Immune regulation of procalcitonin: a biomarker and mediator of infection.

Procalcitonin (PCT) has recently emerged as a powerful biomarker for an early and accurate diagnosis of bacterial infection. Here we summarize our current understanding of the expression pathways of PCT, its potential cellular sources including immune cells, and factors inducing its secretion. Also addressed is the significance of increased blood PCT concentration, which may allow this molecule not only to act as a clinical biomarker but also as an active participant in the development and progression of infectious processes. Experimental approaches to delineate a better understanding of PCT functions, molecular pathways that modulate its expression and therapeutic opportunities to curtail its biological actions are discussed, as well.

Genetics of allergen-induced asthma.

Antigen-induced airway hyperresponsiveness and airway inflammation are features of both human asthma and animal models of this disease. The genesis of these key asthma phenotypes represents the summation of a complex cascade of immune responses. It is hypothesized that multiple cell types are involved in the induction, propagation, and maintenance of these immune processes. Several molecules have been reported to be essential for cell-cell interactions, inflammatory cell recruitment, and effector functions leading to the overall expression of the asthmatic phenotype. This review summarizes the genetic evidence supporting a role for these molecules in antigen-driven airway hyperresponsiveness and inflammation.

Genetics of airway hyperresponsiveness.

Asthma is a disease characterized by intermittent airway obstruction, inflammatory cell infiltrates, increased mucus production, lung epithelial remodeling, and airway hyperreactivity. The genetics of asthma, as investigated in animal models, is poorly understood. Because no animal model of asthma mimics all of the pathologic and physiological features of asthma, genetic studies have focused on several phenotypes, including intrinsic or native airway hyperreactivity. It is generally accepted that both genetic and environmental factors determine the phenotypic expression of this complex disease. The genetics of airway hyperresponsiveness, as investigated in the mouse, are presented in this review. The inbred mouse currently represents the most valuable genetic resource for understanding the factors that control this complex phenotype.

Increased severity of local and systemic anaphylactic reactions in gp49B1-deficient mice.

gp49B1 is an immunoglobulin (Ig) superfamily member that inhibits FcstraightepsilonRI-induced mast cell activation when the two receptors are coligated with antibodies in vitro. The critical question of in vivo function of gp49B1 is now addressed in gene-disrupted mice. gp49B1-deficient mice exhibited a significantly increased sensitivity to IgE-dependent passive cutaneous anaphylaxis as assessed by greater tissue swelling and mast cell degranulation in situ. Importantly, by the same criteria, the absence of gp49B1 also resulted in a lower threshold for antigen challenge in active cutaneous anaphylaxis, in which the antigen-specific antibody levels were comparable in gp49B1-deficient and sufficient mice. Moreover, the absence of gp49B1 resulted in a significantly greater and faster death rate in active systemic anaphylaxis. These results indicate that gp49B1 innately dampens adaptive immediate hypersensitivity responses by suppressing mast cell activation in vivo. In addition, this study provides a new concept and target for regulation of allergic disease susceptibility and severity.

Pathogenesis of herpes simplex virus-induced ocular immunoinflammatory lesions in B-cell-deficient mice.

The role of B cells and humoral immunity in herpes simplex virus (HSV) ocular infections was studied in immunoglobulin mu chain gene-targeted B-cell-deficient mice (muK/O). At doses of virus well tolerated by immunocompetent mice, heightened susceptibility of muK/O mice to herpetic encephalitis as well as to herpetic stromal keratitis (HSK) was observed. An explanation was sought for the increased severity of HSK in the muK/O mice. First, the lack of antibody responses in muK/O mice resulted in longer viral persistence and dissemination to the corneal stroma, the site of inflammation. Prolonged virus expression in the corneal stroma was suggested to cause bystander activation of Th1-type CD4(+) T cells, further contributing to the severity of HSK lesion expression in muK/O mice. Second, muK/O mice generated minimal Th2 cytokine responses compared to wild-type mice. Such responses might serve to downregulate the severity of Th1-mediated HSK lesions.

