Transfer of a T-helper type 2 clone into conjunctiva induces corneal damage in mice.

PURPOSE: We established a T-helper Type 2 (Th2) clone-induced conjunctival eosinophilia model by injecting D10.G.4.1 (D10) cells, a murine Th2 clone, and conalbumin, its specific antigen, into conjunctiva of AKR/J mice. Using this model, we investigated the effect of a coinjection of D10 cells and conalbumin into conjunctiva on corneal damage. METHODS: Corneal fluorescein staining scores and eosinophil peroxidase (EPO) activity in conjunctiva were measured after coinjection of D10 and conalbumin into conjunctiva, and the effects of cyclosporine A, betamethasone, and anti-interleukin-5 antibody on staining scores and EPO activity were examined. RESULTS: Coinjection of D10 and conalbumin induced an increase of the corneal fluorescein staining score after 24, 48, and 96 hours and 10 days. EPO activity in conjunctiva increased time-dependently until 24 hours after coinjection. The increase in the staining score followed the time dependent increase in EPO activity. The instillation of cyclosporine A, an inhibitor of cytokine production from T-cells, and betamethasone significantly inhibited the increase in corneal fluorescein score and EPO activity. Intraperitoneal administration of anti-interleukin-5 monoclonal antibody, which inhibits the infiltration of eosinophils into the conjunctiva, completely inhibited the increase in staining score. CONCLUSION: The transfer of the Th2 clone into the murine conjunctiva induced corneal damage, which may have been caused by Th2 cell-produced interleukin-5 that mediated the activation of eosinophils.

Evaluation of the prophylactic use of ovotransferrin against chlamydiosis in SPF turkeys.

Chlamydophila (C.) psittaci infections are highly prevalent in turkeys and the economical and public health importance of these infections has been recognized since 1950. As there are no vaccines, antibiotic treatment (tetracylines, enrofloxacine) is often needed to allow marketing of poultry. In this study, we explored the use of ovotransferrin (ovoTF), a natural anti-microbial protein, in preventing an experimental C. psittaci infection in specific pathogen free (SPF) turkeys. Turkeys were treated with aerosolized ovoTF prior to the infection. Groups 1 and 2 received a single dose of 10 and 5 mg ovoTF per turkey, respectively. Group 3 received a daily dose of 5mg ovoTF per turkey during 12 days. Group 4 served as untreated, infected control group. Turkeys were aerosol infected using 10(6) TCID(50) of the virulent C. psittaci serovar/genotype D strain 92/1293. Birds were monitored (clinical signs, bacterial excretion) during 12 subsequent days before being necropsied. At necropsy, pathology and C. psittaci replication in various tissues was examined. A single dose of 10mg ovoTF and a repeated daily dose of 5mg ovoTF could not prevent the birds from becoming infected with C. psittaci, but they significantly reduced the outcome of the infection. A single dose of 5mg ovoTF had no influence on the outcome of the infection as compared to the non-treated infected controls. Our results demonstrate the anti-chlamydial effect of ovoTF in vivo and present a base for further research on practical applications of ovoTF on turkey farms.

Role of CD44 in activation-induced cell death: CD44-deficient mice exhibit enhanced T cell response to conventional and superantigens.

T cells upon activation are known to up-regulate CD44 expression. However, the precise function of CD44 on activated T cells is not clear. In this report, we demonstrate that signaling through CD44 plays an important role in activation-induced cell death (AICD). CD44 knockout (KO) mice had an elevated in vivo primary and in vitro secondary response to challenge with conalbumin, anti-CD3 mAb and staphylococcal enterotoxin A (SEA), which correlated with reduced AICD when compared to CD44 wild-type mice. In addition, CD44 KO mice exhibited increased delayed-type hypersensitivity response to dinitrofluorobenzene. In a model examining in vitro AICD, splenocytes from CD44 KO mice showed resistance to TCR-mediated apoptosis when compared to splenocytes from CD44 wild-type mice. In addition, signaling through CD44 led to increased apoptosis in TCR-activated but not resting T cells from CD44 wild-type mice without affecting Fas expression. Injection of SEA into mice deficient in CD44 and Fas (CD44 KO/lpr) led to an increased primary response when compared to mice that expressed CD44 but not Fas (CD44 WT/lpr), suggesting that the enhanced response to SEA was dependent on CD44 but not Fas expression. Administration of anti-CD44 mAb into CD44 wild-type mice caused a significant decrease in antigen-specific T cell response. Together, these data implicate CD44 as an important regulator of AICD in T cells. Furthermore, targeting CD44 in vivo may constitute a novel approach to induce apoptosis in activated T cells, and therefore to treat autoimmune diseases, allograft rejection and graft versus host disease.

