Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.

We have generated mice deficient in the beta2 integrin LFA-1 by targeted disruption of the CD11a gene in embryonic stem cells. In vitro LFA-1 -/- cells exhibit a delayed proliferative response toward alloantigens in the MLR. In vivo the host-vs-graft reaction toward injected allogeneic cells is also reduced. Alloantigen-specific CTLs generated from LFA-1 -/- mice are impaired in their cytotoxic activity toward allogeneic spleen cells as well as cell line targets. The proliferative response of LFA-1 -/- splenocytes following stimulation by LPS, PMA plus ionomycin, or immobilized anti-CD3epsilon mAb is normal, but Con A-stimulated proliferation is greatly diminished. We observe typical edema formation in a delayed type hypersensitivity reaction to SRBC with normal extravasation of leukocytes and demonstrate recruitment of neutrophils to an LPS-induced inflammatory site in these mice, suggesting that LFA-1 does not play an essential role in lymphocyte homing and leukocyte extravasation. We further show that LFA-1 -/- mice are susceptible to metastasis of B16 melanoma tumors, although their in vitro NK cell activity appears normal. A study of LFA-1 -/- mice expressing transgenic TCRs indicates that thymic maturation and selection of T cells are unaffected by the loss of LFA-1. Our results indicate that LFA-1 is important for alloantigen-triggered T cell proliferation and cytotoxicity, for Con A stimulation of T cells, and in tumor rejection. It does not appear to play an essential role in lymphocyte homing and leukocyte extravasation or in T cell maturation and selection in the thymus.

Transgenic mice expressing the human high-affinity immunoglobulin (Ig) E receptor alpha chain respond to human IgE in mast cell degranulation and in allergic reactions.

The high-affinity receptor for immunoglobulin (Ig) E (Fc epsilon RI) on mast cells and basophils plays a key role in IgE-mediated allergies. Fc epsilon RI is composed of one alpha, one beta, and two gamma chains, which are all required for cell surface expression of Fc epsilon RI, but only the alpha chain is involved in the binding to IgE. Fc epsilon RI-IgE interaction is highly species specific, and rodent Fc epsilon RI does not bind human IgE. To obtain a "humanized" animal model that responds to human IgE in allergic reactions, transgenic mice expressing the human Fc epsilon RI alpha chain were generated. The human Fc epsilon RI alpha chain gene with a 1.3-kb promoter region as a transgene was found to be sufficient for mast cell-specific transcription. Cell surface expression of the human Fc epsilon RI alpha chain was indicated by the specific binding of human IgE to mast cells from transgenic mice in flow cytometric analyses. Expression of the transgenic Fc epsilon RI on bone marrow-derived mast cells was 4.7 x 10(4)/cell, and the human IgE-binding affinity was Kd = 6.4 nM in receptor-binding studies using 125I-IgE. The transgenic human Fc epsilon RI alpha chain was complexed with the mouse beta and gamma chains in immunoprecipitation studies. Cross-linking of the transgenic Fc epsilon RI with human IgE and antigens led to mast cell activation as indicated by enhanced tyrosine phosphorylation of the Fc epsilon RI beta and gamma chains and other cellular proteins. Mast cell degranulation in transgenic mice could be triggered by human IgE and antigens, as demonstrated by beta-hexosaminidase release in vitro and passive cutaneous anaphylaxis in vivo. The results demonstrate that the human Fc epsilon RI alpha chain alone not only confers the specificity in human IgE binding, but also can reconstitute a functional receptor by coupling with the mouse beta and gamma chains to trigger mast cell activation and degranulation in a whole animal system. These transgenic mice "humanized" in IgE-mediated allergies may be valuable for development of therapeutic agents that target the binding of IgE to its receptor.

Tepoxalin blocks neutrophil migration into cutaneous inflammatory sites by inhibiting Mac-1 and E-selectin expression.

