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Rheumatoid arthritis (RA) is a chronic inflammatory disease involving dysfunctional lymphatic circulation leading to edema. The adjuvant-induced arthritis (AIA) rat model was used to study the effect of lymphatic pump treatment (LPT) on the expression levels of inflammatory cytokines and lymphangiogenesis within the draining popliteal lymph nodes (PLN). PLN from both control and LPT treatment groups were harvested, and their cDNA were analyzed by qPCR to determine the levels of cytokines, VEGF-C, and VEGFR-3. Additionally, immunohistochemistry was used to visualize the expression of VEGF-C and VEGFR-3 and flow cytometry was used to quantify leukocyte cell types in the PLN. The LPT group showed a decrease in ankle circumference and arthritis grade a few days after the initiation of treatment. Flow cytometry of popliteal lymph nodes showed an increase in CD8, CD4, and CD3 cells in arthritic rats, however cell populations were not significantly different between treatment groups. The qPCR data indicated that the LPT group had a slight reduction in levels of IL-17a, IL-4, IL-6, and IL-1β. IHC showed that the PLN from the LPT group had the highest expression area of VEGF-C and the lowest expression area of VEGFR-3 compared to control. In conclusion, The LPT group showed a reduction in ankle circumference and arthritis score, suggesting that LPT helped alleviate edema and inflammation. When compared to control, the LPT group had lower expression of pro-inflammatory cytokines and elevated levels of VEGF-C, an important lymphatic vessel growth factor, in PLN. Thus, the data suggests that LPT may elevate the swelling and inflammation in rat AIA through the reduction of inflammatory cytokines and the elevation of a lymphatic growth factor.

Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic inflammation mediated by Th-17 cells. Rat adjuvant-induced arthritis (AIA) is a Th-17-dependent RA model with dysfunctional lymphatic circulation. We hypothesized that induction of lymphatic circulation would attenuate arthritic inflammation. We utilized Lymphatic Pump Treatment (LPT), an osteopathic lymphatic manipulation, to determine if inducing lymphatic flow would alleviate arthritic inflammation. LPT was accomplished on AIA rats by gradually increasing compression on the chest wall for 5 seconds followed by a rapid release. This technique was performed on the rats for 1 minute 3X/day over 7 consecutive days following the onset of measurable inflammation on day 14 post adjuvant injection. Ankle caliper measurements, articular index scoring, popliteal lymph nodes, peripheral blood, and spleens were collected. The LPT-treated rats fell into two groups. LPT responders and non-responders. LPT responders had significantly reduced inflammation (as determined by both in ankle circumference and index scoring). These therapeutically treated animals also had significantly decreased IL-17 expression in their draining popliteal lymph nodes. These results along with our previous findings indicate that LPT can reduce arthritis, alter lymphocyte trafficking and differentiation in secondary lymphoid organs during inflammation. These data suggest that induced lymphatic circulation using osteopathic manipulation may serve as a therapy for chronic inflammation in autoimmune disorders and may also be an enhancement to current treatments.

Epstein-Barr virus (EBV) latently infects over 90% of the population and is associated with the development of numerous autoimmune diseases and B cell lymphomas. Therefore, understanding the impact of latency proteins on B cell survival and function is vital. One EBV latency protein, LMP2A, acts as a BCR mimic to promote B cell survival, increase B cell activation, and accelerate tumor development in murine mouse models. Our laboratory identified that LMP2A activates the PI3K/BTK/STAT3 pathway in human B cell lymphomas to increase IL-10 production that promotes survival. However, the ability of LMP2A to activate BTK and STAT3 in non-transformed B cells is unknown. To test this possibility, we returned to the LMP2A transgenic mouse model as a source of resting B cells that express LMP2A. To determine if LMP2A activates STAT3 in the mouse model, resting B cells from wildtype and LMP2A-transgenic mice were isolated and STAT3 phosphorylation was assessed by Western blot analysis. Our data indicate that LMP2A-postitive B cells express higher levels of phosphorylated STAT3 than wildtype controls. We further analyzed whether the LMP2A-dependent activation of STAT3 resulted in an increase in STAT3-dependent targets. Flow cytometric analysis indicates that the addition of STAT3 inhibitors to activated LMP2A-expressing B cells blocked the LMP2A-dependent increase in STAT3 targets in comparison to wildtype controls. These data suggest that LMP2A increases the activation of the oncogene STAT3 in resting B cells, which may contribute to the development of EBV-associated tumors and autoimmune diseases.

