Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF.

TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM-derived or non-BM-derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88(-/-) mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4(-/-)→WT, WT→TLR4(-/-)) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88-related cytokines 4 h post-LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF-related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4(-/-) chimeric mice were resistant to EIU, and this suggests, surprisingly, that non-BM-derived (radiation-resistant) cells in the eye play a greater role than BM-derived cells.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis.

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.

Activation of NOD2 in vivo induces IL-1beta production in the eye via caspase-1 but results in ocular inflammation independently of IL-1 signaling.

Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.

Anterior uveitis accompanies joint disease in a murine model resembling ankylosing spondylitis.

BACKGROUND: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis--features common to the clinical presentations of ankylosing spondylitis. METHODS: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. RESULTS: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. CONCLUSIONS: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.

Endotoxin upregulates CCR7 and its ligands in the lymphatic-free mouse iris.

PURPOSE: Using time lapse intravital microscopy and histology, we previously reported that we could not detect migration of antigen-presenting cells from the iris to the regional lymph node. Dendritic cells (DC) in other peripheral tissues migrate to lymph nodes in response to chemokines, CCL19 (ELC) and CCL21b (SLC), that activate the CCR7 receptor. We hypothesized that DCs in an inflamed iris might show a different chemokine receptor and ligand profile, thus explaining the DC's inability to migrate. METHODS: Eyes of 35 BALB/c mice were injected intravitreally with 2 mul of 250 ng E. coli lipopolysaccharide (LPS) or phosphate buffered saline (PBS). Five mice served as naïve controls. After 3 and 6 h, the iris-ciliary bodies were dissected and pooled in groups of five. Total RNA was isolated, and reverse-transcriptase polymerase chain reaction (RT-PCR) for chemokine receptor and ligand mRNA was performed. In addition, one eye from each of the three animals was taken 6 h after LPS injection for immunohistology (IHC). RESULTS: The naïve iris, the iris after PBS injection, and the iris after LPS injection contained CCR5 mRNA at approximately equal levels and did not have detectable CCR6 mRNA. No CCR7 mRNA expression was found in the naïve iris, but it was weakly expressed in PBS-injected eyes and was approximately 3.4 fold upregulated after LPS injection. This was confirmed by IHC with no staining for CCR7 in the control iris but positive staining in the inflamed eyes. Transcripts for the CCR7 ligands, CCL19 and CCL21b, were found after LPS or PBS injection but not in naïve iris-ciliary bodies. CONCLUSIONS: The clear upregulation of CCR7 and its ligands in the inflamed iris suggests that another mechanism prevents iris DCs from migrating. Other possibilities include the absence of co-factors, inhibitory substances, the lack of lymphatics inside the eye, or inadequate biologicAL activity of these chemotactic factors and ligands.

Modification of the Woodruff-Stamper assay demonstrates binding of Toxoplasma gondii tachyzoites to retinal vascular endothelium.

In the human host, infection with the protozoan parasite, Toxoplasma gondii, most commonly involves the eye and/or the brain. Previous work indicates a relative susceptibility of the human retinal vascular endothelium to infection with the T. gondii tachyzoite, which may contribute to this tissue localization. To facilitate the investigation of potential adhesive interactions between parasite and endothelium in the retina, we have modified the Woodruff-Stamper assay, originally described to study lymphocytic-endothelial binding. Vascular endothelium was identified in sections of human retina by Alexa Fluor 594-tagged anti-von Willebrand factor antibody. Binding of yellow fluorescent protein-expressing tachyzoites to endothelium under conditions of flow, simulated by rotation on an orbital shaker, was quantified in a masked fashion using imaging software. We observed multiple yellow spots in contact with Alexa Fluor 594-positive retina, indicating binding of T. gondii tachyzoites to retinal vascular endothelium. This modification of the Woodruff-Stamper assay provides an opportunity to evaluate potential host receptors for T. gondii on the retinal vascular endothelium. In addition, the assay suggests a methodology that could be used to examine adhesion of other microbes to microvasculature in different tissues.

Cloning, sequencing and expression analysis of the mouse NOD2/CARD15 gene.