Inflammation is characterized by the migration of polymorphonuclear leukocytes from the vasculature into the tissue causing profound injury. Adhesion and migration of neutrophils across the vascular bed are governed by a series of complex events including cytokine/chemokine production which in turn orchestrates the temporal expression of a cohort of adhesion molecules mediating the migration. Many of these adhesion molecules and their inducers are under the control of inflammatory response transcriptional factors such as NF kappa B and AP-1. Recently we showed tepoxalin, previously known as a dual cyclooxygenase/lipoxygenase (CO/LO) inhibitor, to be a potent inhibitor of NF kappa B-induced transcription in vitro. In this study, we demonstrated that when administered in vivo, tepoxalin but not naproxen (a nonsteroidal anti-inflammatory drug, NSAID) or zileuton (an LO inhibitor), effectively inhibits neutrophil migration into inflammatory sites in murine skin stimulated by either lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Immunohistochemical analysis indicates that 10-50 mg/kg of tepoxalin inhibits neutrophil migration. It also effectively blocks the upregulation of Mac-1 (CD11b/CD18) on neutrophils. Quantitative polymerase chain reaction Mac-1 analysis shows that LPS-induced transcription of E-selectin mRNA was dramatically suppressed by both 25 and 50 mg/kg of tepoxalin, whereas the level of ICAM-1 was only affected by 50 mg/kg of tepoxalin. Since it has been documented that the expression of E-selectin and Mac-1 is regulated either directly or indirectly by the transcription factor NF kappa B, our studies provide in vivo evidence that tepoxalin is a potent inhibitor of NF kappa B-mediated events in animal models and this novel molecular mechanism clearly defines it as a new class of anti-inflammatory compounds.

Tepoxalin, a novel immunomodulatory compound, synergizes with CsA in suppression of graft-versus-host reaction and allogeneic skin graft rejection.

Tepoxalin, a dual 5-lipoxygenase and cyclooxygenase inhibitor with nonsteroidal antiinflammatory effects, has recently been shown to suppress NF kappa B transactivation and inhibit T cell proliferation via a mechanism very different from cyclosporine (CsA). In this report, we demonstrate that this novel immunosuppressive effect of tepoxalin is manifested in in vivo transplantation models. Tepoxalin suppressed murine spleen cell proliferation in a mixed lymphocyte reaction (MLR) with an IC50 of 1.3 microM. Coadministration of tepoxalin and CsA in MLR cultures showed an additive inhibitory effect. Oral administration of tepoxalin at 12 mg/kg/day to mice suppressed local graft-versus-host (GVH) responses by about 40% (n = 10). Combination of tepoxalin and CsA at suboptimal doses synergized their immunosuppressive effects on GVH responses (n = 20). In skin transplantation, the median survival time of allogeneic BALB/cByJ (H-2d) mouse skin grafted onto C3H/HeJ (H-2 kappa) mice was 10.5 days (n = 8), and was prolonged to 15.0 days (n = 9) for recipient mice administered tepoxalin at 50 mg/kg/day. Coadministration of suboptimal doses of tepoxalin (12.5 mg/kg/day) and CsA (50 mg/kg/day) prolonged skin graft rejections dramatically (55% of the grafts survived for more than 40 days, n = 9). Taken together, these results demonstrate that tepoxalin is a potent immunomodulatory compound that, when combined with CsA, provides synergistic immunosuppressive activity. The fact that tepoxalin and CsA act on different transcription factors, NF kappa B and NFAT respectively, might explain the synergistic suppressive effects when both compounds were used. Tepoxalin could be an important addition to the cohort of immunosuppressive therapies currently used in solid organ and bone marrow transplantations.

Enhancement of immunostimulatory activity by dual substitution of C8-substituted guanine ribonucleosides: correlation with increased cytokine secretion.

Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine). All of the compounds enhance natural killer (NK) activity, lymphocyte proliferation, and antibody production in dose-dependent fashion. However, the potency and maximal activity of the disubstituted analogues are considerably greater than those of the monosubstituted analogues. Spleen cells stimulated for 48 h with the disubstituted compounds produce immunoreactive interleukin (IL) 1 alpha, IL-6, tumor necrosis factor-alpha (TNF alpha), and interferon-gamma (IFN gamma). Monosubstituted analogues induce lower quantities of IL-6, TNF alpha, and IFN gamma and fail to induce detectable levels of IL-1 alpha. Total IFN activity, assessed by viral inhibition assay, is also lower for the monosubstituted analogues. Augmentation of antibody secretion by B cells is diminished for neither mono- nor disubstituted compounds upon incubation with anti-cytokine antibodies. In contrast, anti-IFN alpha beta markedly reduces the effects of monosubstituted analogues on NK activity but has less marked effects on NK induction by the disubstituted compounds. A similar pattern of differences is seen for lymphocyte proliferation. Thus, although the analogues induce synthesis of several cytokines, to date only IFN alpha beta appears directly involved in enhancement of NK activity and lymphocyte proliferation. The present data do not, however, exclude the existence of an autocrine stimulatory mechanism not susceptible to inhibition by anti-cytokine antibodies.