Distribution fate and mechanism of immune modulation following mucosal delivery of plasmid DNA encoding IL-10.

DNA vaccination has been widely studied in several models of vaccination and in the treatment of inflammatory diseases, even though the mechanism involved is still unclear. This report demonstrates that mucosal administration of plasmid DNA leads to rapid and widespread distribution around the body. Dissemination likely occurred via the bloodstream because plasmid DNA was present in blood plasma. The plasmid DNA was also detectable in several tissues including draining lymph nodes, spleen, liver, bone marrow, and even the dermis of ear pinnae. Except for the site of administration, plasmid DNA was no longer detectable in tissues after 3 wk postadministration. RNA and protein expression was also found in the tissues and bloodstream. Animals previously primed by HSV infection and subsequently given IL-10 DNA via the nasal mucosa, showed diminished Ag-induced delayed type hypersensitivity reactions for up to 5 wk posttreatment. The mechanism of modulation involved diminished the Ag-specific proliferation and production of Th1 cytokines. The Ag-specific silencing effects persisted beyond the duration of detectable plasmid encoded protein and was maintained upon adoptive transfer of T cells into a plasmid-free environment. The silenced T cells were not a source of IL-10, and their anergic state was reversible by exposure to Ag in the presence of exogenous IL-2.

Immune modulation by IL-10 gene transfer via viral vector and plasmid DNA: implication for gene therapy.

In the present report, we have evaluated and compared the modulatory effect of the cytokine interleukin (IL)-10 expressed via viral vector or plasmid DNA on viral antigen-induced cutaneous inflammatory lesions. Our data demonstrate the superior potency of both recombinant vaccinia virus and herpes simplex virus IL-10 expression vectors after single intramuscular administration, but the effects were only short term and only functioned in animals lacking immunity to the viral vectors used for modulation. In contrast, modulatory effects achieved by plasmid DNA expressing IL-10 were delayed in onset and milder in effect but were far more persistent than those achieved by viral vectors. Moreover, plasmid DNA expressing IL-10 provided effective modulation when given repeatedly to animals. Our data also showed that IL-10 gene delivery resulted in a systemic and durable modulatory effect while the effect caused by a single IL-10 protein treatment was transient and confined to the injected site. Our results imply that the viral vector system is superior for obtaining short-term effects, whereas the plasmid DNA approach represents a better strategy to achieve gene therapy to modulate chronic inflammatory lesions.

Resistance to herpetic stromal keratitis in immunized B-cell-deficient mice.

This study evaluates the role of antibody as an indicator of immunity to ocular challenge with herpes simplex virus (HSV). Two genotypes of mice, BALB/c or BALB/c with mu-chain knockout (muK/O; which lack functional B cells), were immunized systemically either with nonvirulent infectious virus or with a eukaryotic expression plasmid encoding glycoprotein B (gB). Whereas naive muK/O mice were 10- to 100-fold more susceptible to HSV infection than BALB/c mice, following immunization both groups showed similar levels of resistance to ocular challenge. Thus both HSV-immunized groups cleared virus within 3 days and showed no signs of ocular lesions. gB DNA-immunized mice cleared virus less rapidly (5 days), and the incidence of lesions was 10 and 25% in BALB/c and muK/O mice, respectively. Since muK/O mice failed to produce detectable anti-HSV antibody, the mechanism of rapid viral removal was assumed to have a T cell basis. However, T cells would likely not mediate any protection directly since such cells were absent in infected corneas during clearance. A likely mechanism of immunity could involve innate defenses, perhaps enhanced by the action of cytokines released from antigen-reactive CD4+ cells in vascularized tissue adjacent to the cornea. Thus an abrupt inflammatory response consisting principally of neutrophils occurred in the corneal stroma in immune mice, and this subsided when virus disappeared. These data reveal that even though the deficiency in generating antibody renders mice more susceptible to HSV infection, once primed, resistance to disease expression is mediated solely by the cellular components and their products.

Virus-induced immunoinflammatory lesions in the absence of viral antigen recognition.

