Search

Neurodegenerative diseases are becoming an increasing social and economical burden as our population ages; but current knowledge of the processes leading to these diseases is still limited, and no effective treatments are available. Neurodegeneration in Alzheimer's disease (AD) is the most common cause of dementia and afflicts an estimated 4 million people in this country alone. Because accumulation of beta-amyloid (Abeta) peptide appears central to AD pathogenesis, large efforts have been directed at understanding and interfering with Abeta production or aggregation. These efforts have largely identified the processes resulting in Abeta production from the larger amyloid precursor protein (APP) and have revealed that Abeta peptide is also produced at low levels in the healthy brain. Interestingly, Abeta production is rapidly increased after neuronal injury, and traumatic brain injury is a known risk factor for AD and Parkinson's disease. In contrast, brain injury in young individuals does not seem to result in AD, and brain injury in animal models can promote Abeta clearance. This suggests that certain factors associated with injury might be able to reduce the accumulation of Abeta. Accumulation of Abeta peptide might be reduced either directly by stimulating phagocytes or other Abeta-degrading processes, or indirectly, by reducing neuronal injury and thus lowering the production of Abeta peptide. Directing the brain's natural mechanisms for clearing Abeta or increasing neuroprotection might therefore be reasonable approaches in interfering with AD pathogenesis., (Copyright 2004 Humana Press Inc.)

TGF-beta1 is a key regulator of diverse biological processes in many tissues and cell types, but its exact function in the developing and adult mammalian CNS is still unknown. We report that lack of TGF-beta1 expression in neonatal Tgfb1(-/-) mice results in a widespread increase in degenerating neurons accompanied by reduced expression of synaptophysin and laminin and a prominent microgliosis. Lack of TGF-beta1 also strongly reduces survival of primary neurons cultured from Tgfb1(-/-) mice. TGF-beta1 deficiency in adult Tgfb1(-/+) mice results in increased neuronal susceptibility to excitotoxic injury, whereas astroglial overexpression of TGF-beta1 protects adult mice against neurodegeneration in acute, excitotoxic and chronic injury paradigms. This study reveals a nonredundant function for TGF-beta1 in maintaining neuronal integrity and survival of CNS neurons and in regulating microglial activation. Because individual TGF-beta1 expression levels in the brain vary considerably between humans, this finding could have important implications for susceptibility to neurodegeneration.

Apolipoprotein (apo) E4 increases the risk and accelerates the onset of Alzheimer's disease (AD). However, the underlying mechanisms remain to be determined. We previously found that apoE undergoes proteolytic cleavage in AD brains and in cultured neuronal cells, resulting in the accumulation of carboxyl-terminal-truncated fragments of apoE that are neurotoxic. Here we show that this fragmentation is caused by proteolysis of apoE by a chymotrypsin-like serine protease that cleaves apoE4 more efficiently than apoE3. Transgenic mice expressing the carboxyl-terminal-cleaved product, apoE4(Delta272-299), at high levels in the brain died at 2-4 months of age. The cortex and hippocampus of these mice displayed AD-like neurodegenerative alterations, including abnormally phosphorylated tau (p-tau) and Gallyas silver-positive neurons that contained cytosolic straight filaments with diameters of 15-20 nm, resembling preneurofibrillary tangles. Transgenic mice expressing lower levels of the truncated apoE4 survived longer but showed impaired learning and memory at 6-7 months of age. Thus, carboxyl-terminal-truncated fragments of apoE4, which occur in AD brains, are sufficient to elicit AD-like neurodegeneration and behavioral deficits in vivo. Inhibiting their formation might inhibit apoE4-associated neuronal deficits.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Abeta deposition, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions, and cease migration upon interaction with immobilized Abeta(1-42). We also show that astrocytes bind and degrade Abeta(1-42). Astrocytes plated on Abeta-laden brain sections from a mouse model of AD associate with the Abeta deposits and reduce overall Abeta levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Abeta deposits, indicate a direct role for astrocytes in degradation of Abeta and implicate deficits in astroglial clearance of Abeta in the pathogenesis of AD. Treatments that increase removal of Abeta by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD.

