Elevated endothelin-1 levels as risk factor for an impaired ocular blood flow measured by OCT-A in glaucoma.

The purpose of this study was to ascertain whether a correlation exists between glaucoma-associated alteration of ocular vascular haemodynamics and endothelin-1 (ET-1) levels exist. Eyes of patients with cataract (n = 30) or glaucoma (n = 68) were examined with optical coherence tomography (OCT) and OCT-angiography (OCT-A; AngioVue™-RTVue-XR; Optovue, Fremont, California, USA). The peripapillary and the macular vessel density (VD) values were measured. Inferior and superior retinal nerve fibre layer (RNFL) thickness loss was used for further OCT staging. Aqueous humour of the examined eye and plasma were sampled during cataract or glaucoma surgery and analysed by means of ELISA to determine their ET-1 level. Glaucoma eyes are characterised by reductions in RNFL thickness and VD that correlate significantly with the OCT GSS score. Peripheral and ocular ET-1 level were significantly elevated in patients with glaucoma and correlate positively with the OCT-GSS score of the entire study population. Peripapillary and macula VD of glaucoma patients correlates negatively with plasma ET-1 levels. Multivariable analysis showed a subordinate role of intraocular pressure predictive factor for impaired retinal blood flow compared with plasma ET-1 level in glaucoma. Peripheral ET-1 level serves as risk factor for detection of ocular blood flow changes in the optic nerve head region of glaucomatous eyes.

Assessment of angiogenesis-related parameters in juvenile idiopathic arthritis-associated uveitis.

PURPOSE: Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be observed clinically. METHODS: Iris tissue (IT), aqueous humor (AH) and serum samples from patients with clinically inactive JIAU (n = 30), acute anterior uveitis (AAU; n = 18), and primary open angle glaucoma (POAG; n = 20) were obtained during trabeculectomy or cataract surgery. Samples were analyzed by RNA-Seq, qRT-PCR, LC-IMS, Western-Blot, and LEGENDplex™ analysis. Pattern of iris vasculature in JIAU patients was assessed qualitatively via fluorescein and indocyanine green angiography (FLA/ICGA). RESULTS: RNA-Seq of IT showed significantly differential expression (DE) of 136 genes between JIAU and POAG, of which 15 were associated with angiogenesis. qRT-PCR, performed to validate RNA-Seq results, showed upregulation of the angiogenesis-related genes Kdr, Angpt-1, Tie-1, Tie-2 and Mmrn2 in IT (JIAU vs POAG, p > 0.05). LC-IMS of IT revealed a total number of 56 DE proteins (JIAU vs POAG), of which Angiopoetin, Lumican and Decorin were associated with angiogenesis and showed increased (p > 0.05) expression on Western-Blot analysis. LEGENDplex™ analysis showed upregulation of ANGPT-2 in AH from JIAU compared to AAU and POAG, whereas VEGF was upregulated in AAU. Iris vascular leakage, hypoperfusion and neovascularization were observed by FLA/ICGA in JIA patients with treatment-refractory complicated course of uveitis. CONCLUSION: Angiogenesis-related factors could play a role in long-standing complicated JIAU, leading to clinically visible alterations in selected cases.

Phenotype of Innate Immune Cells in Uveitis Associated with Axial Spondyloarthritis- and Juvenile Idiopathic Arthritis-associated Uveitis.

Purpose: To analyze circulating immune cells in patients with anterior uveitis (AU) associated to axial spondyloarthritis (SpA), or juvenile idiopathic arthritis (JIA).Methods: Venous blood samples were collected from healthy controls (n = 16), and either SpA (n = 19) or JIA (n = 23) patients with associated anterior uveitis (AU) during active flare, or after ≥3 months of inactivity. Frequencies of CD56+, MHC-I+, and S100A9+ monocytes, CCR7+ dendritic cells, CD56+dim natural killer (NK) cells and CD3+CD56bright T-cells were analyzed via flow cytometry. Serum S100A8/A9 levels were determined via ELISA.Results: SpA patients showed a reduced frequency of CD56+dim NK cells during uveitis activity, a constitutively activated monocyte phenotype, and elevated S100A8/A9 serum levels. In contrast, JIAU patients showed elevated frequencies of CD56+ monocytes and CCR7+ DC.Conclusion: Phenotype of peripheral immune cells differ between patients, probably contributing to different courses of acute onset AU in SpA and insidious onset AU in JIAU patients.Abbreviations: AU: anterior uveitis, AR: arthritis, JIA: juvenile idiopathic arthritis, SpA: axial spondyloarthritis.