Murine strain variation in the natural killer cell and proliferative responses to the immunostimulatory compound 7-allyl-8-oxoguanosine: role of cytokines.

7-Allyl-8-oxoguanosine (loxoribine) is a di-substituted guanine ribonucleoside which has been shown previously to enhance murine NK activity, B lymphocyte proliferation, and antibody synthesis. In this study we examined the relationship among enhancement of NK activity, proliferation, and cytokine synthesis in the responses of different strains of mice to loxoribine to provide insight into the role of cytokines in these biological activities. The NK response of mice was enhanced both in vitro and in vivo in all strains tested with the exception of the NK-deficient beige (BgBg) mouse. However, there was a marked difference in the degree of NK enhancement noted in other inbred strains, with C3H and CBA mice producing the highest responses, C57BL/6, BALB/c, and DBA/2 strains giving intermediate responses, and SJL mice manifesting low responses. Striking enhancement of NK cell activity was seen in SCID mice. A somewhat different effect was seen in humans. Loxoribine treatment enhanced both the NK and LAK activity of cells from individuals with low and high spontaneous NK activity. The degree of enhancement was similar for both groups, and thus the general hierarchy of NK activity among different donors was maintained. There was less interstrain variation in the murine proliferative response to loxoribine although nude (NuNu) mice showed the highest activity and SJL mice produced substantially lower responses than other strains. All strains produced IL-6, TNF alpha, IFN-alpha/beta, and IFN-gamma when spleen cells were cultured for 48 hr with loxoribine. Interstrain variability of cytokine synthesis displayed no consistent pattern from one cytokine to another, and all failed to correlate with interstrain variability of NK cell activity or B lymphocyte proliferation. When anti-cytokine antibodies were tested for the ability to block the immunostimulatory effects of loxoribine, only anti-IFN-alpha/beta and, to a lesser degree, anti-IFN-beta, partially inhibited NK activation. Similarly, only anti-IFN alpha/beta antibodies partially blocked the proliferative response to loxoribine. In both cases, reconstitution of the responses was achieved by adding back IFN-alpha/beta to cultures containing antibodies against IFN-alpha/beta. These data suggest that, although several cytokines are produced in response to loxoribine, only IFN-alpha and IFN-beta are directly involved in the NK activation and proliferative responses. The pattern of strain variation appears to be reflective of variation in NK cell responsiveness to IFN-alpha/beta.

Small-molecule immunostimulants. Synthesis and activity of 7,8-disubstituted guanosines and structurally related compounds.

A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine.

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1.1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.

Loxoribine (7-allyl-8-oxoguanosine) activates natural killer cells and primes cytolytic precursor cells for activation by IL-2.

Guanine ribonucleosides that have been substituted at the C8 position with bromine or thiol groups have been shown previously to activate NK cells and to act as sparing agents for IL-2 in the generation of LAK cells. Herein, we examined a disubstituted guanosine, 7-allyl-8-oxoguanosine (loxoribine), for the ability to activate NK cells and to interact with IL-2 in the generation of LAK cells. Loxoribine enhanced the NK activity of murine spleen cells with optimal activity occurring after 10 h of culture at concentrations ranging from 50 to 150 microM. The response was, however, short lived, approaching baseline levels by 24 h of culture. In contrast, if spleen cells were cultured with a suboptimal concentration of IL-2 (10 U/ml) in combination with loxoribine, a prolonged and enhanced cytolytic activity was seen. The enhancement was greatest if the loxoribine and IL-2 were both added to the cultures at the beginning of the incubation period. Analysis of the expression of the alpha-chain of the IL-2 receptor after loxoribine stimulation indicated that gene transcription was enhanced within 4 h, and cell surface expression was observed on NK1.1+ Thy1+ and NK1.1+ Thy1- cell populations within 24 h of loxoribine treatment. The priming of LAK cell precursors by loxoribine did not appear to be mediated by IFN-alpha/beta, because anti-IFN antibodies did not block either the activation of cytolytic cells by IL-2 or the expression of IL-2 receptors after culture with loxoribine. These data suggest that one mechanism by which cytolytic precursor cells are primed by loxoribine to respond to IL-2 faster and with enhanced cytolytic activity may be through the expression of high affinity IL-2 receptors due to the up-regulation of the alpha-chain.