Cerebrovascular abnormalities, such as reduced blood flow, microvascular fibrosis, and cerebrovascular amyloid angiopathy, are prominent in Alzheimer's disease (AD). However, their etiology is poorly understood and it is unclear whether cerebrovascular changes contribute to functional impairments in the absence of neurodegeneration. In humans with AD, transforming growth factor-beta1 (TGF-beta1) mRNA levels in the midfrontal gyrus correlate positively with the relative degree of cerebrovascular amyloid deposition in that brain region, suggesting a possible role for TGF-beta1 in human cerebrovascular abnormalities. Transgenic mice overexpressing TGF-beta1 in astrocytes develop AD-like cerebrovascular abnormalities, including perivascular astrocytosis, microvascular basement membrane thickening, and accumulation of thioflavin S-positive amyloid in the absence of parenchymal degeneration. Mice overexpressing TGF-beta1 alone or in addition to human amyloid precursor protein (hAPP) show selective accumulation of human beta-amyloid (Abeta) in blood vessels and develop cerebral hemorrhages in old age. In 9-month-old TGF-beta1 transgenic mice, cerebral blood flow (CBF) in the limbic system was significantly less than in nontransgenic littermate controls. Aged TGF-beta1 mice also showed overall reduced cerebral glucose uptake (CGU) as a measure of brain activity. Thus, chronic overproduction of TGF-beta1 in the brain results in structural and functional impairments reminiscent of those in AD cases with amyloid angiopathy.

Abnormal accumulation of beta-amyloid (Abeta) in Alzheimer's disease (AD) is associated with prominent brain inflammation. Whereas earlier studies concluded that this inflammation is detrimental, more recent animal data suggest that at least some inflammatory processes may be beneficial and promote Abeta clearance. Consistent with these observations, overproduction of transforming growth factor (TGF)-beta1 resulted in a vigorous microglial activation that was accompanied by at least a 50% reduction in Abeta accumulation in human amyloid precursor protein (hAPP) transgenic mice. In a search for inflammatory mediators associated with this reduced pathology, we found that brain levels of C3, the central component of complement and a key inflammatory protein activated in AD, were markedly higher in hAPP/TGF-beta1 mice than in hAPP mice. To assess the importance of complement in the pathogenesis of AD-like disease in mice, we inhibited C3 activation by expressing soluble complement receptor-related protein y (sCrry), a complement inhibitor, in the brains of hAPP mice. Abeta deposition was 2- to 3-fold higher in 1-year-old hAPP/sCrry mice than in age-matched hAPP mice and was accompanied by a prominent accumulation of degenerating neurons. These results indicate that complement activation products can protect against Abeta-induced neurotoxicity and may reduce the accumulation or promote the clearance of amyloid and degenerating neurons. These findings provide evidence for a role of complement and innate immune responses in AD-like disease in mice and support the concept that certain inflammatory defense mechanisms in the brain may be beneficial in neurodegenerative disease.

Inflammation is a defense reaction against diverse insults, designed to remove noxious agents and to inhibit their detrimental effects. It consists of a dazzling array of molecular and cellular mechanisms and an intricate network of controls to keep them in check. In neurodegenerative diseases, inflammation may be triggered by the accumulation of proteins with abnormal conformations or by signals emanating from injured neurons. Given the multiple functions of many inflammatory factors, it has been difficult to pinpoint their roles in specific (patho)physiological situations. Studies of genetically modified mice and of molecular pathways in activated glia are beginning to shed light on this issue. Altered expression of different inflammatory factors can either promote or counteract neurodegenerative processes. Since many inflammatory responses are beneficial, directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it.

Recent studies have implicated pro- and anti-inflammatory cytokines as integral to Alzheimer's disease (AD) pathogenesis. Among them, transforming growth factor-beta (TGF-beta) is emerging as an important factor in regulating inflammatory responses. This multifunctional cytokine might be centrally involved in several aspects of AD pathogenesis by regulating beta-amyloid precursor protein synthesis and processing, plaque formation, astroglial and microglial response and neuronal cell death. Among all of these potential roles, studies in transgenic and infusion animal models have shown that TGF-beta may primarily contribute to AD pathogenesis by influencing A beta production and deposition, which in turn might result in damage to the brain microvasculature. The lessons learned from these models are of great interest not only for understanding of the role of TGF-beta in AD, but also for future treatments where testing of anti-inflammatory agents such as ibuprofen and an amyloid vaccine hold great promise. In this regard, further elucidation of the signal pathways by which TGF-beta exerts its effect in AD might lead to specific targets for further therapeutic intervention.

