Dirk Bauer

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.

CD4+ T-cell type 1 and type 2 cytokines in the HSV-1 infected cornea.

Abstract · Purpose: It has been previously shown that CD4+ T-lymphocytes are critical mediators in HSV-1 stromal keratitis (HSK). CD4+ T cell subpopulations (type 1, type 2) can be defined by their capabilities of producing different sets of cytokines. This study was performed to determine the role of type 1 and type 2 cytokines in murine HSK. · Methods: BALB/c mice (n=20) were inoculated with 105 PFU of HSV-1 (KOS strain) and were followed clinically. At various time points post-infection (p.i.), the conjunctival and corneal tissues were analyzed histologically (n=2 each time point), and immunohistochemically (n=5 each time point) for the presence of interleukin-1α (IL-1α), type 1 cytokines (IL-2, interferon-γ) and a type 2 cytokine (IL-4). The expression of cytokine mRNA was tested in eye samples by reverse transcription-polymerase chain reaction (RT-PCR). · Results: Stromal keratitis clinically progressed after day 9. In 15% of the mice, disease regressed until day 14 p.i. Polymorphonuclear neutrophils, lymphocytes and other mononuclear cells infiltrated the conjunctiva by day 2 and rapidly expanded to the central cornea between days 7 and 14. IL-1α, IFN-γ and IL-2 mRNA were found in the eyes at days 1 and 2 p.i. IL-1α protein was detected in the conjunctiva, limbus and corneal epithelium at day 2. The IL-1α staining intensities increased with disease progression. This was paralleled by IL-2 and IFN-γ staining intensities. In contrast, IL-4 mRNA and protein were detected at days 7 through 14 after HSV-1 infection; compared to IL-2 and IFN-γ, IL-4 staining intensities were lower. · Conclusions. The findings suggest that the lymphocytic infiltrate during the development of HSV-1 keratitis is predominantly composed of type 1 cells expressing IL-2 and IFN-γ. Type 2 cytokines participate in the late stage of inflammation and might be useful to improve the course of the disease.

Amniotic Membrane Transplantation Induces Apoptosis in T Lymphocytes in Murine Corneas with Experimental Herpetic Stromal Keratitis

PURPOSE To investigate the effect of human amniotic membrane transplantation (AMT) on T-cell immune response in murine corneas with herpetic stromal keratitis (HSK). METHODS Herpes simplex virus (HSV)-1-infected BALB/c mice with necrotizing HSK were treated with AMT. CD3(+) cell apoptosis was determined in treated corneas and in vitro by flow cytometric analysis using the annexin V/7-AAD system. The effect of interleukin (IL)-2, cyclosporine, rapamycin, or Fas on T-cell survival was measured. Activation phenotype was measured by (3)H-thymidine uptake and flow cytometry (CD25, CD69, major histocompatibility complex class II). Cytokine/chemokine secretion from amniotic membrane (AM)-treated corneas or draining lymph node cells was measured. The immune-modulating capacity of long-term AMT treatment and adoptive transfer of AM-treated splenocytes was tested. RESULTS After AMT, HSK and corneal inflammatory cell infiltration improved, and T-lymphocyte apoptosis occurred. T-cell apoptosis was also induced in vitro, independently of rIL-2, cyclosporine, rapamycin, or Fas. AMT-treated corneas and cultured lymphocytes had reduced IL-2, IL-10, IL-12, CRG-2, and CCL-2 content. Long-term AMT treatment decreased the proliferative response and type 1 helper T-cell cytokine level in draining lymph node cells. The improvement in HSK did not persist. Delayed-type hypersensitivity or HSV-1-specific cytotoxicity was not altered CONCLUSIONS The results suggest that murine HSK improves after AMT through reduced local T-helper cell immune responses by inducing apoptosis in T lymphocytes, independently of passive apoptosis or activation-induced cell death. AM also reduces local T-helper cytokine and chemokine levels but does not result in immune deviation. Immunologic memory against HSV-1 is not affected by AMT, and long-term protection or tolerance is not induced.

Conjunctival macrophage-mediated influence of the local and systemic immune response after corneal herpes simplex virus-1 infection

