New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations.

BACKGROUND: Cathepsins are endosomal/lysosomal proteases that play important roles in regulating cell physiological processes in cardiovascular, neurological, musculoskeletal, and immunological systems. Pathophysiological processes are often associated with a change in cathepsin expression and activity, leading to the possibility of using cathepsins as disease markers for diagnosis and prognosis. METHODS: We describe a new assay utilizing an argon laser flow cytometer to measure activities of cysteine cathepsins B, L, and S in live cells using cell permeable fluorogenic cresyl violet-conjugated peptides as selective substrates. Substrate concentration dependency and time kinetics studies were performed. The activity assay was combined with immunofluorescence staining to detect cell lineage-specific molecules and assess cathepsin activities in a heterogeneous cell population. RESULTS: Substrate concentrations utilized were not limiting, because MFI significantly increased in a macrophage cell line stimulated with bacterial lipopolysaccharide. Selective cathepsin inhibitors demonstrated the selectivity of substrate cleavage. Cells fixed and stored before analysis had no loss of fluorescence product. Activities of cathepsins B, L and S in splenic B cells, T cells and macrophages identified by immunofluorescence staining were analyzed. CONCLUSION: This novel technique determines cathepsin activities on a per cell basis without requiring purification of different cell types from a heterogeneous cell population.

Immunotoxicity of gallium arsenide on antigen presentation: comparative study of intratracheal and intraperitoneal exposure routes.

Gallium arsenide (GaAs) is a semiconductor utilized in electronics and computer industries. GaAs exposure of animals causes local inflammation and systemic immune suppression. Mice were administered 2 to 200 mg/kg GaAs. On day 5, intratracheal instillation increased lung weights in a dose-dependent manner and induced pulmonary inflammation exemplified by mononuclear cell infiltration and mild epithelial hyperplasia. No fibrosis, pneumocyte hyperplasia, proteinosis, or bronchial epithelial damage was observed in the lungs. Splenic cellularity and composition were unaffected. GaAs' effect on antigen presentation by macrophages was similar after intratracheal and intraperitoneal exposure, although the lowest observable adverse effect levels differed. Macrophages from the exposure site displayed an enhanced ability to activate an antigen-specific CD4(+) helper T-cell hybridoma compared with vehicle controls, whereas splenic macrophages were defective in this function. The chemical's impact on peritoneal macrophages depended on the exposure route. GaAs exposure augmented thiol cathepsins B and L activities in macrophages from the exposure site, but decreased proteolytic activities in splenic macrophages. Alveolar macrophages had increased expression of major histocompatibility complex (MHC) Class II molecules, whereas MHC Class II expression on splenic and peritoneal macrophages was unaffected. Modified thiol cathepsin activities statistically correlated with altered efficiency of antigen presentation, whereas MHC Class II expression did not. Our study is the first one to examine the functional capability of alveolar macrophages after intratracheal GaAs instillation. Therefore, thiol cathepsins may be potential target molecules by which GaAs exposure modulates antigen presentation.

Gallium arsenide exposure impairs processing of particulate antigen by macrophages: modification of the antigen reverses the functional defect.

Gallium arsenide (GaAs), a semiconductor used in the electronics industry, causes systemic immunosuppression in animals. The chemical's impact on macrophages to process the particulate antigen, sheep red blood cells (SRBC), for a T cell response in culture was examined after in vivo exposure of mice. GaAs-exposed splenic macrophages were defective in activating SRBC-primed lymph node T cells that could not be attributed to impaired phagocytosis. Modified forms of SRBC were generated to examine the compromised function of GaAs-exposed macrophages. SRBC were fixed to maintain their particulate nature and subsequently delipidated with detergent. Delipidation of intact SRBC was insufficient to restore normal antigen processing in GaAs-exposed macrophages. However, chemically exposed cells efficiently processed soluble sheep proteins. These findings suggest that the problem may lie in the release of sequestered sheep protein antigens, which then could be effectively cleaved to peptides. Furthermore, opsonization of SRBC with IgG compensated for the macrophage processing defect. The influence of signal transduction and phagocytosis via Fcgamma receptors on improved antigen processing could be dissociated. Immobilized anti-Fcgamma receptor antibody activated macrophages to secrete a chemokine, but did not enhance processing of unmodified SRBC by GaAs-exposed macrophages. Restoration of normal processing of particulate SRBC by chemically exposed macrophages involved phagocytosis through Fcgamma receptors. Hence, initial immune responses may be very sensitive to GaAs exposure, and the chemical's immunosuppression may be averted by opsonized particulate antigens.

Role of cannabinoid receptors in inhibiting macrophage costimulatory activity.

Delta(9)-tetrahydrocannabinol (THC) inhibits several immunologic functions of macrophages. THC's impact on peritoneal macrophages to deliver costimulatory signals to a helper T cell hybridoma was investigated by T cell interleukin-2 production stimulated with immobilized anti-CD3 antibody. The drug's inhibition of costimulatory activity depended on the macrophages. THC decreased costimulation provided by peritoneal cells elicited with polystyrene beads and thioglycollate, but the drug had no influence with macrophages elicited with thioglycollate alone. Bead administration induced CB2 mRNA expression in macrophages, while CB1 mRNA was not detected. Although inhibition was associated with functional heat-stable antigen, a costimulatory molecule, on macrophages, THC exposure did not alter cell surface heat-stable antigen expression. Inhibition by THC and anti-heat-stable antigen antibody was not additive suggesting the inhibitory mechanisms may overlap. Cannabinoid suppression was stereoselective; low affinity synthetic isomer CP56,667 did not diminish the T cell response. CB1-selective antagonist SR141716A completely reversed, and CB2-selective antagonist SR144528 partially blocked THC's inhibition. Both antagonists appeared to behave as inverse agonists in a receptor-selective manner. Although T cells expressed a low level of CB2 mRNA, neither THC nor SR141716A affected T cell activation in a system independent of macrophages, while SR144528 was inhibitory. High affinity synthetic agonist CP55,940, but not partial agonist THC, impaired costimulation by macrophages from mice lacking CB2 receptor. Although CB1 mRNA was not detected in CB2 null macrophages, CP55,940 reversed the inverse agonist activity of SR141716A. Hence, CB2 and possibly another receptor subtype may be involved in mediating cannabinoid suppression of macrophage costimulation.

Gallium arsenide exposure impairs splenic B cell accessory function.

Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4(+) T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.

Impact of in vitro gallium arsenide exposure on macrophages.

The semiconductor gallium arsenide (GaAs) is classified as an immunotoxicant and a carcinogen. We previously showed that GaAs in vivo induces several phenotypic changes in macrophages located at the exposure site, indicative of an activated state. These physiological alterations may be a primary or secondary consequence of chemical exposure. To discern primary influences, our current study examined the in vitro effects of the chemical on macrophage cell lines and murine peritoneal macrophages. GaAs augmented cathepsins L and B proteolytic activities in all three sources of macrophages. Expression of the two mature isoforms of invariant chain and its cleavage fragment was also significantly increased, indicating that the chemical directly affects macrophages. However, GaAs did not alter the overall cell surface expression of major histocompatibility complex class II molecules on macrophages nor influence their ability to stimulate antigen-specific helper T cell hybridomas to respond to intact antigens that require processing. These findings raise the possibility that the chemical's complete in vivo impact may involve cytokines. Further, GaAs in vitro enhanced steady-state cathepsin L protein, and cathepsins L and B mRNA expression in macrophages, indicating that GaAs may alter gene expression, which may contribute to the chemical's adverse biological effects.