Primary Research: Short Communication: Evidence Supporting Rare AIDS-Kaposi's Sarcoma Metastasis In Keeping With Their Vascular Endothelial Evolution

Background It is postulated that the unusual manifestations of Kaposis's sarcoma cells in nonendothelial brain tissues and on eyeballs in advanced acquired immune deficiency syndrome (AIDS) cases are metastasized AIDS-Kaposi's sarcoma cells arising from vascular endothelial cells. Methods Experiments were performed to explore the above hypothesis by testing for intercellular adhesion molecule-1 (CD54 antigens) on cutaneous AIDS-Kaposi's sarcoma cells as well as on AIDS-Kaposi's sarcoma cells isolated from eyeballs as studies have illustrated that, unlike localized Kaposi's sarcoma cells of primary lesions, proliferating Kaposi's sarcoma cells in proximity to primary lesions express a negative or diminished phenotype when evaluated for identical surface antigens. Parallel CD54 antigen tests were done on vascular endothelial cells and monocytes/macrophages as endothelial cells are considered evolutionarily related to Kaposi's sarcoma cells and monocytes/macrophages are ideal CD54 antigen positive controls. Results Our data showed that only AIDS-Kaposi's sarcoma cells of the eyes did not express CD54 antigens. Conclusions We therefore report that our findings support the postulation suggesting AIDS-Kaposi's sarcoma dissemination in advanced AIDS patients in keeping with their vascular endothelial heredity.


Introduction
Recent evidence documenting the preponderance of endothelial cells (ECs) in and about Kaposi's sarcoma (KS) lesions is just one factor that is indicative of primary KS formation from EC defects [1,2]. Consistent with this knowledge is the postulation that the occasional appearances of Kaposi's sarcoma cells (KSCs) in nonendothelial cerebral tissues and on eyeballs in advanced acquired im-mune deficiency syndrome (AIDS) patients are secondary AIDS-KS aggregates, that owe their existence to migrated AIDS-KSCs [1][2][3]. This observation is further refined to the understanding that these seldom and, if any, disseminating AIDS-KSCs are small in population as compared to the majority of AIDS-KSCs, which remain localized, but also that there is the need to characterize the difference between these two populations of AIDS-KSCs in order to an-alytically show that dissemination, possibly, has taken place [4].
According to an extensive histological determination, a direct correlation exists between surface ligand (Ag) expression and proliferation among KSCs [5]. The same analysis also documents a similar correlation between Ag expression and metastasis for an endothelial tumor [5]. These findings suggest that because of correlations and ancestral connections, Ag integrity on AIDS-KSCs is crucial in establishing merit of the metastasis theory particularly as Ag sensitivity is frequently utilized in histopathology not only to diagnose tumor dissemination but also to predict benign to cancerous transformation [5]. Now, when peripheral blood lymphocytes (PBLs) are allosensitized with recombinant interleukin-2 (rIL-2) they develop into cytotoxic lymphokine-activated killer (LAK) cells [6]. These LAK-cells (effectors) are capable of lysing a wide variety of tumors (targets) both in vitro and in vivo through Ag mediated interactions [6,7]. In addition, the Ag mediated interactions of LAK effectors also target and cause cytotoxicity to vascular ECs and monocytes/macrophages (MΦ) [8,9]. It was therefore proposed that the presence of heterogeneous LAK-cell Ags on vascular ECs, MΦ and tumor cells would make it possible to obviate of the complicating injury to ECs and MΦ in LAK-cell immunotherapy via the application of monoclonal antibodies (mAbs) to selectively inhibit EC and MΦ damage [10].
Pursuant to this approach, we incorporated the cold-target inhibition technique [11] in chromium release assays to detect possible heterogeneity of LAK-cell Ags on AIDS-KSCs, vascular ECs and MΦ [10]. Data accumulated then showed that unlike cutaneous AIDS-KS10 cells, AIDS-KS21 cells collected from ocular lesions did not cold-target inhibit EC lysis [10]. This suggested differences in LAKcell Ags of AIDS-KS21 cells versus AIDS-KS10 cells and ECs, specifically, pointing to intercellular adhesion molecule-1 (ICAM-1) or CD54 Ags as studies have repeatedly elucidated ICAM-1 on targets binding to lymphocyte function associated antigen-1 (LFA-1) on LAK effectors in LAK-cell attacks [12]. In this project we explore the metastasis postulation by testing for CD54 Ag presentations on vascular ECs, MΦ and AIDS-KS21/-KS10 cells. We report that our combined data support the postulation suggesting AIDS-KS or epidemic-KS dissemination within the confines of their vascular endothelial heredity.