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Delta 272--299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.

Abnormal accumulation of the amyloid-beta peptide (Abeta) in the brain appears crucial to pathogenesis in all forms of Alzheimer disease (AD), but the underlying mechanisms in the sporadic forms of AD remain unknown. Transforming growth factor beta1 (TGF-beta1), a key regulator of the brain's responses to injury and inflammation, has been implicated in Abeta deposition in vivo. Here we demonstrate that a modest increase in astroglial TGF-beta1 production in aged transgenic mice expressing the human beta-amyloid precursor protein (hAPP) results in a three-fold reduction in the number of parenchymal amyloid plaques, a 50% reduction in the overall Abeta load in the hippocampus and neocortex, and a decrease in the number of dystrophic neurites. In mice expressing hAPP and TGF-beta1, Abeta accumulated substantially in cerebral blood vessels, but not in parenchymal plaques. In human cases of AD, Abeta immunoreactivity associated with parenchymal plaques was inversely correlated with Abeta in blood vessels and cortical TGF-beta1 mRNA levels. The reduction of parenchymal plaques in hAPP/TGF-beta1 mice was associated with a strong activation of microglia and an increase in inflammatory mediators. Recombinant TGF-beta1 stimulated Abeta clearance in microglial cell cultures. These results demonstrate that TGF-beta1 is an important modifier of amyloid deposition in vivo and indicate that TGF-beta1 might promote microglial processes that inhibit the accumulation of Abeta in the brain parenchyma.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Alzheimer's disease (AD) is frequently associated with cerebrovascular changes, including perivascular astrocytosis, amyloid deposition, and microvascular degeneration, but it is not known whether these pathological changes contribute to functional deficits in AD. To characterize the temporal relationship between amyloid deposition, cerebrovascular abnormalities, and potential functional changes, we studied transgenic mice that express transforming growth factor-beta 1 (TGF-beta 1) at low levels in astrocytes. TGF-beta 1 induced a prominent perivascular astrocytosis, followed by the accumulation of basement membrane proteins in microvessels, thickening of capillary basement membranes, and later, around 6 months of age, deposition of amyloid in cerebral blood vessels. At 9 months of age, various AD-like degenerative alterations were observed in endothelial cells and pericytes. Associated with these morphological changes were changes in regional cerebral glucose utilization. Preliminary results showed that TGF-beta 1 mice had significantly decreased glucose utilization in the mammillary bodies, structures involved in mnemonic and learning processes. Glucose utilization tended to be decreased in several other brain regions as well; however, in the inferior colliculus, it was markedly higher in TGF-beta 1 mice than in controls. We conclude that chronic overproduction of TGF-beta 1 triggers a pathogenic cascade leading to AD-like cerebrovascular amyloidosis, microvascular degeneration, and local alterations in brain metabolic activity. Similar mechanisms may be involved in AD pathogenesis.

Cerebrovascular amyloid deposition and microvascular degeneration are frequently associated with Alzheimer's disease (AD), but the etiology and pathogenetic role of these abnormalities are unknown. Recently, transforming growth factor-beta1 (TGF-beta1) was implicated in cerebrovascular amyloid formation in transgenic mice with astroglial overproduction of TGF-beta1 and in AD. We tested whether TGF-beta1 overproduction induces AD-like cerebrovascular degeneration and analyzed how cerebrovascular abnormalities develop over time in TGF-beta1-transgenic mice. In cerebral microvessels from 3- to 4-month-old TGF-beta1-transgenic mice, which display a prominent perivascular astrocytosis, levels of the basement membrane proteins perlecan and fibronectin were severalfold higher than in vessels from nontransgenic mice. Consistent with this increase, cortical capillary basement membranes of TGF-beta1 mice were significantly thickened. These changes preceded amyloid deposition, which began at around 6 months of age. In 9- and 18-month-old TGF-beta1 mice, various degenerative changes in microvascular cells of the brain were observed. Endothelial cells were thinner and displayed abnormal, microvilli-like protrusions as well as occasional condensation of chromatin, and pericytes occupied smaller areas in capillary profiles than in nontransgenic controls. Similar cerebrovascular abnormalities have been reported in AD. We conclude that chronic overproduction of TGF-beta1 triggers an accumulation of basement membrane proteins and results in AD-like cerebrovascular amyloidosis and microvascular degeneration. Closely related processes may induce cerebrovascular pathology in AD.