In vivo activation of natural killer cells and priming of IL-2 responsive cytolytic cells by loxoribine (7-allyl-8-oxoguanosine).

Guanine ribonucleosides which have been substituted at the N7 and/or C8 positions have been shown previously to activate natural killer (NK) cells and to act as sparing agents for interleukin-2 (IL-2) in the in vitro generation of lymphokine activated killer (LAK) cells. In this paper we examined a disubstituted guanosine, 7-allyl-8-oxoguanosine (loxoribine), for the ability to activate NK cells and to interact with IL-2 in the generation of LAK cells in vivo. Following iv administration, loxoribine enhanced murine splenic NK activity in a dose-related fashion, with optimal responses occurring at 3 mg/mouse. Enhanced lysis of YAC-1 cells was seen within 6 hr of injection and NK activity remained elevated for over 96 hr. Mature B and T cells were not required for NK activation since SCID mice responded to loxoribine within the same dose range as did the normal, immunocompetent mice. Both effector and precursor cells were eliminated by the administration of anti-asialo GM1 antibodies and NK activation was totally blocked in mice injected with anti-NK 1.1 antibodies. To test whether loxoribine would act as a sparing agent for IL-2 stimulated LAK activation, mice were injected with 2 mg loxoribine followed by twice daily administration of 10,000 units IL-2. In assays performed 48, 72, and 96 hr after injection of loxoribine, the cytolytic activity with the combination therapy exceeded the activity expected from the algebraic sum of the responses to the individual agents. Single injections of 2 mg loxoribine and 25,000 units IL-2 also stimulated NK/LAK activity, but the greatest enhancement was seen when loxoribine was administered 24 hr before the IL-2. Analysis of mRNA transcripts for the alpha chain of the IL-2 receptor indicated that gene transcription was enhanced within hours of loxoribine administration.

In vivo enhancement of murine natural killer cell activity by 7-allyl-8-oxoguanosine (loxoribine).

7-Allyl-8-oxoguanosine (loxoribine) is a novel immunostimulatory compound which has been shown previously to enhance the antibody synthesis of antigen-stimulated B-lymphocytes. In this report, loxoribine was tested for the ability to activate murine natural killer (NK) cells. In studies in which mice were given a single subcutaneous (s.c.) or intravenous (i.v.) injection of loxoribine, splenic NK cell activity was increased in a dose-related manner with clear enhancement seen within 2 h of drug administration. The enhancement was optimal at 48 h but persisted for a minimum of 4 days. Slow and continuous administration of loxoribine via subcutaneously implanted infusion pumps successfully enhanced the NK activity for several days after all of the pump contents had been delivered. Peak NK responses were seen following s.c. or i.v. administration of 2-3 mg loxoribine per mouse in sesame oil, intralipid, or saline vehicles. Significant oral activity was seen after the administration of 8-10 mg/mouse in sesame oil or intralipid. The in vivo enhancement of NK activity was observed in spleen, blood, and bone marrow but was negligible in lymph nodes and thymus. Multiple injections of optimal concentrations of loxoribine did not tend to enhance the NK activity above that seen with a single injection, suggesting that the timing of injections was critical for optimal responsiveness.

Ex vivo spermine dialdehyde treatment prevents lethal GVHD in a murine bone marrow transplantation model.

Spermine dialdehyde (SDA), an oxidized product of spermine which irreversibly suppresses T cell and NK cell activities, was evaluated as an ex vivo purging agent in the prevention of graft-versus-host disease (GVHD) in mice transplanted with SDA-treated allogeneic bone marrow. In this model, lethally irradiated C3H (H-2k) mice received BALB/c (H-2d) bone marrow and spleen cell mixtures which had been treated ex vivo for 10 min with SDA. Mice receiving SDA-treated cells survived past 100 days whereas mice in the control group died between days 25-35 suffering from severe GVHD. Surviving mice from the SDA-treated groups exhibited full chimerism at day 120. In vitro assays indicated that SDA inhibited T cell and NK cell activities at concentrations that spared myeloid cell growth. When a minimum number of bone marrow cells were used for reconstitution, SDA-treated marrow reconstituted lethally irradiated mice as effectively as control marrow suggesting that SDA had little impact on early myeloid cells which are required for engraftment. SDA may have clinical application as a purging agent in allogeneic bone marrow transplantation.