BACKGROUND: Mutations in the human NOD2/CARD15 gene have been associated with Crohn's disease and Blau syndrome. The objective of the present study was to clone the murine form of NOD2 and characterize its tissue distribution, function and response to inflammatory stimuli. METHODS: Murine NOD2 was isolated using anchored polymerize chain reaction (PCR). Sequence analysis confirmed the identification of full-length cDNA representing the murine NOD2 gene. Using this sequence to search a Mus musculus supercontig database, NOD2 genomic DNA was identified. NOD2 was transfected into human embryonic kidney (HEK) cells and nuclear factor kappa B (NF-kappaB) activation was measured using a reporter assay. Tissue distribution and changes in transcription in mouse monocytes in response to inflammatory stimuli was determined by real time PCR. RESULTS: The NOD2 gene spans 39 KB and contains 12 coding exons on chromosome 8. Expression of mouse NOD2 into HEK cells resulted in NF-kappaB activation. NOD2 was found to be expressed in all mouse tissues analyzed except skin, with highest levels in lung, thymus and spleen. NOD2 mRNA levels increased greater than two-fold in a monocyte cell line in response to lipopolysaccharide, lipoteichoic acid, interferon-g and tumor necrosis factor-alpha. CONCLUSIONS: Common structural and functional features between human and mouse NOD2 were identified. This should allow for development of relevant animal models to evaluate the role of NOD2 in chronic inflammatory disorders.

Simultaneous in vivo imaging of leukocyte migration: heterogeneity among iris, limbus, and choroid vessels.

PURPOSE: Endothelial cells of different vascular systems may express site-specific adhesion molecules to attract leukocyte subsets. This study describes a method to visualize and compare leukocyte-endothelial interactions in three vascular beds within the same eye in mice. METHODS: Digital in vivo fluorescence microscopy was used to record a trans-corneal iris view, a superficial limbus view, and a trans-scleral anterior choroid view of mouse tissue. Uveitis was induced by intravitreal injection of E. coli endotoxin into BALB/c mice. Leukocytes were labeled systemically with SYTO-16 or rhodamine 6G. Leukocyte rolling and sticking were quantified at baseline and 4, 6, and 24 hours after endotoxin injection. RESULTS: In a normal animal, the limbus had 18 times the number of rolling leukocytes and 6 times the number of sticking leukocytes relative to the iris. All three vascular beds were affected by intravitreal injection of endotoxin. Although they each showed increased numbers of rolling and sticking cells, the levels and kinetics of these increases differed. Rolling peaked at 6 hours in the iris (34-fold increase from baseline) and limbus (7-fold increase) but was maximal in the choroid earlier with a 16-fold increase. Sticking was maximal at 4 hours for iris (96-fold increase) and choroid (19-fold increase) but peaked in the limbus at 6 hours (47-fold increase from baseline). CONCLUSIONS: This study demonstrates that leukocyte-endothelial dynamics are not the same in different vascular beds in the normal mouse eye. Furthermore, site-specific differences in responses to intravitreally injected endotoxin, beyond what can be readily explained by differential distribution of endotoxin, were observed. The methodology can be used to test the hypothesis that endothelial cells within the eye have site-specific patterns of adhesion molecule expression.

Cell adhesion molecule expression in cultured human iris endothelial cells.

PURPOSE: To develop a method to isolate human iris microvascular endothelial cells (HIECs) for exploring their constitutive and inflammatory agent-modulated expression of intercellular adhesion molecules (ICAM)-1 and -2, vascular cell adhesion molecule (VCAM)-1, and E-selectin. METHODS: Endothelial cells from collagenase-digested irises were isolated on the basis of their expression of platelet endothelial cell adhesion molecule (PECAM)-1, using antibody-coupled magnetic beads. Cells were characterized as endothelial based on morphologic criteria, their expression of PECAM-1 and von Willebrand factor, their uptake of acetylated low-density lipoprotein, and their ability to form capillary-like networks on a synthetic basement membrane. Constitutive and inflammatory agent-modulated expression of ICAM-1 and -2, VCAM-1, and E-selectin was evaluated by the reverse transcription-polymerase chain reaction, enzyme-linked immunocellular assays (ELICAs), Western blot analysis, and functional studies of leukocyte adhesion to HIEC monolayers. RESULTS: HIECs constitutively expressed mRNA and protein for ICAM-1 and -2, but only low to nondetectable levels of VCAM-1 or E-selectin. When stimulated with endotoxin- or tumor necrosis factor (TNF)-alpha, ICAM-1, VCAM-1, and E-selectin were potently and time- and dose-dependently upregulated at both the message and protein levels. By contrast, ICAM-2 message and protein were slowly downregulated by inflammatory agents over time, but nonetheless remained present and functional. Overall, cytokine- or endotoxin-activation of HIECs resulted in enhanced adhesiveness for leukocytes. CONCLUSIONS: ICAM-1, VCAM-1, and E-selectin have been previously implicated in mediating anterior ocular inflammation. This is a report of the selective isolation of HIECs, with a demonstration of differential expression and regulation of these adhesion molecules in them. In addition, this is the first demonstration of the regulated expression of ICAM-2 in any ocular microvascular cells.