The class A scavenger receptor (SR) is expressed on reactive microglia surrounding cerebral amyloid plaques in Alzheimer's disease (AD). Interactions between the SR and amyloid beta peptides (Abeta) in microglial cultures elicit phagocytosis of Abeta aggregates and release of neurotoxins. To assess the role of the SR in amyloid clearance and Abeta-associated neurodegeneration in vivo, we used the platelet-derived growth factor promoter to express human amyloid protein precursors (hAPPs) in neurons of transgenic mice. With increasing age, hAPP mice develop AD-like amyloid plaques. We bred heterozygous hAPP (hAPP(+/-)) mice that were wild type for SR (SR(+/+)) with SR knockout (SR(-/-)) mice. Crosses among the resulting hAPP(+/-)SR(+/-) offspring yielded hAPP(+/-) and hAPP(-/-) littermates that were SR(+/+) or SR(-/-). These second-generation mice were analyzed at 6 and 12 months of age for extent of cerebral amyloid deposition and loss of synaptophysin-immunoreactive presynaptic terminals. hAPP(-/-)SR(-/-) mice showed no lack of SR expression, plaque formation, or synaptic degeneration, indicating that lack of SR expression does not result in significant accumulation of endogenous amyloidogenic or neurotoxic factors. In hAPP(+/-) mice, ablation of SR expression did not alter number, extent, distribution, or age-dependent accumulation of plaques; nor did it affect synaptic degeneration. Our results do not support a critical pathogenic role for microglial SR expression in neurodegenerative alterations associated with cerebral beta amyloidosis.

Amyloid precursor proteins (APPs) are expressed in multiple organs and cell types in diverse species. Their conservation across species and high abundance in brain and the association of various APP missense mutations with autosomal dominant forms of familial Alzheimer's disease (FAD) suggest important roles for APP in the central nervous system. However, the basic functions of APP in the central nervous system remain largely unknown. To assess potential effects of APP on neuronal death and survival, we transfected APP-deficient rat neuroblastoma cells (B103) with DNA constructs encoding wild-type or FAD-mutant human APP. Wild-type, but not FAD-mutant, APP effectively protected cells against apoptosis induced by ultraviolet irradiation, staurosporine, or p53. Wild-type APP also strongly inhibited p53 DNA-binding activity and p53-mediated gene transactivation, whereas FAD-mutant APP did not. We conclude that APP protects neuronal cells against apoptosis by controlling p53 activation at the post-translational level. Disruption of this function by mutations or alterations in APP processing could enhance neuronal vulnerability to secondary insults and contribute to neuronal degeneration.

Apolipoprotein (apo) E isoforms are key determinants of susceptibility to Alzheimer's disease. The apoE4 isoform is the major known genetic risk factor for this disease and is also associated with poor outcome after acute head trauma or stroke. To test the hypothesis that apoE3, but not apoE4, protects against age-related and excitotoxin-induced neurodegeneration, we analyzed apoE knockout (Apoe-/-) mice expressing similar levels of human apoE3 or apoE4 in the brain under control of the neuron-specific enolase promoter. Neuronal apoE expression was widespread in the brains of these mice. Kainic acid-challenged wild-type or Apoe-/- mice had a significant loss of synaptophysin-positive presynaptic terminals and microtubule-associated protein 2-positive neuronal dendrites in the neocortex and hippocampus, and a disruption of neurofilament-positive axons in the hippocampus. Expression of apoE3, but not of apoE4, protected against this excitotoxin-induced neuronal damage. ApoE3, but not apoE4, also protected against the age-dependent neurodegeneration seen in Apoe-/- mice. These differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of Alzheimer's disease and to the poor outcome after head trauma and stroke associated with apoE4 in humans.

The human CD4 molecule (hCD4) is expressed on T lymphocytes and macrophages and acts as a key component of the cellular receptor for HIV. At baseline, hCD4 transgenic mice expressed hCD4 on microglia, the resident mononuclear phagocytes of the brain, and showed no neuronal damage. Activation of brain microglia by peripheral immune challenges elicited neurodegeneration in hCD4 mice but not in nontransgenic controls. In post-mortem brain tissues from AIDS patients with opportunistic infections, but without typical HIV encephalitis, hCD4 expression correlated with neurodegeneration. We conclude that hCD4 may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system.