The intrasplenic circulation of three formulations of the same protein antigen.

This paper presents a kinetic study of the intrasplenic circulation of three formulations of the protein antigen conalbumin including the soluble form and two liposomal formulations, one encapsulated in the internal aqueous milieu and one surface-linked to the liposomal vehicle. These formulations differ not only in their physical status but also in their immunostimulating properties and were chosen in an attempt to correlate the movements of antigen in lymphoid tissues with the immune response elicited. The presence of conalbumin was followed over a period of 21 days using, as a detection system, an antibody that we developed and which allows for the visualization of antigenic peptides such as those presented at the surface of antigen-presenting cells (APC). The results demonstrate that the amount of antigen accessing to the spleen, its time of residency and the pathway it follows are all profoundly influenced by the form under which it penetrates the immune system. The results also indicate that the marked initial preferences of an antigen for either B cells, marginal zone macrophages (MZM) or metallophilic macrophages (MM) are of fundamental importance in determining the fate of this antigen in the spleen. It is concluded that the exact formulation of an antigen is as crucial to the regulation of the immune response as is the nature of this antigen. It is further concluded that liposomes can be used efficiently to modify the formulation of an antigen and can contribute as such to the induction of specific immune functions by driving the antigen towards some privileged immune cell populations.

CpG oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma.

CpG oligodeoxynucleotides (CpG-ODN) administered during Ag sensitization or before Ag challenge can inhibit allergic pulmonary inflammation and airway hyperreactivity in murine models of asthma. In this study, we investigated whether CpG-ODN can reverse an ongoing allergic pulmonary reaction in a mouse model of asthma. AKR mice were sensitized with conalbumin followed by two intratracheal challenges at weekly intervals. CpG-ODN was administered 24 h after the first Ag challenge. CpG-ODN administration reduced Ag-specific IgE levels, bronchoalveolar lavage fluid eosinophils, mucus production, and airway hyperreactivity. We found that postchallenge CpG-ODN treatment significantly increased IFN-gamma concentrations and decreased IL-13, IL-4, and IL-5 concentrations in bronchoalveolar lavage fluids and spleen cell culture supernatants. Postchallenge CpG-ODN treatment also increased B7.1 mRNA expression and decreased B7.2 mRNA expression in lung tissues. These results suggest that CpG-ODN may have potential for treatment of allergic asthma by suppressing Th2 responses during IgE-dependent allergic airway reactions. The down-regulation of Th2 responses by CPG-ODN may be associated with regulation of the costimulatory factors B7.1 and B7.2.

Bruton's tyrosine kinase deficiency in macrophages inhibits nitric oxide generation leading to enhancement of IL-12 induction.

We show that macrophages of X-linked immunodeficient mice with a mutant nonfunctional Bruton's tyrosine kinase produce less NO than wild-type macrophages in response to a variety of stimuli. Induction of the inducible NO synthase (iNOS) protein, the transcription factor IFN regulatory factor-1 involved in iNOS expression, and the transcription factor STAT-1 involved in regulating IFN regulatory factor-1 induction are all poorer in X-linked immunodeficient than in wild-type macrophages. On the other hand, induction of IL-12 is higher in X-linked immunodeficient than in wild-type macrophages. Macrophage IL-12 induction is enhanced by iNOS inhibitors such as aminoguanidine and thiocitrulline and is inhibited by NO generation via sodium nitroprusside. There is relative enhancement of IFN-gamma production by immune T cells from mice immunized under aminoguanidine cover. Our data thus suggest that Bruton's tyrosine kinase participates in signaling for iNOS induction via IFN regulatory factor-1 in macrophages and that NO is an inhibitor of IL-12 induction.

Induction of pulmonary allergic responses by antigen-specific Th2 cells.

The development of pulmonary allergic responses was examined in mice following pulmonary transfer of Ag (conalbumin)-specific Th2 cells. The levels of serum-specific IgE, cellular infiltrates, airway mucus goblet cells, and airway responsiveness were analyzed and compared with those in Ag-sensitized and -challenged mice. Pulmonary transfer of the conalbumin-specific Th2 clone (D10) induced, in an Ag-specific manner, high levels of the Th2 cytokines IL-4 and IL-5 in the bronchoalveolar lavage fluids and mucosal eosinophils, concomitant with an increase in airway responsiveness. The D10 cell-induced responses were seen in the absence of serum specific IgE. In the presence of Ag, the transferred D10 cells not only remained in the lungs, but also increased in number 72 h post-cell transfer. Although significantly higher levels of IL-4 and IL-5 in the bronchoalveolar lavage fluids were found in D10-transferred mice, the levels of pulmonary eosinophilia, mucus goblet cells, and airway responsiveness were significantly lower than those in Ag-sensitized and -challenged mice. These results demonstrate that although Ag-specific activation of Th2 cells at mucosal sites is able to mediate the recruitment of eosinophils and the subsequent induction of airway hyper-responsiveness, the more severe pulmonary allergic responses were observed only in mice sensitized and challenged with Ag.

