TIM-1 regulates macrophage cytokine production and B7 family member expression (1)
The T-cell immunoglobulin mucin (TIM) family of cell surface proteins has attracted much attention as potential regulators of the immune system. Individual TIM family members may serve as susceptibility markers for asthma, allergies and autoimmune diseases, as well as potential cell surface markers for Th1 and Th2 T cells.
In allergen-induced airway hyperactivity (AHR), a mouse model of asthma, BALB/c mice develop severe AHR after sensitization and challenge with allergen. In contrast, DBA/2 mice challenged with allergen have normal airway responses.When the sequence of the TIM-1 protein in BALB/c and DBA/2 mice are compared, there is a 15 amino acid difference. In vivo administration of anti-mouse TIM-1 increases T cell proliferation and IL-4 production in response to antigen stimulation. LPS or IFN-g activated macrophages are able to bind TIM-1-immunoglobulin fusion protein (TIM-1-Ig).
RAW 264.7 cells were harvested and placed in Falcon polystyrene round-bottom tubes at 5 × 105 cells per tube. The cells were washed with ice cold assay buffer (PBS contain- ing 1% BSA and 0.1% NaAzide), then blocked with normal mouse Ig at 10 ug per tube for 30 min at 4 ◦C. Recombi- nant mouse TIM-1/his or rmTIM-4/his was added at 10ug per tube, and the cells were incubated for 30 min at 4 ◦ C. Cells were then washed twice with ice cold assay buffer, and stained using a mouse anti-polyhistidine PE-conjugated monoclonal antibody at 10ul/tube. A total of 10,000 events were collected and analyzed on a FacsCalibur flow cytometer using CellQuest software.
rmTIM- 1/his but not rmTIM-4/his bound to RAW 264.7 cells. Stimulation of RAW 264.7 cells with LPS did not increase rmTIM-1/his or rmTIM-4/his binding.
Both LPS and rmTIM-1/his treatment stim- ulated high levels of nitric oxide production versus untreated cells (6.8- and 8.6-fold increases, respectively) that was statistically significant (p < 0.05) as compared to unstimulated cells.
RAW 264.7 cells were cultured in CoStar 96-well tissue cul- ture plates in DMEM high glucose media containing 2% FCS, P/S, and l-glutamine at a density of 5 × 104 cells per well, overnight at 37 ◦C in a 5% CO2 humidified chamber. The cultured cells were then stimulated with rmTIM-1/his at 3 ug/ml or LPS at 8 ng/ml for an additional 24 h. Supernatants were collected and evaluated by ELISA kits for TNF-a, IL-6, and IL-10 production following the manufacturers instructions.
Both rmTIM-1/his and LPS induced high levels of the pro- inflammatory cytokines TNF-a and IL-6 that was statistically significant (p < 0.05) as compared to unstimulated cells. Specifically, rmTIM-1/his stimulation of RAW 264.7 cells increased TNF-a secretion by 20-fold and IL-6 by 10-fold. A moderate increase (2–3-fold) that was statistically significant (p < 0.05) in the immuno-modulatory cytokine IL-10 was also seen following stimulation with rmTIM-1/his or LPS.
TIM-1 stimulation can also alter macrophage function perhaps leading to a type II-activated phenotype characterized by increased secretion of IL-10 and intact expression of IL-6 and TNF-a. Production of IL-4 (a Th2 cytokine) correlated with the pro- duction of IL-10 by type II-activated macrophages. An increase in IL-4 and IL-10 production is seen in splenocytes from PLP-immunized or OVA-immunized mice treated with TIM-1- Ig. IL-10 is produced in response to TIM-1 stimu- lation of RAW 264.7 cells with a corresponding decrease in their expression of B7-H2.