Deposition of amyoid-beta peptide in the central nervous system is a hallmark of Alzheimer's disease and a possible cause of neurodegeneration. The factors that initiate or promote deposition of amyloid-beta peptide are not known. The transforming growth factor TGF-beta1 plays a central role in the response of the brain to injury, and increased TGF-beta1 has been found in the central nervous system of patients with Alzheimer's disease. Here we report that TGF-beta1 induces amyloid-beta deposition in cerebral blood vessels and meninges of aged transgenic mice overexpressing this cytokine from astrocytes. Co-expression of TGF-beta1 in transgenic mice overexpressing amyloid-precursor protein, which develop Alzheimer's like pathology, accelerated the deposition of amyloid-beta peptide. More TGF-beta1 messenger RNA was present in post-mortem brain tissue of Alzheimer's patients than in controls, the levels correlating strongly with amyloid-beta deposition in the damaged cerebral blood vessels of patients with cerebral amyloid angiopathy. These results indicate that overexpression of TGF-beta1 may initiate or promote amyloidogenesis in Alzheimer's disease and in experimental models and so may be a risk factor for developing Alzheimer's disease.

Cerebral expression of the injury response cytokine transforming growth factor-beta 1 (TGF-beta 1) has been found to be increased in several neurological diseases but it remains unclear whether its function is primarily beneficial or detrimental. Here we show that transgenic (tg) mice that overexpress bioactive (TGF-beta 1 in the central nervous system (CNS) and show no overt phenotype in the unmanipulated state, are more susceptible to the immune-mediated CNS disease experimental autoimmune encephalomyelitis (EAE). TGF-beta 1 tg mice with EAE showed an earlier onset of clinical symptoms, more severe disease and increased mononuclear cell infiltration in their spinal cords compared with non-tg littermate controls with EAE. Whereas previous observations indicated that increased peripheral levels of TGF-beta 1 can suppress EAE, our findings demonstrate that local expression of TGF-beta 1 within the CNS parenchyma can enhance immune cell infiltration and intensify the CNS impairment resulting from peripherally triggered autoimmune responses.

beta-Amyloid precursor protein (beta APP), transforming growth factor beta (TGF beta), and tumor necrosis factor-alpha (TNF alpha) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGF beta and TNF alpha in characteristic ways when the brain is injured. TGF beta, TNF alpha and secreted forms of beta APP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNF alpha and the fibrillogenic amyloid beta-peptide (A beta) derivative of beta APP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGF beta, TNF alpha or beta APP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGF beta (serine/threonine kinase cascades), TNF alpha (p55 receptor linked to a sphingomyelin-ceramide-NF kappa B pathway), and secreted forms of beta APP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K+ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.

A number of important neurological diseases, including HIV-1 encephalitis, Alzheimer's disease, and brain trauma, are associated with increased cerebral expression of the multifunctional cytokine transforming growth factor-beta 1 (TGF-beta 1). To determine whether overexpression of TGF-beta 1 within the central nervous system (CNS) can contribute to the development of neuropathological alterations, a bioactive form of TGF-beta 1 was expressed in astrocytes of transgenic mice. Transgenic mice with high levels of cerebral TGF-beta 1 expression developed a severe communicating hydrocephalus, seizures, motor incoordination, and early runting. While unmanipulated heterozygous transgenic mice from a low expressor line showed no such alterations, increasing TGF-beta 1 expression in this line by injury-induced astroglial activation or generation of homozygous offspring did result in the abnormal phenotype. Notably, astroglial overexpression of TGF-beta 1 consistently induced a strong upmodulation of the extracellular matrix proteins laminin and fibronectin in the CNS, particularly in the vicinity of TGF-beta 1-expressing perivascular astrocytes, but was not associated with obvious CNS infiltration by hematogenous cells. While low levels of extracellular matrix protein expression may assist in CNS wound repair and regeneration, excessive extracellular matrix deposition could result in the development of hydrocephalus. As an effective inducer of extracellular matrix components, TGF-beta 1 may also contribute to the development of other neuropathological alterations, eg, the formation of amyloid plaques in Alzheimer's disease.