Inhibition of leukocyte sticking and infiltration, but not rolling, by antibodies to ICAM-1 and LFA-1 in murine endotoxin-induced uveitis.

PURPOSE: Cell-adhesion molecules are critical elements in intravascular rolling and sticking of leukocytes during acute inflammation. In this process, selectins are thought to be involved in initial adhesion and rolling, and integrin-Ig superfamily interactions are believed primarily to mediate stronger adhesion and transendothelial migration. This study clarifies the role of two adhesion molecules, intercellular adhesion molecule (ICAM)-1 and leukocyte functional antigen (LFA)-1, in endotoxin-induced uveitis (EIU). METHODS: Intravital microscopy was used to record the movement and location of leukocytes in the irises of mice with uveitis induced by intravitreal injection of 250 ng Escherichia coli endotoxin. Each mouse concurrently received an intraperitoneal injection of monoclonal neutralizing antibodies for ICAM-1, LFA-1, or both or control irrelevant antibodies. RESULTS: Mice treated with endotoxin and control antibodies had an inflammatory response that was clearly present at the 6- and 24-hour time points and was mostly resolved by 48 hours. Mice that received anti-ICAM-1 or anti-LFA-1 had significantly fewer cells infiltrating their irises at 6 and 24 hours. Detailed analysis of the 6-hour time point recordings revealed that neither anti-ICAM-1 nor anti-LFA-1 significantly reduced the number of leukocytes rolling on venule endothelial surfaces, but the treatments reduced the number of firmly adherent cells. CONCLUSIONS: These data confirm previous reports that ICAM-1 and LFA-1 are important mediators of EIU. The dynamic in vivo images clearly support the hypothesis that integrin-mediated cell adhesion is more critical for the firm adhesion of sticking cells than for leukocyte rolling.

Intraocular in vivo imaging of activated T-lymphocytes expressing green-fluorescent protein after stimulation with endotoxin.

BACKGROUND: Intravital microscopy allows imaging of specific cell populations in vivo. The value of this technique is well established, but would be enhanced if one could distinguish functional states of cells in vivo. Interleukin-2 (IL-2) is expressed upon stimulation of T-cells and is a commonly used marker for T-cell activation. This study tests the use of enhanced green fluorescent protein (GFP) as a reporter gene for interleukin-2 (IL-2) expression in vivo. METHODS: Characterization of mice that have the GFP gene under the control of IL-2 regulatory sequences has previously been published. Uveitis was induced by injection of E. coli endotoxin into the vitreous of these IL-2/GFPki transgenic mice. Four hours later, 3 microg of recombinant mouse IL-2 was injected into the anterior chambers of one group of mice. In vivo imaging of infiltrating cells in the iris stroma was performed with fluorescence microscopy at 6, 24, 48, and 72 h after endotoxin injection. The absolute number of fluorescent cells per mm2 was evaluated. RESULTS: Eyes with endotoxin-induced uveitis had cells that expressed GFP and were identifiable by intravital microscopy. The fluorescent cells were exclusively seen in the subset of cells that had infiltrated the iris stroma or arrested along the vascular endothelium. The number of GFP-positive infiltrating cells in the iris increased from undetectable at baseline to 0.5 cells/mm2 at 6 h and 1.3 cells/mm2 at 72 h. The animals that received endotoxin as well as IL-2 tended to have more GFP-positive cells at the 48-h and 72-h time points, but these differences were not statistically significant CONCLUSIONS: GFP is commonly used as a reporter gene for in vitro expression assays. The results presented here document that transgenic mice with GFP under the control of IL-2 regulatory elements can be used with intravital microscopy for in vivo expression assays that allow detection of activated T-cells at multiple time points within the same animal. This provides a novel method for temporal and spatial studies on the state of cell activation in inflammatory responses.

Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis.

The process of inflammation is accompanied by an alteration of leukocyte-endothelial dynamics. Reciprocal changes in the endothelium and the white cell permit the leukocyte to relinquish its normal free-flowing state in order to roll, arrest, and emigrate through the endothelium. Although intravital microscopy is an established method to observe this process, the eye has been under-utilized for this purpose. Iris vasculature can be videophotographed without the artifact of trauma. We used rhodamine 6G in vivo staining of leukocytes from BALB/c mice in a model of inflammation induced by intravitreally injected endotoxin. Digital video technology was used to record observations at baseline, 2 h, and 4 h after the endotoxin injection. Off-line analysis of microhemodynamic parameters established that the percentage of venules exhibiting rolling increased significantly from 4% at baseline to 34% at 2 h and 82% at 4 h after endotoxin injection. We found a marked increase in leukocyte arrest within 4 h (601+/-119 cells per mm(2) vs. 2+/-1 cells per mm(2) in control animals). Although shear stress differs minimally between iris arterioles and venules, both rolling and arrest occurred preferentially in venules indicating that shear stress is not the dominant factor for determining cell adhesion. Compared to previous reports on intravital microscopy, our methodology includes refinements or advantages in visualizing cells that have transmigrated as well as the avoidance of surgical trauma. The resolution and quantifiable nature of this technique are such that the methodology can be applied to repetitive observation of leukocyte-endothelial dynamics during an immune response.

Reduced leukocyte migration, but normal rolling and arrest, in interleukin-8 receptor homologue knockout mice.

PURPOSE: To determine the role of the murine interleukin-8 receptor homologue (mIL-8Rh, neutrophil chemokine CXC receptor 2) in leukocyte migration using intravital microscopy in a standardized model of eye inflammation, endotoxin-induced uveitis (EIU). METHODS: Two hundred fifty nanograms of E. coli endotoxin was injected into the vitreous of knockout mIL-8Rh(-/-) (n = 7) mice or heterozygous littermate mIL-8Rh(+/-) controls (n = 7). Intravital microscopic examination of iris microvasculature was performed at baseline and 6 and 24 hours after endotoxin injection. The numbers of rolling (cells/mm2 endothelial surface/min), sticking (cells/mm2 endothelial surface), and infiltrating cells (cells/mm2 iris tissue) were evaluated by digital off-line quantification. RESULTS: The number of infiltrating cells was significantly reduced in mIL-8Rh(-/-) mice: 406 +/- 77 cells/mm2 at 6 hours and 242 +/- 50 cells/mm2 at 24 hours in mIL-8Rh(+/-) mice versus 14 +/- 4 cells/mm2 at 6 hours and 38 +/- 11 cells/mm2 at 24 hours in mIL-8Rh(-/-) mice (P < 0.001). In contrast, the absence of the IL-8 receptor homologue did not reduce rolling or sticking. CONCLUSIONS: Iris rhodamine angiography allows precise quantification of leukocyte-endothelial dynamics in the absence of surgical trauma. IL-8 and its homologues are known to be potent signals for leukocyte migration. Although IL-8 has previously been implicated in cell adhesion, video imaging in vivo demonstrated that deletion of the IL-8 receptor homologue had minimal effect on rolling or arrest in this model of inflammation.

Anti-rat ICAM-1 antibody does not influence the course of experimental melanin-induced uveitis.

PURPOSE: Experimental melanin-induced uveitis (EMIU) is a T cell-mediated rat model of acute anterior uveitis. We investigated the possibility of preventing the inflammation with monoclonal antibody directed against rat intercellular adhesion molecule 1 (ICAM-1). METHODS: To induce EMIU, Lewis rats were immunized with bovine ocular melanin extract (250-500 microg). Each day from day 6 post-immunization, rats were injected intraperitoneally with anti-ICAM-1 monoclonal antibody (IA29) or normal mouse serum, and examined with a slit-lamp biomicroscope. On the first day of clinical inflammation, intravital microscopy of iris vasculature was performed on each animal following intraperitoneal injection of rhodamine 6G. At the peak of clinical inflammation, rats were killed, and eyes were examined histologically. Binding potency of IA29 was tested by flow cytometry using concanavalin A-stimulated rat T cells. Immunohistochemical staining was used to detect IA29 on rat uveal vascular endothelium. RESULTS: The ability of IA29 to bind T cell blasts was present to a 1:2000 dilution, and IA29 was readily detectable on uveal vascular endothelium following systemic administration. However, there was no significant difference (p > 0.05) in incidence, time of onset, or severity or histological appearance of EMIU for the rats treated with IA29 when compared with the control rats. Intravital microscopy revealed sticking of leukocytes in the iris vasculature in both groups. CONCLUSIONS: We were unable to demonstrate an inhibitory effect of anti-rat ICAM-1 antibody on the outcome of EMIU. Our observations may reflect a redundancy in the adhesion molecule profile responsible for this uveitis.