Herpetic stromal keratitis (HSK) is a CD4+ T cell-controlled immunopathologic lesion in the eye that results from infection with herpes simplex virus (HSV). Target Ags involved in HSK remain undefined. In this study, we determined if HSK could be induced in animals genetically incapable of generating HSV Ag-specific CD4+ T cells. Mice bearing transgenic TCR specific to OVA peptide 323-339 (DO11.10) were crossed to SCID mice whose offspring (Tg-SCID) possessed CD4+ T cells, >98% of which expressed the OVA peptide-specific TCR. HSV infection of Tg-SCID mice was lethal, and mice failed to generate detectable T cell responses even after repeated immunization with a mutant avirulent virus (AN-1). Immunization with AN-1 virus followed by ocular challenge with HSV resulted in ocular inflammation before encephalitis, in contrast to the protection conferred in the control BALB/c and DO11.10 mice. These results indicate that clinical HSK may not require viral Ag recognition by CD4+ T cells and that T cells of irrelevant specificity can be recruited, activated, and driven into effector function in the HSV-infected cornea. This is suggested to represent a bystander activation effect resulting from the presence of proinflammatory mediators resulting from HSV replication.

Modulation of virus-induced delayed-type hypersensitivity by plasmid DNA encoding the cytokine interleukin-10.

This report evaluates the efficacy of eukaryotic expression plasmids encoding cytokines at modulating the induction and expression of cutaneous delayed-type hypersensitivity (DTH) responses to virus infections. Mice given a single intramuscular administration of cytokine DNA were subsequently infected with either herpes simplex virus (HSV) or vaccinia virus, then tested for DTH. Responses in animals given interleukin-10 DNA were markedly suppressed for at least 5 weeks after pretreatment. Animals also expressed diminished T-cell proliferative responses and modest changes in the balance of T helper type 1 and 2 T-cell reactions. Treatment of animals already sensitized to express DTH, also showed inhibited responses, these taking 6-7 days after treatment to become apparent. Our results show the potency and convenience of plasmid DNA encoding cytokines to modulate inflammatory reactions. Advantages and risks of the cytokine DNA approach are briefly discussed.

Immune induction and modulation by topical ocular administration of plasmid DNA encoding antigens and cytokines.

In this article, the authors investigated if administration of eukaryotic expression plasmid DNA delivered to the ocular surface provided a means of inducing and modulating the immune response to herpes simplex virus (HSV). Topical application of gB DNA led to the development of HSV specific systemic humoral and cellular immunity. In addition, mucosal antibody was induced at both proximal and distal locations. Topically gB DNA immunized animals were protected against lethal challenge via either the systemic or the vaginal mucosal routes. Ocular pre-exposure to DNA encoding the cytokines interleukin (IL)-4 or IL-10, but not IL-2 or interferon-gamma, modulated the severity of the immunoinflammatory response to subsequent corneal infection with HSV. The present results indicate that the ocular surface provides a readily accessible site for DNA immunization and is suitable for both immune induction and modulation of the nature of the immune response that is induced.

Immunomodulation by mucosal gene transfer using TGF-beta DNA.

This report evaluates the efficacy of DNA encoding TGF-beta administered mucosally to suppress immunity and modulate the immunoinflammatory response to herpes simplex virus (HSV) infection. A single intranasal administration of an eukaryotic expression vector encoding TGF-beta1 led to expression in the lung and lymphoid tissue. T cell-mediated immune responses to HSV infection were suppressed with this effect persisting as measured by the delayed-type hypersensitivity reaction for at least 7 wk. Treated animals were more susceptible to systemic infection with HSV. Multiple prophylactic mucosal administrations of TGF-beta DNA also suppressed the severity of ocular lesions caused by HSV infection, although no effects on this immunoinflammatory response were evident after therapeutic treatment with TGF-beta DNA. Our results demonstrate that the direct mucosal gene transfer of immunomodulatory cytokines provides a convenient means of modulating immunity and influencing the expression of inflammatory disorders.

