Claudia Schwab

Generation of C-reactive protein and complement components in atherosclerotic plaques.

C-reactive protein (CRP) and complement are hypothesized to be major mediators of inflammation in atherosclerotic plaques. We used the reverse transcriptase-polymerase chain reaction technique to detect the mRNAs for CRP and the classical complement components C1 to C9 in both normal arterial and plaque tissue, establishing that they can be endogenously generated by arteries. When the CRP mRNA levels of plaque tissue, normal artery, and liver were compared in the same cases, plaque levels were 10.2-fold higher than normal artery and 7.2-fold higher than liver. By Western blotting, we showed that the protein levels of CRP and complement proteins were also up-regulated in plaque tissue and that there was full activation of the classical complement pathway. By in situ hybridization, we detected intense signals for CRP and C4 mRNAs in smooth muscle-like cells and macrophages in the thickened intima of plaques. By immunohistochemistry we showed co-localization of CRP and the membrane attack complex of complement. We also detected up-regulation in plaque tissue of the mRNAs for the macrophage markers CD11b and HLA-DR, as well as their protein products. We showed by immunohistochemistry macrophage infiltration of plaque tissue. Because CRP is a complement activator, and activated complement attacks cells in plaque tissue, these data provide evidence of a self-sustaining autotoxic mechanism operating within the plaques as a precursor to thrombotic events.

Presence of reactive microglia in monkey substantia nigra years after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration.

This report describes the presence of reactive microglia, the accumulation of extracellular melanin, and the extensive loss of dopaminergic neurons in the substantia nigra of monkeys administered 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) 5 to 14 years before death. This evidence of chronic neuroinflammation years after MPTP exposure is similar to that previously reported in humans. The monkeys were drug free for at least 3 years before death, indicating that a brief exposure to MPTP had instituted an ongoing inflammatory process. The mechanism is unknown but could have important implications regarding the cause of Parkinsons disease and possible approaches to therapy.

Up-regulated production and activation of the complement system in Alzheimer's disease brain.

We used reverse transcriptase-polymerase chain reaction and Western blotting techniques to measure the levels of complement mRNAs and their protein products in Alzheimers disease (AD) brain compared with non-AD brain. mRNAs for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 were detected in the 11 regions of brain that were investigated. The mRNA levels were markedly up-regulated in affected areas of AD brain. In the entorhinal cortex, hippocampus, and midtemporal gyrus, which had dense accumulations of plaques and tangles, C1q mRNA was increased 11- to 80-fold over control levels, and C9 mRNA 10- to 27-fold. These levels were substantially higher than in the livers of the same cases. Western blot analysis of AD hippocampus established the presence of all of the native complement proteins as well as their activation products C4d, C3d, and the membrane attack complex. These data indicate that high levels of complement are being produced in affected areas of AD brain, that full activation of the classical complement pathway is continuously taking place, and that this activation may be contributing significantly to AD pathology.

Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease.

C-reactive protein (CRP) and amyloid P (AP) are pentraxins which are associated with many pathological lesions, including the amyloid deposits and neurofibrillary tangles (NFTs) of Alzheimer disease (AD). It has always been assumed that they are generated by liver and delivered to their sites of action by serum. Here we report by in situ hydridization, reverse transcriptase-polymerase chain reaction analysis, Western blotting and immunohistochemistry that the mRNAs and proteins of both CRP and AP are concentrated in pyramidal neurons and are upregulated in affected areas of AD brain. Controlling pentraxin production at the tissue level may be important in reducing inflammatory damage in AD.

Distribution of cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins in human brain and peripheral organs

We used the techniques of reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry to evaluate the expression of cyclooxygenase (COX)-1 and -2 in brain and peripheral organs of Alzheimer disease (AD) and control cases. We found both COX-1 and COX-2 to be constitutively expressed in all organs tested, i.e., brain, heart, liver, kidney, spleen and intestine. COX-2 was substantially upregulated in affected areas of AD brain and in infarcted areas of human heart. COX-1 was only mildly upregulated in AD brain. Immunohistochemically, COX-2 was strongly expressed in the perinuclear, dendritic and axonal areas of pyramidal neurons, with enhanced staining in AD. These data suggest a special role for COX-2 in neuronal function.

Inflammatory Aspects of Alzheimer Disease and Other Neurodegenerative Disorders

Alzheimer and a number of other neurodegenerative diseases are characterized by the presence of reactive microglia and reactive astrocytes in association with the lesions. The classic view that microglia exist primarily in either a resting or activated state needs to be broadened in view of recent results. Resting microglia are in constant activity sampling their surround. Activated microglia may be pro-inflammatory, releasing inflammatory cytokines and other inflammatory mediators, or anti-inflammatory, promoting the healing process. There is evidence that microglial phagocytosis is more powerful in the anti-inflammatory state. Activated astrocytes also have pro-inflammatory and anti-inflammatory properties. In the pro-inflammatory state they release inflammatory cytokines. In the anti-inflammatory state they release various growth factors. In AD and other neurodegenerative diseases, both microglia and astrocytes are in a pro-inflammatory state. From a therapeutic point of view it is desirable to find methods of tipping the balance towards an anti-inflammatory state for both types of glia.