Activated T-cells expressing MHC class II surface antigens are able to present antigen and thus function as peptide-presenting cells (T-APCs). In this study we investigated whether antigen presentation by T-cells induced programmed cell death. As a model we used tetanus p30 peptide (aa 947-967)-specific, noncytotoxic CD4+ T-cell clones (C11 and C31). For experimental purposes these T-cell clones were stimulated (a) with p30 peptide-pulsed and fixed EBV-transformed antigen-presenting cells (B-APCs), (b) with p30-pulsed and fixed activated T-cells as APCs (as T-APCs we used either the T-cell clones themselves or an autologous T-cell clone (CT3) with p30 unrelated specificity), or (c) with soluble p30 peptide. The efficiency of antigen presentation was monitored by measuring proliferation as [3H]thymidine uptake. Apoptosis was measured by quantifying fragmented, cytoplasm DNA with the fluorescent dye 4,6-diamidino-2-phenylindole or by visualizing fragmented DNA by gel electrophoresis. Stimulation with p30-pulsed and fixed B-APCs or T-APCs induced proliferation but no apoptosis of the responding T-cells. However, stimulation of cloned T-cells with soluble peptide induced up-regulation of the FAS surface molecules and apoptosis, which was dependent on the peptide doses. Because cloned T-cells express HLA class II molecules, they can theoretically exert both functions at once: antigen presentation and antigen response when they are stimulated with soluble peptide. Because death by apoptosis is only seen under such circumstances, we suggest that T-cells simultaneously presenting and responding to an antigen die of apoptosis and thus contribute to the down-regulation of the immune response. Such phenomena might occur in HIV infection when activated CD4+ T-cells take up gp120 via their CD4 molecules, present it on their HLA class II surface antigens, and are simultaneously stimulated via their TCR.

T cells recognize peptides in association with major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). To sensitize APC for antigen presentation in vitro and in vivo, high concentrations of synthetic peptides can be added from the outside and bind to the MHC molecules, thereby mimicking naturally processed peptides. In this report we investigated whether the transferrin (Tf) molecule could be used as a carrier to introduce antigenic peptides into the antigen presentation pathway of APC. We coupled to Tf various MHC class II DR1 restricted peptides and compared the sensitization of DR1+ APC by the Tf-bound or by the soluble peptide, using peptide-specific T cell clones (TCC). The presentation of the Tf-bound peptides was MHC restricted and could be blocked by the fixation of the APC with glutaraldehyde or by the addition of an excess of Tf. Tf-bound peptides were more efficiently presented than soluble peptides, since smaller concentrations were required to sensitize APC. Moreover, they could compete with a soluble peptide for MHC restricted presentation with a very high efficiency if compared to soluble competing peptides. Tf peptide conjugates could even compete with the presentation of a native antigen like tetanus toxoid. Peptides bound to the transferrin molecule might be useful for immunization strategies, as the relevant bound peptides are efficiently presented to peptide-specific TCC.

In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, outer root sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular outer root sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.

Human T cells express major histocompatibility complex (MHC) class II antigens and adhesion molecules characteristic of antigen-presenting cells (APCs), and recent in vitro and in vivo evidence supports an antigen-presenting function for T cells. In this guise, T cells provide downregulatory signals for the immune response by inducing anergy in T cells that have already been activated and cytotoxicity in resting T cells. Here, Werner Pichler and Tony Wyss-Coray suggest that this may represent an important negative mechanism for T-cell homeostasis.