IL-1 and TNF receptor-deficient mice show decreased inflammation in an immune complex model of uveitis.

PURPOSE: To determine the role of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in the induction of uveitis by a reverse passive Arthus reaction (RPAR). METHODS: Human serum albumin (HSA) antiserum was injected into the vitreous of "knockout" or "double knockout" mice genetically deficient in IL-1 receptor type I (IL-1RI-/-), TNF receptors p55 and p75 (TNFR p55-/-/p75-/-), IL-1RI and TNFR p55 (IL-1RI-/-/TNFR p55-/-), and controls. Twenty-four hours later, animals were challenged with intravenous HSA. Eyes were enucleated 4 hours after antigen challenge, and inflammation was quantitated by counting cells on histologic sections. Interleukin-6 in aqueous humor was measured with a B9 cell bioassay. The distribution of immune complexes in eyes was observed by immunohistochemical staining for IgG and complement component C3. RESULTS: Four hours after antigen challenge, immune complexes were localized at the ciliary body and iris of receptor-deficient mice. A transient uveitis was most severe at this time. A significant reduction in the median number of infiltrating cells was found in TNFR p55-/-/p75-/- mice (4.8, n = 15), compared with controls (14.2, n = 20, P < 0.05). The median number of infiltrating cells was significantly reduced in IL-1RI-/- mice (knockout 2.6, n = 11; controls 7.4, n = 8, P < 0.005). Interleukin-1RI-/-/TNFR p55-/- mice had a strong reduction in infiltrating cells (knockout 1.6, n = 11; controls 27.3, n = 12, P = 0.002). Interleukin-6 activity in aqueous humor was reduced in IL-1RI-/-/TNFR p55-/- mice (P = 0.03) but not in TNFR p55-/-/p75-/- (P = 0.40) mice. Most IL-1RI-/-mice had no detectable aqueous humor IL-6, but this group was not statistically different from controls. CONCLUSIONS: In contrast to endotoxin-induced uveitis, both IL-1 and TNF appear to have critical roles in RPAR uveitis. When receptors for these cytokines were deleted, the severity of immune complex-induced uveitis was profoundly reduced.

Murine endotoxin-induced uveitis, but not immune complex-induced uveitis, is dependent on the IL-8 receptor homolog.

PURPOSE: To determine the roles of the murine interleukin-8 receptor homolog (mIL-8Rh, neutrophil chemokine CXC receptor 2) and macrophage inflammatory protein-1alpha (MIP-1alpha, a CC chemokine) in two eye inflammation models: endotoxin-induced uveitis (EIU) and immune complex-induced uveitis (reverse passive Arthus reaction (RPAR) uveitis). METHODS: For the EIU model, 250 ng E.coli endotoxin was injected into the vitreous of mIL-8Rh-/- mice or heterozygous littermate mIL-8Rh+/- controls and into MIP-1alpha-/- mice or congenic MIP-1alpha+/+ controls. Eyes were harvested after 24 h for histologic characterization of infiltrating cells and IL-6 bioassays. For the RPAR model, mouse antiserum against human serum albumin (HSA) was injected into the vitreous of mIL-8Rh-/-, mIL-8Rh+/-, MIP-1alpha-/-, and MIP-1alpha+/+ mice. Twenty-four hours later, animals were challenged with intravenous HSA. Eyes were harvested after 4 h for analysis. RESULTS: RPAR resulted in the deposition of immune complexes at the ciliary area and iris with the subsequent development of uveitis. Genetic deficiency of mIL-8Rh reduced the median number of infiltrating cells in EIU by 63% (p < 0.01) but had no effect on RPAR-induced inflammation. In the EIU model, macrophages comprised a much higher percentage (45%) of infiltrating cells in mice lacking mIL-8Rh than in controls (17%). Loss of the MIP-1alpha gene had no apparent effect on RPAR uveitis and a 39% reduction of infiltrating cells in EIU that was not statistically significant. IL-6 activity in aqueous humor was much less in mice with RPAR uveitis than in those with EIU. Neither gene deletion had a significant impact on IL-6 levels in either disease model. CONCLUSIONS: Chemokines acting via mIL-8Rh have a significant role in the induction of neutrophil infiltration during EIU but not during RPAR uveitis. MIP-1alpha is not critical for either EIU or RPAR-induced uveitis. The differential dependence on IL-8-like chemokines is in accord with the two forms ofuveitis having different etiologies and, therefore, potentially different optimal therapies.