Role of ICAM-1 in persisting inflammation in Parkinson disease and MPTP monkeys

It has been established that neuroinflammation is present in the substantia nigra (SN) of Parkinson disease (PD) cases but the factors responsible are as yet unknown. One contributing protein may be the intercellular adhesion molecule-1 (ICAM-1, CD54). ICAM-1 with its counter receptor, the lymphocyte function-associated antigen 1 (LFA-1) is known to play a key role in inflammatory processes and in T-cell mediated host defense mechanisms. We detected large numbers of ICAM-1-positive reactive astrocytes in the SN of a series of 14 patients with neuropathologically confirmed PD, including 3 of familial origin, compared with 11 age-matched controls. In PD SN, these ICAM-1-positive reactive astrocytes were particularly concentrated around many residual neurons in areas of heavy neuronal loss and extracellular melanin accumulation. LFA-1-positive reactive microglia gathered in areas of intense ICAM-1 expression, and LFA-1-positive leukocytes were identified infiltrating the tissue. Double immunostaining for ICAM-1 and LFA-1 revealed aggregates of reactive microglia embedded in areas of diffuse ICAM-1. Leukocyte counts were 5 fold higher in PD SN compared to controls (P < 0.001). Similar over-expression of ICAM-1 was found in monkeys that had been exposed to MPTP from 5.5 to 14 years previously compared with control monkeys. The presence of ICAM-1-positive reactive astrocytes in Parkinson disease and MPTP-treated monkeys is indicative of a sustained inflammatory process and suggests that antiinflammatory agents may have a place in PD therapy.

Astrocytes are GABAergic cells that modulate microglial activity.

GABA is assumed to function in brain only as an inhibitory neurotransmitter. Here we report a much broader CNS role. We show that human astrocytes are GABAergic cells, and that human microglia are GABAceptive cells. We show that in adult human brain tissue, astrocytes immunostain for the GABA synthesizing enzyme GAD 67, the GABA metabolizing enzyme GABA‐T and the GABAA and GABAB receptors. The intensity of staining is comparable or greater to that observed for known inhibitory neurons. We show that cultured human astrocytes strongly express the mRNA and protein for GAD 67, as well as GABA‐T, and the GABAA and GABAB receptors. We further show that cultured human microglia express the mRNA and protein for GABA‐T, in addition to the GABAA and GABAB receptors characterizing them as GABAceptive cells. We demonstrate that GABA suppresses the reactive response of both astrocytes and microglia to the inflammatory stimulants lipopolysaccharide (LPS) and interferon‐γ by inhibiting induction of inflammatory pathways mediated by NFκB and P38 MAP kinase. This results in a reduced release of the inflammatory cytokines TNFα and IL‐6 and an attenuation of conditioned medium neurotoxicity toward neuroblastoma SH‐SY5Y cells. These inhibitory reactions are partially mimicked by the GABAA receptor agonist muscimol and the GABAB receptor agonist baclofen, indicating that GABA can stimulate both types of receptors in astrocytes as well as microglia. We conclude that the antiinflammatory actions of GABA offer new therapeutic opportunities since agonists should enhance the effectiveness of other antiinflammatory agents that operate through non‐GABA pathways.

Distinct isoforms of tau aggregated in neurons and glial cells in brains of patients with Pick's disease, corticobasal degeneration and progressive supranuclear palsy

Abstract. We investigated isoform composition of aggregated tau protein in brains with Picks disease (PiD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) by immunoblot analysis of sarkosyl-insoluble fractions of brain homogenates. We also examined the adjacent brain tissues immunohistochemically with a rabbit antibody, Ex10, which specifically recognizes exon 10 of tau. The Ex10 recognizes tau isoforms with four microtubule-binding repeats (4Rtau) but not those with three microtubule-binding repeats (3Rtau). Sarkosyl-insoluble tau from the brains of patients with CBD and PSP consisted of 4Rtau. Insoluble tau from the PiD brains contained both 3Rtau and 4Rtau, where 3Rtau predominated over 4Rtau. In brain tissues of CBD and PSP, Ex10 immunostained all neuronal and glial tau-positive structures. They included pre-tangles, astrocytic plaques, tuft-shaped astrocytes, and oligodendroglial coiled bodies. In PiD brains, astrocytic inclusions were also positive for 4Rtau. However, the majority of, if not all, Pick bodies and oligodendroglial tau inclusions were negative for 4Rtau. Such results suggest that, in neurons and oligodendroglia, tau isoforms involved in the pathological processes differ between CBD/PSP and PiD, and are thus disease specific. This contrasts with the astrocytic tau isoforms that accumulate similarly in all three disorders.

Human Heart Generates Complement Proteins That Are Upregulated and Activated After Myocardial Infarction

In human heart, we detected mRNAs and proteins for C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9 with the use of reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical techniques. We found an upregulation of both mRNAs and proteins in areas of recent and old myocardial infarctions. In both situations, the classical complement pathway was activated, with C4d, C3d, and the membrane attack complex (C5b-9) being deposited on damaged cardiac myocytes. These activated complement components were also identified on Western blots of infarcted tissue. Complement mRNAs in infarcted heart tissue were higher than those in liver, and liver complement mRNAs were not upregulated in cases with infarcted hearts. Our results establish that (1) complement proteins are endogenously produced by human heart; (2) the classical complement pathway is fully activated after myocardial infarction; (3) complement activation is directly involved in myocardial damage after ischemic insults; and (4) damage from complement activation may be chronically sustained. These data suggest that inhibition of the complement system should be effective in treating myocardial infarction.