Increased Hydrostatic Pressure Promotes Primary M1 Reaction and Secondary M2 Polarization in Macrophages.

Patients with chronic anterior uveitis are at particularly high risk of developing secondary glaucoma when corticosteroids [e.g., dexamethasone (Dex)] are used or when inflammatory activity has regressed. Macrophage migration into the eye increases when secondary glaucoma develops and may play an important role in the development of secondary glaucoma. Our aim was to evaluate in vitro if increased hydrostatic pressure and corticosteroids could induce changes in macrophages phenotype. By using a pressure chamber cell culture system, we assessed the effect of increased hydrostatic pressure (HP), inflammation, and immunosuppression (Dex) on the M1/M2 phenotype of macrophages. Bone marrow-derived macrophages (BMDMs) were stimulated with medium, lipopolysaccharide (LPS, 100 ng/ml), Dex (200 ng/ml), or LPS + Dex and incubated with different HP (0, 20, or 60 mmHg) for 2 or 7 days. The numbers of CD86+/CD206- (M1 phenotype), CD86-/CD206+ (M2 phenotype), CD86+/CD206+ (intermediate phenotype), F4/80+/TNF-α+, and F4/80+/IL-10+ macrophages were determined by flow cytometry. TNF-α and IL-10 levels in cell culture supernatants were quantified by ELISA. TNF-α, IL-10, fibronectin, and collagen IV expression in BMDMs were detected by immunofluorescence microscopy. Higher HP polarizes macrophages primarily to an M1 phenotype (LPS, 60 vs. 0 mmHg, d2: p = 0.0034) with less extra cellular matrix (ECM) production and secondary to an M2 phenotype (medium, 60 vs. 0 mmHg, d7: p = 0.0089) (medium, 60 vs. 20 mmHg, d7: p = 0.0433) with enhanced ECM production. Dex induces an M2 phenotype (Dex, medium vs. Dex, d2: p < 0.0001; d7: p < 0.0001) with more ECM production. Higher HP further increased M2 polarization of Dex-treated macrophages (Dex, 60 vs. 0 mmHg, d2: p = 0.0417; d7: p = 0.0454). These changes in the M1/M2 phenotype by high HP or Dex treatment may play a role in the pathogenesis of secondary uveitic glaucoma- or glucocorticoid (GC)-induced glaucoma.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

The Phenotype of Monocytes in Anterior Uveitis Depends on the HLA-B27 Status.

HLA-B27 is the allele most frequently associated with human anterior uveitis. The majority of HLA-B27-positive [acute anterior uveitis (AAU)] patients develop clinically distinct symptoms with acute symptomatic onset of flare and a recurrent disease course characterized by a massive cellular ocular infiltrate during uveitis relapse. By contrast, uveitis in HLA-B27-negative [idiopathic anterior uveitis (IAU)] patients tends to develop a clinically less fulminant, more chronic, and typically asymptomatic disease course. To analyze systemic immune responses in the different uveitis entities, we analyzed peripheral blood cells by flow cytometry. In addition, as a pro-inflammatory biomarker serum, S100A8/A9 levels were quantified by ELISA from patients with AAU (n = 27) and IAU (n = 21), and in healthy controls (n = 30). Data were obtained either during active uveitis flare or after 3 months of inactivity. IAU patients showed a transiently increased frequency of CD56- and CD163-positive monocytes and of both granulocytic myeloid-derived suppressor cells and Th17 cells during active uveitis. By contrast, AAU patients showed an elevated frequency of monocytes, activated T cells, and elevated S100A8/A9 serum levels during clinically quiescent disease. The differentially regulated response of both innate and adaptive immune cells in the blood may be related to the clinically distinct characteristics of the two different uveitis entities.