We hypothesized that reactive arthritis can be induced in the knees and ankles of wild-type C57BL/6 mice following infection with Chlamydia muridarum through the involvement of inflammatory mediators. Reactive arthritis is a chronic form of arthritis, which can also result in conjunctivitis and inflammation of the genital, urinary, or gastrointestinal systems. C. muridarum is a gram-negative obligate intracellular pathogen which can infect the epithelium of the cervix, urethra, and rectum. The exact pathological mechanism of reactive arthritis is unknown. It is understood to result from CD4+ and CD8+ T cells, and factors including TNF-alpha and perforin. Five groups of C57BL/6J mice were used including a positive control group, CD4+ T cells knockout mice, TNF-alpha knockout mice, perforin knockout mice and TNFRSF knockout mice. Mice from each group were intra-vaginally infected with 5×104 inclusion forming units of C. muridarum, marked as day 0. The mice were swabbed a few days after infection to confirm C. muridarum. The mice from each group were euthanized on day 7 and day 14, and sera was collected and analyzed for cytokine expression using an antibody array. Ankles collected from C57BL/6J positive control mice were histologically processed and graded to determine pathology. C57BL/6J positive control mice showed increased synovial lining thickening 14 days following infection. Cytokine expression from TNF-alpha knockout mice, CD4+ T cells knockout mice, perforin knockout mice, and TNFRSF knockout mice was generally reduced. The results show that chlamydial induced reactive arthritis can be detected in the ankles of C57BL/6J mice and suggest that TNF-alpha and CD4+ T cells may be involved in this inflammatory process.

Rheumatoid arthritis (RA) is characterized by synovial inflammation, cytokine production and lymph node collapse. Lymphatic pump treatment (LPT) is an osteopathic manipulative treatment used to reduce edema through increasing lymphatic circulation. This study examined the therapeutic potential of LPT on rats with adjuvant-induced arthritis (AIA). We hypothesize that LPT would lessen joint inflammation through increasing lymphatic drainage resulting in the reduction of cytokines in synovial space. AIA was induced in female Lewis rats and randomly placed into a LPT groups or sham groups. Experiments were repeated using different LPT protocols varying the frequency and number of LPT treatments. However in each experiment LPT consisted of rhythmically pressing below the rib cage once a second for 30 seconds, while the sham group was held for 30 seconds. Joint inflammation was determined by measuring ankle circumferences and by articular index scoring. Ankles were collected and homogenized to determine changes in synovial cytokine levels by magnetic immunoassays and ELISA. Clustering LPT treatments three-times a day at the peak of joint inflammation had the greatest impact on arthritis. Animals treated with LPT in this manner exhibited significantly reduced ankle edema and inflammation. Additionally, synovial levels of inflammatory cytokines (GRO, IL17, and IL-6) were reduced with LPT. These results indicate that LPT can reduce joint edema and inflammation in a rat model of RA.

Isolated from sponges on the ocean floor, MZA has anti-inflammatory properties which might be beneficial in treating rheumatoid arthritis (RA) or osteoarthritis (OA). Preliminary data suggested that MZA causes RA fibroblast-like synoviocytes (FLS) to detach from gelatin. We hypothesized that MZA decreased the adhesive properties of synovial fibroblasts isolated from RA, OA, or normal joints. All fibroblasts were isolated from tissues collected from patients with IRB approval. Photos of cells were taken at various time points (1, 2, 4, 8, 12, and 24 hrs) and counted for adherence after treatment with various concentrations of MZA or vehicle on gelatin, collagen, or laminin substrates. Flow cytometry was used to determine α1 integrin subunit expression on various cell types, since this subunit is important in binding to adhesive substrates. RA FLS on gelatin appeared most sensitive to MZA, in that 1 µM could significantly detach cells in as little as 8 hrs (n=4, p

Mucin 3 (MUC3) is elevated in rheumatoid arthritis (RA) synovial tissues (STs). We hypothesized that MUC3 is expressed by RA synovial fibroblasts, that it is cytokine inducible and chemotactic for RA synovial fibroblasts. To test this, RA synovial fibroblast MUC3 expression was determined by immunohistochemistry, flow cytometry and Western blot analysis. Levels of MUC3 from arthritic synovial fluids (SFs) were measured by a novel MUC3 ELISA. RA synovial fibroblast chemotaxis to RA SFs was assessed using a modified Boyden chamber assay. RA ST cells stained for both MUC3 and vimentin indicating that RA synovial fibroblasts express MUC3. Flow cytometry experiments showed that most of the RA synovial fibroblast MUC3 is cytoplasmic. Cytokine stimulation of these cells significantly increased MUC3 production (P

MZA is a complex alkaloid compound with anti-inflammatory properties that has recently been synthesized. OA has been characterized to have an inflammatory component involving increased cytokine release by synovial tissue fibroblasts. We hypothesized that MZA treatment of OA fibroblasts could decrease their production of pro-inflammatory cytokines at non-toxic concentrations. OA fibroblasts were isolated with IRB approval, treated with MZA or vehicle, and viability was assessed using multiple methods. OA fibroblast condition media (CM) was collected at various time points after MZA or control treatment, screened by antibody microarrays, and verified by ELISAs. Toxicity studies on OA fibroblasts suggested that concentrations up to 2.5 µM MZA were not toxic using a sensitive flow cytometry kit (results varied in a method-, time-, and concentration-dependent manner). Antibody microarray performed on CM pooled from 5 primary OA fibroblast lines treated with 10 µM MZA for 24 hrs suggested that monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6 were two of the most prominent proteins detected, and MZA appeared to decrease MCP-1 production. ELISAs suggested that concentrations of 0.5 µM MZA and higher significantly decreased production of MCP-1 (n=5; p