Tumor necrosis factor-alpha is not essential in endotoxin induced eye inflammation: studies in cytokine receptor deficient mice.

OBJECTIVE: Anterior uveitis frequently occurs in association with specific systemic inflammatory diseases. Interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have been implicated in the pathogenesis of these diseases. We evaluate the need for these cytokines in a model of anterior uveitis. METHODS: Endotoxin was injected into the vitreous of mice deficient in IL-1 receptor type I, TNF receptors p55 and p75, both IL-1R1 and TNFR p55, or controls. Eyes were harvested after 24 h for histology and IL-6 bioassays or after 3 h for reverse transcriptase-polymerase chain reaction analysis of mRNA for specific cytokines or enzymes. RESULTS: No significant difference in the number of infiltrating cells was found in TNFR p55/p75 deficient mice compared to controls in any of 4 separate experiments or in the combined data (p = 0.8). The number of infiltrating cells was significantly reduced in 2 of 4 experiments with IL-1R1 deficient mice (p < 0.001 based on combined data from 4 studies). IL-1R1/TNFR p55 deficient mice had a reduction in infiltrating cells in 2 of 3 experiments (p < 0.001 based on combined data from all studies). IL-6 levels were not significantly reduced in either of 2 experiments with TNFR p55/p75 deficient mice, but were reduced in one of 2 experiments with IL-1R1-/- mice (p = 0.02) and in one experiment with IL-1R1/TNFR p55 deficient mice (p = 0.01). In response to endotoxin, all 3 receptor deficient lines increased mRNA levels for IL-1-alpha, IL-10, TNF-alpha, IL-1 receptor antagonist, and inducible nitric oxide synthase. CONCLUSIONS: IL-1 appears to have a more pivotal role in endotoxin induced uveitis than TNF-alpha, although neither cytokine is essential. Deletion of receptors for both cytokines has the most consistent effect, which is in accord with the hypothesis that these cytokines are, at least in part, functionally redundant.

Ultrastructural immunolocalization of basic fibroblast growth factor in mast cell secretory granules. Morphological evidence for bfgf release through degranulation.

We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation.

The effect of genetic deficiency of adhesion molecules on the course of endotoxin-induced uveitis.

PURPOSE: Multiple adhesion molecules of the selectin, integrin, and immunoglobulin-like families are involved in the migration of leukocytes out of the bloodstream into inflamed tissues. This study addresses the question of which adhesion molecules are specifically involved in endotoxin-induced uveitis. METHODS: Mice genetically deficient in p-selectin, ICAM-1, beta2-integrin, or controls received intravitreal injections of endotoxin. Eyes were harvested 24 h later and inflammation was evaluated by histologic and immunohistochemical assays of infiltrating cells. RESULTS: Mice lacking either P-selectin or beta2-integrin had less inflammation than controls (median cells/section: 64 for P-selectin knockout vs 130 for controls, p=0.02, n=17 per group: 244 for beta2-integrin knockouts, n=14, vs 355 for controls, n=17, p=0.05). Neither gene deletion significantly changed the ratio of infiltrating neutrophils to macrophages. ICAM-1 knockouts tended to have fewer infiltrating cells (median 22 cells/section) compared to controls (median 132 cells/section), but this difference was not statistically significant (p=0.25, n=9 per group). CONCLUSIONS: P-selectin, beta2-integrin, and possibly ICAM-1 are involved in the ocular inflammatory response to endotoxin. The lack of complete inhibition of leukocyte infiltration with the complete loss of each adhesion molecule is in accord with the notion that alternative adhesion molecules also participate in this process.