Central to the disease process, in the absence of a demonstrated toxin, is the interaction between the microorganism and host cells. In order to examine whether cells undergo detectable, cytopathic changes in response to pathogens, we have undertaken to assess possible interactions between several human pathogens and cultured human cells. Cytoadhesive receptors expressed on cell surfaces are essential for cell-cell interaction and adhesion to the extracellular matrix. Many microorganisms have evolved the ability to use these surface receptors, called integrins, to bind and then to invade host cells. This ability often becomes an important virulence determinant.Lyme disease is the most common arthropod-borne infection in the United States. Over 100,000 cases have been reported to date. The disease is a multisystem disorder with dermatologic, neurologic and rheumatologic manifestations. The spirochete, Borrelia burgdorferi, is the causative agent. Given the neurological complications of Lyme disease, normal human astrocyte cultures were used to assess the possible interaction between cells of the central nervous system and Borrelia burgdorferi. Scanning electron microscopy was used to monitor and to characterize the initial interaction. Cell cultures, determined to be >95% astrocytes using the specific glial fibrallary acidic protein (GFAP) assay, were co-incubated with early log phase Borrelia burgdorferi, washed extensively to discourage non-specific attachment, glutaraldehyde fixed, critical point dried and mounted for field emission scanning electron microscopy at various time points after mixing. A time dependent association between cultured astrocytes and spirochetes was found to range from an 18% interaction after one hour to a 96% interaction after 18 hours. In most cases many spirochetes were visible attached to the surface of each astrocyte. However, this interaction could be considerably reduced by the addition of the RGD- specific disintegrin, flavoridin, to the co-incubation mixture. While scanning electron microscopy revealed the initial surface binding of spirochete to astrocyte, transmission electron microscopy of ultra thin sections was used to assess astrocyte invasion and a fluorescent light microscopy (live/dead kit) assay was used to score astrocyte viability following spirochete attachment and invasion. Although internalized spirochetes were clearly detectable within astrocytes several hours after mixing, neither detectable cytopathic effect nor significant loss of cell viability could be demonstrated when infected cultures were compared with uninfected controls over the same time periods. These laboratory experiments have demonstrated that normal human astrocytes are aggressively targeted and effectively invaded by Borrelia burgdorferi, however, cell damage over the short term appears minimal. This would support the notion that human glial cells may represent a potentially important target of infection by the Lyme disease spirochete. This target may, at the very least, represent a protected site useful to the organism in maintaining chronic infections, if not a site to initiate neural pathology. - Lyme disease, electron microscopy, astrocytes, neural pathology, Borrelia burgdorferi
