either anti-Thy-1.2 alone or both anti-Thy-1.2 and anti-L3T4 exhibited complete absence of Thy-1+ cells within their inguinal lymph nodes, the proliferative responses to Con A by LN cells from mice treated with the combination of anti-Thy-1.2 and anti-L3T4 were drastically reduced compared to the responses of either the anti-Thy-1.2 or anti-L3T4-treated groups. Finally, daily treatments with cyclosporin A, an immunosuppressive drug which preferentially acts on T cells, were also effective in modifying the clinical course of the arthritic disease in mice with established CIA. These results suggest that immunocompetent cells which express the Thy-1 surface marker, most likely Thy-1+ T cells, may play a role in maintaining and perpetuating the inflammatory disease of established CIA.

Murine T cells reactive to type II collagen. I. Isolation of lines and clones and characterization of their antigen-induced proliferative responses.

Mice of the DBA/1 strain develop arthritis after immunization with native chick type II collagen. Although both a humoral and a cell-mediated response specific to type II collagen are associated with the disease, the underlying immunologic basis remains to be established. As an initial step to analyzing the involvement of cellular immunity in collagen-induced arthritis, we isolated and characterized T cell lines and clones specific to type II collagen. Two sets of T cell lines were obtained by limiting dilution. One set was found to react exclusively with denatured type II collagen, whereas the other set responded to both native and denatured type II collagen. The specificity of such T cells was demonstrated by their inability to respond to other soluble proteins such as type I collagen, HGG, KLH, or OVA. Cells from these lines recognized type II collagen only in an MHC (H-2q)-restricted fashion. Furthermore, the collagen-specific T cells were found to respond to type II collagens obtained from various species, including chick, bovine, and rat. Finally, each set of cells displayed a phenotype characteristic of T helper cells, namely Thy-1+, L3T4+, Lyt-2-.

Murine T cells reactive to type II collagen. II. Functional characterization.

DBA/1 mice immunized with native chick type II collagen (NcII) emulsified in complete Freund's adjuvant develop arthritis, whose underlying mechanisms are still undefined. As an initial step to studying the role of T cells in collagen-induced arthritis (CIA), we have established T cell lines specific to type II collagen. Characterizations of the antigen-induced proliferative responses mediated by these T cells have been reported. In essence, two major populations of collagen-reactive T cells were isolated: those that responded mainly to denatured type II collagen (DcII) and another group that reacted with both DcII and NcII. As shown in the present study, all of the collagen-reactive T cell lines isolated were found to be functional, although they differed in their capacity to mediate helper activities assessed by different assays. Hence, both populations of T cells exhibited the ability to trigger B cell proliferation, whereas only the population that recognized both DcII and NcII was capable of activating the synthesis of immunoglobulins by B cells. T cells from this latter group also provided specific help for the generation of a secondary anti-DNP antibody response. In addition, these T cells were capable of activating NcII-specific B cells to produce anti-collagen antibodies. By contrast, the T cell lines that reacted exclusively to DcII failed to mediate such specific helper functions. The inability of such T cells to activate DNP-primed B cells upon challenge with DNP-DcII did not appear to be due to a modification of antigenic sites on DcII by haptenation. Inasmuch as the DcII-specific T cell lines also proliferate less well in response to DcII than the T cell lines that recognize both DcII and NcII, a difference in the nature of the antigen receptors expressed by the two populations of collagen-specific T cells may partly explain the above observations. However, the inability to generate appropriate factors required for further differentiation of B cells to produce antibodies may also account for the failure of DcII-specific T cells to activate DNP-primed B cells. Finally, both populations of T cells were capable of mediating specific delayed-type hypersensitivity response.

Decreased syngeneic mixed lymphocyte reaction in autoimmune susceptible mice.

The syngeneic mixed lymphocyte reaction (SMLR) is a proliferative response mediated by murine splenic thymus-derived (T) lymphocytes after stimulation by syngeneic non-T cells. Several murine strains genetically susceptible to autoimmune diseases have decreased SMLR compared with normal strains. This diminished SMLR occurs before the onset of clinical disease and is most pronounced in those autoimmune mice that express a severe form of the disease. For example, in NZB/NZW F1 hybrid (B/W) mice, the SMLR is lower at 7 than at 2 months of age. The SMLR is lower in BXSB males, which develop more accelerated autoimmune disease, than in BXSB females. The defect in these autoimmune strains resides within the responding Lyt 1+,2,3-T-cell population. These results suggest the presence of a common T-cell defect in several murine strains that spontaneously develop autoimmunity.