Modulation of viral immunoinflammatory responses with cytokine DNA administered by different routes.

The efficacy of plasmid DNA encoding cytokine administered by different routes, systemic or surface exposure, was evaluated and compared for their modulating effects on subsequent lesions caused by infection with herpes simplex virus (HSV). Systemic or topical administration of both interleukin-4 (IL-4) and IL-10 DNA but not IL-2 DNA caused a long-lasting suppression of HSV-specific delayed-type hypersensitivity response. IL-4 or IL-10 DNA preadministration also modulated the expression of immunoinflammatory lesions associated with corneal infection of HSV. Suppression of ocular lesions required that the DNA be administered to the nasal mucosa or ocular surfaces and was not evident after intramuscular administration. The modulating effect of IL-10 DNA was most evident after topical ocular administration, whereas the effects of IL-4 DNA given by both routes appeared to be equal. Preexposure of IL-4 DNA, but not IL-10 DNA, resulted in a significant change in Th subset balance following HSV infection. Our results indicate that the modulating effect of IL-4 or IL-10 DNA may proceed by different mechanisms. Furthermore, our results suggest that surface administration of cytokine DNA is a convenient means of modulating immunoinflammatory lesions.

Role of mucosal immunity in herpes simplex virus infection.

This study evaluates whether the vaginal mucosal surface of immunized mice can prevent invasion by herpes simplex virus (HSV) and aims to identify immune components that affect immunity after challenge at the vaginal mucosa. Despite the induction of both IgA and IgG vaginal Ab following immunization with recombinant vaccinia virus vectors expressing either glycoproteins B or D, viral infection occurred in most animals even after minimal viral dose challenge. Challenged immune animals, including those genetically unable to generate anti-HSV Ab, survived and showed few if any clinical signs of infection. Experiments with T cell subtype knockout animals and depletion with T cell subset-specific MAb indicated that immunity following vaginal challenge was principally dependent on the function of CD4+ T cells. Our results indicate that anti-HSV vaccines may not provide barrier immunity at the vaginal mucosal site but may be adequate to minimize clinical expression of disease.

Modulation of mucosal and systemic immunity by enteric administration of nonreplicating herpes simplex virus expressing cytokines.

In this report the ability of enteric immunization with recombinant replication deficient (ICP4-/-) HSV expressing IFN gamma to generate protection and modulate mucosal and systemic immunity was evaluated. ICP4-/-HSV, ICP4-/-HSV expressing IL4, live replicating, and uv HSV were used as controls. Following enteric administration of live HSV, a Th1 cytokine response was induced in the spleen, while both Th1 and notable Th2 cytokine production were detected at mucosal sites. Modulation of mucosal and systemic immune response was achieved when nonreplicating recombinant HSV viruses expressing cytokines were used. Compared to the control replication defective viruses, decreased frequency of Th2 cytokine producing cells in Peyer's patches was observed following enteric administration of nonreplicating HSV expressing IFN gamma. When IFN gamma expressing virus was given enterically, modulation was observed at the systemic level, measured by ELISPOT for cytokine producing cells, ELISA from the in vitro restimulated splenic cell cultures, and by the increase of the IgG2a/IgG1 ratio in the serum. This report provides evidence that replication defective viruses expressing cytokine genes in contrast to uv HSV, are immunogenic when administered enterically and can generate significant immunomodulatory effects at the mucosal and systemic levels.

Production of key molecules by ocular neutrophils early after herpetic infection of the cornea.

Herpes simplex virus infection of the eye can result in a blinding inflammatory lesion that is a T cell mediated immunopathological reaction. A prominent early event following HSV infection is neutrophil invasion of the corneal stroma. These cells may be involved in viral clearance and may influence the nature of the anti HSV T cell response which subsequently occurs. This article measures the expression of some key molecules which could participate in viral clearance and immune modulation. Using RT-PCR and in-situ hybridization, both corneal and peritoneal neutrophils were shown to be sources of iNOS and TNF alpha molecules which likely contribute to antiviral activity. Neutrophils also produce the cytokine IL-12, a key molecule which modulates the CD4+ T cell response to a type which mediates immunopathology. The present results indicate that neutrophils play an important role in the pathogenesis of herpetic ocular lesions.