CD4+ T cell clones were generated to tetanus toxin or to two tetanus toxin-derived peptides p2 (AA 830-834) and p30 (AA 947-976). 11 of the 24 p30-specific clones reacted to shorter p30 subunits (p301 or p302), and only 14 of the p2 or p30-specific clones reacted with TT presented by EBV-transformed B cell lines (B-LCL). The p30-specific clones were HLA-DP4 restricted. In contrast to autologous B cell lines, the majority of allogeneic, but HLA-DP4-positive cell lines failed to present p30 to the specific clones. We concluded that T cell clones are highly specific and that both, small alterations of the peptide length as well as discrete differences of the HLA-molecule may abrogate recognition of the peptide HLA complex by T cells. Moreover, use of peptides as stimulators of T cells may recruit and activate T cells which fail the "original" peptide, derived from normal antigen processing. Clones could usually be maintained in culture for 4-6 months, but with the help of freezing and thawing some clones are now available for over 2 years and still specific. Comparison of different autologous antigen-presenting cells, namely B-LCL and activated MHC class II-positive T cells revealed that not all clones were able to mount a proliferative response to peptide presentation by T cells, while all clones proliferated to B cells as APC. If stimulated with peptide and B-LCL, the clone proliferating to T cells as APC (so-called T responder clones) secreted a broad spectrum of cytokines (Th0-like) and were easier to maintain in culture. In contrast, clones which were unable to proliferate to peptide presentation, so-called T-nonresponder clones, showed a more restricted cytokine pattern and elevated or very low IL4/IFN gamma ratio upon antigen specific stimulation. However, all clones secreted at least small amounts of IL2, IL4, IFN gamma and TNF alpha, if stimulated by PMA and ionomycin. Thus, both chemical and antigen-specific stimulations should be considered if T cell clones are classified as Th1 or Th2, whereby those clones, which secrete a limited cytokine pattern after antigen stimulation only, might be named Th1 or Th2 like clones, while clones which even after PMA/ionomycin do not secrete all cytokines, might represent "real" Th1 or Th2 clones.

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57-68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8+ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s,r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen-presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM-1, LFA-3 and B7, they might play a role as APC and be involved in the regulation of T-Tcell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide- or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B-LCL) or HTLV-1-transformed T cells, or with non-classical APC, like activated keratinocytes or eosinophils. CD4+, DP-restricted T cell clones specific for a tetanus toxin peptide (amino acids 947-967) and CD4+, DR-restricted allospecific T cell clones produced interleukin (IL)-2, IL-4, tumor necrosis factor-alpha and interferon-gamma (IFN-gamma) after phorbol 12-myristate 13-acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose-response curves revealed that the antigen-presenting capacity of activated, MHC class II+, B7+ T cells was comparable to the one of B-LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN-gamma/IL-4 ratio. Cytokine-treated, MHC class II+ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM-1-positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM-1 and LFA3), provided a reduced signal resulting in a diminished IFN-gamma/IL-4 ratio. We conclude that non-classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM-1) expressed seems to determine the efficacy of this presentation.

The B cell antigen B7 delivers a strong co-stimulatory signal for the activation of T cells by binding to its ligands CD28 and CTLA4. Here we demonstrate the surface expression of the B7 molecule on activated human T cells in vitro and under certain conditions in vivo and its functional importance in T-T cell interactions. B7 was detected by flow cytometry on antigen-specific CD4+ and allospecific CD8+ cloned T cells from different donors with anti-B7 monoclonal antibody (mAb) or a soluble CTLA4-C gamma 1 chimera molecule and by reverse transcription-polymerase chain reactions. The expression of B7 was up-regulated following restimulation of the T cell clones and peaked after 7-9 days. Moreover, we show that the B7 molecule on T cells is functionally involved in T-T cell interactions: mAb to CD28 and the CTLA4-Ig fusion protein could inhibit the proliferation of specific T cell clones in response to T cells as antigen-presenting cells (APC) or the proliferation of peripheral blood mononuclear cells in a primary allostimulation with activated T cells as stimulator cells. Finally, we found that B7 can be expressed on freshly isolated circulating T cells since in a preliminary study with a limited number of patients, B7 was present on a subset of CD3+ cells. B7 was expressed on activated T cells (CD4+ and CD8+) of certain human immunodeficiency virus (HIV)-infected individuals (0.5-20% B7+CD8+ cells) or some patients with autoimmune diseases whereas CD3+ cells of healthy individuals did not express B7. The coexpression of major histocompatibility complex class II molecules and B7 may be relevant for the capacity of activated T cells to function as APC. The expression of B7 on T cells in vivo in autoimmune diseases and in HIV infection may be important for a better understanding of these diseases.