Differential biodistribution of encapsulated and surface-linked liposomal antigens.

The biodistribution of liposomal antigens either encapsulated in or surface-linked to liposomes of similar composition was studied over time following intravenous injection and the results analyzed in relation to adjuvanticity. The two formulations were shown to behave very differently in vivo. While encapsulated antigen was rapidly focused to liver and spleen as expected, surface-linked antigen exhibited a more disseminated distribution which parallels that of the free protein. In dual-labelling experiments, it was also shown that encapsulated antigen remains associated with its liposomal vehicle in contrast to surface-linked antigen which is rapidly dissociated. This dissociation was apparently neither due to an exchange with plasma lipoproteins nor to a direct action of blood constituents. Besides, it was found that surface-linked antigen was rapidly accumulated in the carcass. We propose that the retention of the surface-linked antigen in the carcass results from a pre-processing of the protein involving more probably mononuclear phagocytes. This pre-processing might in turn favor the dissociation of the protein from the liposomes in a form that allows its dissemination in the whole organism and its interaction with more efficient antigen presenting cells such as for example Langerhans or dendritic cells.

Liposomal adjuvanticity: effect of encapsulation and surface-linkage on antibody production and proliferative response.

The immunoadjuvanticity of liposomal antigens, namely encapsulated and surface-linked conalbumin, was studied at different levels of an immune response including immunoglobulin production, blastogenic response and lymphokine production in sensitization conditions compatible with vaccine designs. The results demonstrated that both liposomal formulations stimulate all properties analyzed with respect to free antigen but significantly differ in some of their inductive capabilities, suggesting that they follow different routes in the immune network. Thus, although both liposomal antigens are capable of inducing potent humoral responses characterized by increased production of IgM and IgG2a, covalently linked antigen stimulates a quasi-polyclonal blastogenic response accompanied by the simultaneous secretion of IL-2 and IFN gamma, while encapsulated antigen which is less blastogenic mainly induces IL-2 secretion. We can conclude that: first, both antigenic formulations induce a Th1 type of activation and might therefore potentiate cell-mediated immunity, but surface-linkage favors a more rapid maturation of the response and a much more intense help induction. Second, although a strong adjuvanticity can be observed whatever the route of sensitization, namely intraperitoneally, intravenously or subcutaneously, the intravenous injections induce the better potentiation. The in vitro data were all compared to those obtained with naïve mice, allowing dissociation of the contribution of the in vitro rechallenge from that of the in vivo immunization. These differences observed between the liposomal antigens might be taken advantage of while formulating vaccines specifically suited to meet required needs and suggest that covalently linked antigen might be particularly useful in situations where induction of cell-mediated immunity is of prime importance.

Correlation between in vitro and in vivo behaviour of liposomal antigens.

Using conalbumin as a model antigen, we demonstrate in this paper that liposomal antigen differently influences the activation of the immune system depending on the mode of association of the antigen with the liposomal vehicle whether it is by encapsulation or surface linkage. This conclusion is based on in vivo data showing that encapsulated antigen induces a short-lasting response dominated by IgG1 production while surface-linked antigen has a longer-lasting effect characterized by increased production of IgM, IgG2a, IgG3 as well as of IgG1. The in vivo data were complemented by in vitro proliferation studies carried out on spleen cells or macrophage-depleted spleen cells obtained from mice sensitized in vivo and rechallenged in vitro on day 4 following sensitization. Rechallenge was carried out in the absence or presence of anti-IL1. The data indicate that, in contrast to what is generally observed in vivo, liposomes alone potentiate spleen cell proliferative response in a dose-dependent manner. This liposomal effect totally obscures the antigen-specific proliferation that was expected with encapsulated antigen without masking that induced by surface-linked antigen. The mode of antigen association also influences anti-cytokine responsiveness as demonstrated by the insensitivity of the surface-linked antigen response to the presence of anti-IL1 and the significantly decreased response observed with encapsulated antigen under identical conditions. The response to both liposomal antigenic formulations was almost totally abolished in adherent cell-depleted cultures. The overall results therefore suggest that encapsulated and surface-linked antigens activated different immune pathways.