Recently it has been shown that selective subconjunctival macrophage depletion reduced the incidence and severity of stromal herpes simplex virus (HSV) keratitis in mice. In this study, we examined the effect of conjunctival macrophage depletion on the corneal and systemic T‐cell‐mediated immune response. BALB/c mice were treated with subconjunctival injections of dichloromethylene diphosphonate (Cl2MDP)‐liposomes (Cl2MDP‐LIP) or phosphate‐buffered saline (PBS) 7 and 2 days before corneal infection with 105 plaque‐forming units (PFU) of HSV‐1 (KOS strain). Interferon (IFN)‐γ, interleukin (IL)‐2, and IL‐4 production in the cornea was analysed by enzyme‐linked immunosorbent assay (ELISA), and cytokine mRNA levels (IFN‐γ, IL‐4) were measured by semiquantitative reverse transcription–polymerase chain reaction (RT‐PCR). Cell culture supernatants from submandibular lymph nodes were analysed by ELISA for expression of IFN‐γ, IL‐2, and IL‐4 and by bioassay for IL‐6. The HSV‐1‐specific proliferative response of lymphocytes from regional lymph nodes and the delayed‐type hypersensitivity (DTH) response were tested after corneal infection. Virus‐neutralizing antibody titres and HSV‐1‐specific immunoglobulin G (IgG)2a/IgG1‐ratios were measured. Cytokine mRNA expression (IFN‐γ, IL‐4) and secretion (IFN‐γ, IL‐2, IL‐4) in the corneas were decreased after HSV‐1 corneal infection in the macrophage‐depleted mice. The secretion of IFN‐γ and IL‐2 was decreased in the regional lymph nodes from Cl2MDP‐LIP‐treated animals (P<0·05). Furthermore, Cl2MDP‐LIP‐treated mice had decreased HSV‐1 specific proliferative responses (P<0·05) and DTH response after corneal HSV‐1 infection (P<0·05). The virus‐neutralizing serum‐antibody levels (P<0·05) increased while the HSV‐1 specific IgG2a/IgG1‐ratio was unaffected after macrophage depletion. Macrophage depletion did not induce a shift between the T helper 1 (Th1) and Th2 response in this HSK model. The data suggest that conjunctival macrophage functions are enhancing the T‐cell‐mediated immune response after corneal infection. This effect is at least in part responsible for the impaired course of herpetic keratitis after macrophage depletion.

Amniotic membrane induces peroxisome proliferator-activated receptor-γ positive alternatively activated macrophages.

PURPOSE Amniotic membrane transplantation (AMT) reportedly improves herpetic stromal keratitis (HSK). Here we studied the role of the amniotic membrane (AM) on macrophages. METHODS BALB/c mice with necrotizing HSK received an AMT or tarsorrhaphy (TAR) as control. Apoptosis of F4/80+ cells was determined using the annexinV/7-AAD system. Macrophage invasion was determined using a cornea invasion assay. Cytokine secretion was quantified by ELISA. Arginase activity was measured by bioassay. Expression of nuclear factor (NF)-κB or peroxisome proliferator-activated receptor (PPAR)-γ related proteins was detected by Western blot analysis, and the expression of costimulatory surface molecules or PPAR-γ by flow cytometry. Lipid accumulation was observed by Oil red O and Sudan B staining. RESULTS After AMT apoptotic features of corneal macrophages, but also macrophage invasion increased. IL-6, IL-10, IL-12, TNF-α, and NF-κB content in HSK corneas had decreased with AMT. AMT increased expression of PPAR-γ, arginase 1 and 2, and arginase activity in AM-treated HSK corneas. In vitro, NF-κB, cytokine production, costimulatory molecules (CD80, CD86, CD40), phagocytic capacity, proliferation, viability, and accessory function to herpes simplex virus (HSV)-1 specific draining lymph node (DLN) cells were reduced in bone marrow derived macrophages (BM) cocultured with AM, while CD206, CD204, CD163, and CD68, lipid accumulation in the cytoplasm, PPAR-γ expression, and arginase activity was increased. An increase in viability and proliferation was observed in the presence of AM combined with apoptotic cells, compared with AM alone. CONCLUSIONS Based on these results it can be concluded that the action mechanism of AM is associated with modulation of classically activated macrophages into alternatively activated macrophages or macrophage cell death, probably by engaging lipid metabolism and activating the PPAR-γ pathway, consequently curtailing effector T cell functions. Apoptotic cells induced in the environment with AM support the presence and survival of such macrophages.

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

Correlation Between Disease Severity and Presence of Ocular Autoantibodies in Juvenile Idiopathic Arthritis- Associated Uveitis

PURPOSE The pathogenesis of juvenile idiopathic arthritis-associated uveitis (JIAU) is undefined. This study intended to analyze the presence of antiocular autoantibodies in serum and their correlation with disease course. METHODS Serum samples from children with JIAU (n = 47); JIA without uveitis (n = 67); idiopathic anterior uveitis (IAU; n = 12); and healthy controls (n = 52) were collected. The binding patterns of serum antibodies to ocular cryosections from swine eyes were analyzed by indirect immunohistochemistry, and were correlated to epidemiological, clinical, and laboratory test results. RESULTS The patient groups differed with respect to their presence of antibody binding to the sections: JIAU (94%), JIA (75%), IAU (75%), and healthy controls (29%) to uveal and/or retinal structures. Serum antibodies of JIAU patients predominantly bound at iris (74%), and ciliary body (79%). Iris/ciliary body positive staining correlated with the presence of uveitis complications (P < 0.005) in JIAU patients, but not with positivity of serum antinuclear antibodies (ANA), rheumatoid factor (RF), or HLA-B27, and was independent from uveitis activity or type of anti-inflammatory therapy. CONCLUSIONS In JIAU patients, antiocular serum antibodies can be detected more frequently than in control groups. Binding patterns to ocular tissue correlate with complicated uveitis course but not with uveitis activity and anti-inflammatory treatment. Antibody binding is not specific for this uveitis entity, and does not correlate with ANA positivity.

