Abstract
The great majority of intracellular signaling pathways involve reversible phosphorylation of component proteins. Recently, phosphorylation state-specific antibodies (PSSAs) have been developed that allow the identification of specific phosphorylated proteins in tissue sections. This provides a novel technique for arresting and localizing the dynamic process of signal transduction. Presently there are over 300 commercially available PSSAs against transmembrane receptor tyrosine kinases, intracellular kinases, transcription factors, and other nuclear proteins. Most are affinity-purified polyclonal antibodies raised against synthetic phosphorylated peptides. PSSAs require rigorous specificity testing. False negative reactions are the most common problems encountered with these antibodies; antigen retrieval may be useful. Because phosphorylation is a transient phenomenon, it is critical that tissues be fixed as rapidly as possible. Moreover, when interpreting the result of staining with PSSAs, it is important to recognize that these antibodies provide at best a snapshot of a very short-lived phenomenon. Still, PSSAs are providing pathologists with tantalizing glimpses into signaling dynamics in normal and diseased tissue.
Mandell JW: Phosphorylation state-specific antibodies: applications in investigative and diagnostic pathology. Am J Pathol 163:1687–1698, 2003
CD8+ T cells are important antiviral effector cells; however, their effector mechanisms are active only when the cells are in contact with their immune targets. Thus, recruitment of CD8+ T cells to sites of viral replication is critical for a successful antiviral response. Researchers in Denmark examined the role of the chemokine macrophage inflammatory protein-1α (MIP-1α) in generating CD8+ T lymphocytes and recruiting these cells to sites of lymphocytic choriomeningitis (LCM) viral replication. Their studies showed that MIP-1α was chemotactic for virus-activated CD8+ lymphocytes and CD8+ cells were also the main producers of MIP-1α at sites of viral infection. However, wild-type and MIP-1α knockout mice were equally able to clear LCM virus in vivo and to generate a cellular immune response against the virus in a footpad swelling assay. Furthermore, CD8+ lymphocyte recruitment to sites of LCM replication in the brain was similar in wild-type and knockout animals. Thus, MIP-1α is not required either for CD8+ lymphocyte homing to virus-infected sites or for the development of a specific effector response to viral antigens.
Madsen AN, Nansen A, Christensen JP, Thomsen AR: Role of macrophage inflammatory protein-1alpha in T-cell-mediated immunity to viral infection. J Virol 77:12378–12384, 2003
Investigators at the National Center for Toxicologic Research compared several methods for evaluating proliferation in normal paraffin-embedded rat tissues, including immunohistochemistry for 5-bromodeoxyuirdine (BrdU), proliferating cell nuclear antigen (PCNA), and Ki-67, as well as in situ hybridization for histone mRNA. Immunohistochemistry for the pyrimidine analogue BrdU clearly identifies cells in S-phase; however, this technique has limitations. Living animals must be injected ante mortem with BrdU, and BrdU is also incorporated into DNA during repair. Ki-67 is a nuclear protein expressed at all phases of the cell cycle except G0. If the appropriate antibody (MIB-5) was employed, Ki-67 immunohistochemistry gave nuclear labeling indices very similar to those obtained with BrdU immunohistochemistry. Immunohistochemistry for PCNA, a DNA polymerase accessory protein, gave extensive nuclear labeling; the labeling indices were not correlated with the indices seen for BrdU staining. In situ hybridization for histone mRNA gave strong cytoplasmic labeling; the number and location of positive cells correlated very well with BrdU immunohistochemistry results. A limitation of in situ hybridization for histone mRNA is the loss of signal seen in tissues fixed for more than 24 hours. Based on these findings, Ki-67 immunohistochemistry was recommended for assessing cell proliferation in formalin-fixed paraffin-embedded tissues in the absence of ante mortem BrdU injection.
Muskhelishvili L, Latendresse JR, Kodell RL, Henderson EB: Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA. J Histochem Cytochem 51:1681–1688, 2003
French researchers carried out detailed morphologic studies of 30 separate regions in the brains of two cows infected with the bovine spongiform encephalopathy (BSE) agent, evaluating vacuolation, gliosis, amyloidosis, neuronal changes, prion expression, and apoptosis. Vacuolar changes were widespread and appeared to be bilaterally symmetrical; the extent of gliosis was related to the severity of vacuolation. Rare apoptotic cells were detected in both animals. Only a single animal had evidence of amyloid deposition. Immunohistochemistry for prion protein revealed several different patterns of staining: scattered granular staining in the neuropil, plaque-like deposits in the neuropil, discontinuous staining of the peripheral neuronal perikaryon or neuronal processes, staining of granular or aggregated material within central neuronal cell bodies, and stellate (glial-like) staining. The most heavily stained brain regions were the brainstem, hypothalamus, and thalamus. Positive staining for prion protein was seen in some areas in the absence of vacuolation.
Debeer S, Baron T, Bencsik A: Neuropathological characterisation of French bovine spongiform encephalopathy cases. Histochem Cell Biol 120:513–521, 2003
The immunologic synapse is a stable membrane structure formed between an antigen-presenting cell and a T lymphocyte. It consists of a central core (C-SMAC) of T cell receptors (TCRs) enclosed in a ring of adhesion molecules (P-SMAC). The function of the immunologic synapse has not been precisely defined. Scientists investigated the role of the synapse by comparing signaling through TCRs in T lymphocytes of wild-type mice and mice lacking CD2AP, a molecule required for receptor segregation into the C-SMAC. Based on in vitro studies and computer modeling, it was shown that C-SMAC formation enhanced TCR signaling by concentrating TCR and kinases in a small area where kinases could act on clustered rather than individual TCRs to yield a very high rate of complete TCR phosphorylation. However, greater TCR phosphorylation also enhanced receptor degradation, because only fully phosphorylated TCRs were degraded intracellularly. Thus, strong signaling through TCRs was actually attenuated when these TCRs formed part of a wild-type immunologic synapse.
Lee KH, Dinner AR, Tu C, Campi G, Raychaudhuri S, Varma R, Sims TN, Burack WR, Wu H, Wang J, Kanagawa O, Markiewicz M, Allen PM, Dustin ML, Chakraborty AK, Shaw AS: The immunological synapse balances T cell receptor signaling and degradation. Science 302:1218–22, 2003
The lpr mouse develops a lupus erythematosus–like disease characterized by interferon-γ-induced activation of T helper lymphocytes, lymphadenopathy, autoantibody production, and immune complex glomerulonephritis. To test the role of interleukin-18 (IL-18), a potent interferon inducer in the autoimmune disease in lpr mice, European scientists immunized young lpr mice against IL-18 using a genetic vaccine (DNA of a plasmid containing cloned IL-18 coding sequences). These mice developed autoantibodies against IL-18, had reduced interferon production, and exhibited substantial amelioration of autoimmune disease. Thus, IL-18 appears to play a major role in the development of autoimmune disease and to be an important potential therapeutic target.
Bossu P, Neumann D, Del Giudice E, Ciaramella A, Gloaguen I, Fantuzzi G, Dinarello CA, Di Carlo E, Musiani P, Meroni PL, Caselli G, Ruggiero P, Boraschi D: IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease. Proc Natl Acad Sci U S A. 100:14181–14186, 2003