In this report, we describe the discrimination of human T cell clones based on their reactivity with activated T cells as antigen-presenting cells (APC). CD4+ T cell clones specific for peptide P30 of tetanus toxin (amino acids 947-967) and restricted to the DP4 molecule were established and tested for proliferation to peptide presented either by peripheral blood mononuclear cells (PBMC), Epstein-Barr virus (EBV)-transformed B cells or major histocompatibility complex (MHC) class II-expressing T cells. We found two sets of T cell clones: one set proliferated to peptide presentation by PBMC, EBV-transformed B cell lines (EBV-B cells) and MHC class II+ T cells (termed T-responder clones), while the other set of clones was only stimulated to proliferate, if the peptide was presented by PBMC or EBV-B cells, but not by T cells (T-nonresponder clones). Nevertheless, these T-nonresponder clones recognized P30 also on T cells, as revealed by Ca2+ influx. The discrimination of the clones was not due to different avidities of the T cell receptors (TcR) of individual clones for the MHC-peptide complex as T-responder and T-nonresponder clones had similar dose-response curves to P30 presented by fixed EBV-B cell lines. Addition of cytokines [interleukin (IL)-1, IL-2, IL-4 and interferon gamma] did not change the proliferative response of the clones, which was consistent throughout an observation period of greater than 4 months. T-nonresponder clones, exposed to P30 on MHC class II-expressing T cells, became not anergic, as they could be restimulated by P30 presented on EBV-B cells. The measurement of a panel of T cell activation markers and adhesion molecules on T-responder and T-nonresponder clones revealed a higher expression of the CD28 molecule on the T-nonresponder clones. The data suggest that freshly cloned T cells can be differentiated by peptide presentation on classical (PBMC, EBV-B cells) or non-classical APC (class II+ T cells), and that this discrimination is further underlined by different levels of adhesion molecules.

Human CD4+ T-cell clones specific for AA 947-67 of tetanus toxin (P30) can be differentiated into clones that proliferate to peptide presentation by activated, MHC class II expressing T cells (T-responder clones) and clones which do not proliferate to peptide-pulsed T cells (T nonresponder). An analysis of the cytokine pattern of these clones revealed that T-responder clones correspond to the Th0 helper phenotype, as these T cells secreted IL-2, IL-4, IFN-γ and TNF-α upon stimulation with autologous EBV-transformed B (EBV-B) cells pulsed with P30. In contrast, T-nonresponder clones secreted a Th2 (IL-4, low IL-2 and low IFN-γ) or a Th1 cytokine pattern (low IL-4, high IL-2, IFN-γ levels) to peptide presentation by B cells. A comparison of EBV-B cells with activated, MHC class II expressing T cells as antigen-presenting cells (APC) revealed that T-cell clones, which failed to proliferate to T cells as APC, secreted cytokines upon peptide stimulation with T-APCs. This demonstrates that the peptide-MHC complex is recognized but that T-APC do not provide sufficient signals to induce proliferation of Th1- or Th2-like clones. Moreover, the use of T cells as APC induced a shift in the cytokine pattern of Th0 clones, as the clones produced less IL-2 and IFN-γ, but still substantial amounts of IL-4, thus favoring a cytokine profile inducing an allergic inflammation., (© 1992 S. Karger AG, Basel.)

Human T cells can express MHC-class II products and were shown to be potential antigen-presenting cells. However, they are unable to capture the antigen and only antigens, which bind to T cell membranes such as the gp120 glycoprotein of HIV, are internalized, processed, and presented by T cells. To better understand the role of T cells as antigen-presenting cells, we established a method which overcomes the lack of antigen capture by T cells. Antigen (tetanus toxoid, TT) or an antigenic peptide of TT (residue 830-843, P2) was coupled to antibodies directed to T cell surface molecules such as CD2, CD4, CD8. Antibody/TT and antibody/P2 constructs stimulated P2-specific T cell clones in the absence of accessory cells, if the antibody recognized a T cell surface structure. Compared to the peptide alone, a 100-500 times lower molar concentration of the antibody/peptide construct was required to achieve a similar proliferative response. T cell stimulation via the constructs involved intracellular processing, as nonspecific, glutaraldehyde fixed T cell lines pulsed with the constructs could present the peptide and processing inhibitors like Leupeptin or Chloroquine inhibited the development of a proliferative response to the constructs. Our data underline the ability of T cells to function as antigen-processing and -presenting cells and show that antibody/antigen or antibody/peptide constructs are able to direct a certain antigen or peptide to a T cell. Antibody/peptide constructs may be interesting tools to better understand antigen processing and to study the consequences of antigen presentation by different cells.