Effects of Systemic and Intravitreal TNF-α Inhibition in Experimental Autoimmune Uveoretinitis

PURPOSE To investigate the effect of systemic or local TNF-α inhibition with etanercept on experimental autoimmune uveoretinitis (EAU). METHODS EAU was induced by immunizing B10.RIII mice with IRBPp161-180 or by adoptively transferring uveitogenic splenocytes. Mice received systemic or local treatment with etanercept in the afferent or efferent phase. For systemic treatment, mice were injected intraperitoneally. For local treatment, etanercept was injected intravitreally or subconjunctivally. Control mice received PBS. EAU scores were determined histologically. Splenic cells were assessed for [(3)H]thymidine incorporation. ELISA was performed to measure levels of cytokines produced by splenocytes. Vitreous cavity-associated immune deviation (VCAID) was induced by intravitreally injecting ovalbumin and evaluated by measuring DTH reaction. RESULTS After systemic treatment with etanercept in the afferent phase, EAU disease scores, IRBP-specific cell proliferation, and production of Th1, Th2, and Th17 cytokines were reduced. EAU also improved after intravitreal etanercept treatment in the afferent phase, with unaltered IRBP-specific proliferation, reduced IFN-γ, but increased IL-6 and IL-10 secretion. VCAID induction was impaired after intravitreal etanercept treatment. No amelioration of EAU or reduction in IRBP-specific cell response was found after systemic or intravitreal treatment in the efferent phase or after subconjunctival treatment. After adoptive transfer, etanercept- and PBS-treated recipients showed similar disease severity and antigen-specific proliferation of splenocytes. CONCLUSIONS It can be concluded that TNF-α participates mainly in the immunopathology in the induction phase of EAU. The mechanism of action underlying EAU improvement may be different for local and systemic etanercept treatment.

Prevalence of parvovirus B19-specific antibodies and of viral DNA in patients with endogenous uveitis.

BackgroundParvovirus B19 causes erythema infectiosum (fifth disease) in children. The virus has been associated with several autoimmune diseases and has been found in synovium or serum from patients with long-standing arthritis or other rheumatic diseases. We investigated serum and aqueous fluid samples of patients with endogenous uveitis.MethodsSera of 70 consecutive uveitis patients were investigated for IgG against structural proteins VP1 and VP2 and non-structural protein NS1 by western blot technique. Viral DNA in serum was assessed by quantitative PCR. Simultaneous DNA analysis was also performed on aqueous fluid samples from 11 uveitis patients and 10 patients without uveitis during routine cataract extraction.ResultsIn 15 (21.4%; healthy population: 16 of 10,000) of the 70 serum samples (mean age 42.69 years, range 9–82 years; 48 women, 22 men), viral DNA was detected. IgG directed against structural protein VP1 or VP2 was found in 60 patients (85.7%; healthy population: 80%) and IgG against non-structural protein NS1 in 33 patients (47.1%; healthy population: 22%). In none of the patients was IgM antibody as a serological marker for acute parvovirus infection found. Patients with posterior uveitis (n=11) had detectable serum DNA in 54.5% (p=0.021). This group displayed the highest rate of deterioration of visual acuity (>2 Snellen lines), at 72.7% (all patients: 58.6%; p=0.219). In 2 of the 11 aqueous fluid samples, viral DNA was detected. Antibodies against parvovirus B19 were found in both samples, whereas viral DNA was absent in the serum. In the control group of 10 healthy cataract patients, 3 had detectable DNA in the aqueous humor; 1 of these 3 also had detectable levels of viral DNA in serum.ConclusionsPrevalence of IgG antibodies against NS1 protein in serum samples and DNA in serum is increased in uveitis patients compared with the normal population, suggesting a persistent infection. The presence of DNA in aqueous humor samples shows, for the first time, persistence of parvovirus B19 in the eye in uveitis and also in healthy patients. The role of parvovirus DNA in the aqueous fluid and of IgG antibodies against NS1 in the pathogenesis of uveitis cannot be defined at present.

Amniotic Membrane Transplantation for the Treatment of Corneal Ulceration in Infectious Keratitis

■ Corneal ulceration may occur in diverse types of infectious keratitis, e.g., in herpetic, bacterial, or parasitic infections