Multiplex Cytokine Analysis of Aqueous Humor in Juvenile Idiopathic Arthritis-Associated Anterior Uveitis With or Without Secondary Glaucoma.

Patients with juvenile idiopathic arthritis often develop chronic anterior uveitis (JIAU). JIAU patients possess a particularly high risk for developing secondary glaucoma when inflammatory inactivity has been achieved. By using multiplex bead assay analysis, we assessed levels of pro- and anti-inflammatory cytokines, chemokines, or metalloproteinases in the aqueous humor (AH) of patients with clinically inactive JIAU with (JIAUwG) or without secondary glaucoma (JIAUwoG), or from patients with senile cataract as controls. Laser-flare photometry analysis prior to surgery showed no significant differences between JIAUwG or JIAUwoG. Compared with the control group, levels of interleukin-8, matrix metalloproteinase-2, -3, -9, serum amyloid A (SAA), transforming growth factor beta-1, -2, -3 (TGFß-1, -2, -3), and tumor necrosis factor-alpha in the AH were significantly higher in patients with clinically inactive JIAUwG or JIAUwoG. Samples from JIAwoG patients displayed significantly higher levels of SAA (P < 0.0116) than JIAUwG patients. JIAUwG patients showed an increased level of TGFß-2 in AH samples compared with JIAUwoG (P < 0.0009). These molecules may contribute to the clinical development of glaucoma in patients with JIAU.

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.

Intravitreal treatment with antisense oligonucleotides targeting tumor necrosis factor-α in murine herpes simplex virus type 1 retinitis.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine known to participate in intraocular inflammatory disease. This study investigated whether treatment with intravitreal antisense-oligonucleotides (ASON) targeting TNF-α mRNA affects the progression of herpes simplex virus 1 (HSV-1) retinitis in mice. METHODS: The in vivo uptake of the oligonucleotid after intravitreal injection was determined with FITC-labeled TNF-α ASON. HSV-retinitis was induced on day 0 by the injection of HSV-1 (KOS strain) into the anterior chamber (AC) of the right eyes of BALB/c mice (von Szily model). The left contralateral eyes were injected intravitreally on day 7 with TNF-α ASON, sequence-unspecific control ASON (CON), or buffer. The clinical course of retinitis, ocular inflammatory cell-infiltration, TNF-α expression in the eye by ELISA, delayed-type hypersensitivity (DTH) reaction, virus-neutralizing antibody titers in the serum, uptake of [3H]thymidine from regional lymph node (rln) cells, and viral content in the eyes were determined. RESULTS: In vivo, strong fluorescence of FITC- TNF-α ASON was detected in the choroid and retina up to 3 days after intravitreal injection, but none in the rln. After treatment of eyes with ASON, decreased expression of TNF-α in the eye, and reduced incidence and severity of retinitis on day 10 after infection (P < 0.05) could be found. The other parameters were not significantly influenced after TNF-α ASON treatment. CONCLUSIONS: TNF-α participates in the pathology of HSV-1 retinitis. Local inhibition of TNF-α mRNA by intraocular TNF-α ASON injection did not influence the systemic HSV-specific immune response or the antiviral response in the eye, but reduced ocular inflammatory bystander damage.

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.

Subconjunctival antisense oligonucleotides targeting TNF-alpha influence immunopathology and viral replication in murine HSV-1 retinitis.