Herpes simplex virus latency and the immune response.

Following infection, herpes simplex virus establishes latency in the nervous system and recurrences of lytic replication occur periodically. Molecular events which may determine how virus enters latency, how it is maintained and what occurs during reactivation have been investigated. The role of the immune response in limiting infection of the nervous system, influencing the latent state and removing virus from peripheral sites following reactivation has also been studied.

Suppression of ongoing ocular inflammatory disease by topical administration of plasmid DNA encoding IL-10.

Ocular infection with herpes simplex virus leads to an inflammatory lesion in the cornea orchestrated by CD4+ Th1 lymphocytes. This immunopathologic disease, called herpetic stromal keratitis, is an important cause of impaired vision. In this study, we set out to determine whether established lesions of herpetic stromal keratitis could be controlled by topically administering naked plasmid DNA encoding cytokines to the corneal surface. A single topical administration of DNA encoding IL-10 was beneficial to the majority (75%) of treated animals, and 50% (vs 10% in controls) resolved their lesions completely over a 23-day observation period. Topical ocular application of DNA encoding foreign proteins was also shown to be an effective means of inducing systemic and mucosal immune responses. The direct application of DNA encoding cytokines may represent an additional therapeutic option for the management of immunoinflammatory disease.

Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunization.

The ability of mucosally delivered plasmid DNA encoding glycoprotein B (gB) of herpes simplex virus type 1 (HSV-1) to generate systemic as well as distal mucosal immunity was evaluated. BALB/c mice were immunized intranasally (i.n.) with gB DNA or DNA expressing beta-galactosidase (beta-Gal). Two days following immunization, gB and beta-Gal gene expression was detected by reverse transcription (RT)-PCR in lungs and cervical lymph nodes (CLN). Histological analysis showed that beta-Gal protein was expressed in vivo in the lungs and the CLN of animals immunized with i.n. administered beta-Gal DNA. The immune responses generated by i.n. administration of gB DNA with or without cholera toxin (CT) were compared to those generated by intramuscular (i.m.) gB DNA and i.n. live HSV administration. Three i.n. doses of gB DNA over a 3-week period resulted in a distal mucosal immunoglobulin A (IgA) response. In addition, the mucosal IgA response was enhanced by coadministration of CT with gB DNA. The i.m. route of immunization induced a strong IgG response in the serum and vagina but was inefficient in generating a mucosal IgA response. Antigen-specific cytokine ELISPOT analyses as well as the serum IgG1/IgG2a ratio indicated induction of stronger Th2 responses following the additional i.n. administration of CT compared to i.n. or i.m. gB DNA or i.n. live HSV immunization. In addition, mucosal immunization with gB DNA induced anti-HSV cell-mediated immunity in vivo as measured by delayed-type hypersensitivity. Although i.n. DNA immunization was an effective means of inducing mucosal antibody, it was inferior to i.m. DNA delivery in providing protection against lethal HSV challenge via the vaginal route. In addition, both i.m. and i.n. plasmid immunizations failed to generate an immune barrier to viral invasion of the mucosa.

Disease in the scurfy (sf) mouse is associated with overexpression of cytokine genes.

The murine X-linked lymphoproliferative disease scurfy is similar to the Wiskott-Aldrich syndrome in humans. Disease in scurfy (sf) mice is mediated by CD4+ T cells. Based on similarities in scurfy mice and transgenic mice that overexpress specific cytokine genes, we evaluated the expression of cytokines in the lesions of sf mice by Northern blotting, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and by hybridization in situ. Overall, the phenotypic characteristics of scurfy disease correlated well with increased interleukin (IL)-4 (lymphadenopathy), IL-6 (B cell proliferation, hypergammaglobulinemia), IL-7 (dermal inflammatory cell infiltration), and high levels of tumor necrosis factor-alpha (wasting).