BACKGROUND: To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in immunopathology and viral replication in the contralateral eye in the von Szily model of herpes simplex virus (HSV)-1 acute retinitis. METHODS: In vivo distribution was analyzed after subconjunctival injection of FITC-labeled antisense oligonucleotides (ASON). After HSV-1 (KOS) was injected in the right anterior chamber (AC) in BALB/c mice, the course of the contralateral retinitis was evaluated. The left eyes were treated with either TNF-alpha ASON, sequence-unspecific control (CON), or buffer. The ocular TNF-alpha content was quantified by ELISA. The delayed-type hypersensitivity (DTH) reaction, uptake of [3H]thymidine from regional lymph nodes (rln)- and spleen cells, serum-neutralizing antibodies, and viral titer in the eyes were evaluated. RESULTS: After subconjunctival injection, FITC-labeled ASON were found in the choroid and retina. In the TNF-alpha ASON-treated eyes, TNF-alpha expression and the incidence and severity of retinitis were reduced on day 8 postinfection (PI) (p < 0.05). On day 10 PI, higher viral titers were only seen in the eyes of the TNF-alpha ASON group (p < 0.05), and retinitis was slightly more severe on day 12 PI. While the HSV-1 specific [3H]thymidine uptake from rln cells was higher in the TNF-alpha ASON mice (p < 0.05), the [3H]thymidine uptake from spleen cells, the DTH response, and the neutralizing-antibody titers did not differ between the groups. CONCLUSIONS: After regional blockade of TNF-alpha in experimental HSV-1 retinitis TNF-alpha seems to possess an antiviral capacity against HSV-1 in the contralateral eye and participates in the immunopathology of HSV-1-induced acute retinitis.

On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation.

Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl(2)MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1alpha, IL-2, interferon (IFN)-gamma, CXCL1, CXCL2, and tumor necrosis factor (TNF)-alpha production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant (r) cytokines as rIL-1alpha, rIL-2, and rTNF-alpha or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells (P<0.01), but a higher ratio of apoptotic to viable PMN-resembling cells (P<0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl(2)MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1alpha, IL-2, CXCL1, and TNF-alpha were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea.

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.

Topical treatment with antisense oligonucleotides targeting tumor necrosis factor-alpha in herpetic stromal keratitis.

PURPOSE: Tumor necrosis factor (TNF)-alpha is a pleiotropic factor that is critical for the development of inflammation. The authors investigated whether topical application of TNF-alpha-antagonizing molecules, antisense oligonucleotides (ASON), might be an effective way of modifying the course of immune-mediated herpetic stromal keratitis (HSK). METHODS: ASON targeting TNF-alpha mRNA were examined for their efficiency in interfering with the production of this cytokine in vitro. In vivo uptake was determined by FITC-labeled ASON. HSV-1 corneally infected mice were injected three times with ASON. Mice from the control groups received unrelated control oligonucleotides (CON) or buffer. The clinical course of HSK, the delayed-type hypersensitivity (DTH) reaction, the uptake of [(3)H]thymidine from cells derived from the spleen, virus-neutralizing antibody titers in the serum, and viral replication in the infected eyes were determined. The eyes were examined histologically. The corneal TNF-alpha content was measured by ELISA. RESULTS: The TNF-alpha ASON reduced the lymphocytic cytokine expression in vitro. In vivo, the FITC-labeled molecules were detected in the cornea even after 10 days. In the TNF-alpha ASON mice the incidence of HSK decreased, and the severity of the disease was diminished. The corneal content of TNF-alpha was reduced, and the number of inflammatory cells was decreased. The other investigated parameters were not significantly altered by TNF-alpha ASON treatment. CONCLUSIONS: The data suggest that TNF-alpha ASON diminishes the release of TNF-alpha from cultured lymphocytes and from lymphocytes in the HSV-1-infected cornea. This topical treatment mitigates the course of HSK, whereas the systemic antiviral effector functions were not impaired.

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)-gamma, interleukin (IL)-2, and IL-4 production in the cornea was analysed by enzyme-linked immunosorbent assay (ELISA), and cytokine mRNA levels (IFN-gamma, 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-gamma, 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-gamma, IL-4) and secretion (IFN-gamma, IL-2, IL-4) in the corneas were decreased after HSV-1 corneal infection in the macrophage-depleted mice. The secretion of IFN-gamma 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.