Results and Discussion
LAK-cell cytotoxicities to tumor cells, virally or microbially infected cells, vascular ECs and MΦ occur via the adhesion of effectors to targets facilitated by LFA-1 Ags on effectors interacting with ICAM-1 or CD54 moieties (Ags) on targets [12]. RH1-38 is a murine mAb, which inhibits LAK-cell cytotoxicity by binding to the homologous β chains or CD18 Ags of LFA-1 [13]. In our present study we treated LAK effectors with RH1-38 in chromium release cytotoxicity assays to test for the presence of CD54 Ags on MΦ, vascular ECs and on both AIDS-KS21 and AIDS-KS10 cells. Our results herein showed that although RH1-38 did not inhibit killing of AIDS-KS21 cells, it concomitantly inhibited rupture of AIDS-KS10 cells, vascular ECs and MΦ (Table 1). Combining this finding with our previous data, where AIDS-KS21 cells failed to cold-target inhibit lysis of vascular ECs, as otherwise demonstrated by AIDS-KS10 cells [10], summarize that AIDS-KS21 cells bore either diminished or negative CD54 Ag composition. Published papers to date have reported CD54 Ags on several other AIDS-KS cell lines unaffected following multiple passages in vitro [14], and that no significant immunophenotyping differences exist between AIDS-KS3, -KS6 and -KS8 cells at comparable stages [15].
In our earlier paper, we specified that AIDS-KS21 cells originated from an eye lesion, whereas, AIDS-KS10, -KS8 and -KS3 cells were cutaneous, visceral and pleural fluid isolates, respectively [10]. Juxtaposed was put the observation that the differences in reactions of AIDS-KS21 cells may be related to location, however, a plausible explanation could not be proposed [10]. In light of recent evidence, we suspect the following histopathogenesis. Kaposi's sarcoma (KS) are widely accepted as EC derivatives of either vascular or lymphatic lineage, but the precise EC progenitor remains unestablished [12,16,17]. Nevertheless, evidence illustrating histological similarities between KSCs and ECs, coupled with EC Ag detections on KSCs, lend credence to this evolution [1, 2,16,17]. KS, by itself, does not behave like other soft tissue sarcomas, which can proceed to metastasize after growing locally; it is a stationary tumor, which arises in numerous sites via multicentric development [1,2,4,16,17]. However, AIDS-KS or epidemic-KS presents an aggressive form capable of drastic histopathogenesis [1, 2,4,16,17]. One manifestation of such aggressiveness is that on rare instances AIDS-KS can involve brain and, or, eyes in advanced AIDS patients [1,2]. It is widely held that since eyes and brain are devoid of lymphatic endothelium, these organs are unlikely sources of primary KS if KSCs are indeed derived from lymphatic endothelium [1,2]. Aside from this insight, research has shown AIDS-KS capable of angiogenesis, metastasis and tumorigenicity in mice [18,19]. Owing to such detail, the occurrences of AIDS-KS in brain and on eyes in propinquity to cranial nerves, minute capillaries and associated blood vessels are attributed to vascular endothelial alteration [1 -3]. And in keeping with this evolutionary profile, occurrences of AIDS-KS in nonendothelial, nonvascularized cerebral sites and on conjunctiva are postulated to be products of AIDS-KS metastasis [1-3]. One investigation reported that al-though AIDS-KS skin biopsies expressed the cell surface proenzyme, procathepsin L, neighboring AIDS-KSCs, which were characterized predominantly by vascular endothelial cell proliferation, as in an angiomatous lesion, did not express the protease, eventhough, adjoining blood vessels stained positively [20]. Another examination found diminished or negative reactions for UEA-1 lectin, HLA-DR and OKM5 Ags on KSCs, which were in adjacent proliferating areas slightly distant from their ECmix surrounding capillaries [5]. In addition, cells of the vascular endothelial tumor, malignant angioendothelioma, which bore the blood group H Ag in premetastatic stage had somehow lost the Ag when metastasis occurred [5]. Further probing revealed lymphatic tumors not to display this differentiating property [5]. Therefore, this invasive phenomenon possibly explains the paucity of CD54 Ags on AIDS-KS21 cells that rendered them unprotected following mAb treatment. Such a course would be consistent with the view that the failure of KSCs to exhibit uniform staining for Ags suggests differences in their proliferation stages particularly as mutated cells are understood to be capable of altering their surface Ag expression [2,17].

rIL-2 activation
In accordance with described protocols, 1 million MNCs/ ml were treated with 1000 units of rIL-2 (kindly donated by Cetus, Emeryville, CA, USA) and immediately incubated under 5% CO 2 /37°C for 4 days to generate LAK-cells [6]. These LAK-cells were then harvested, washed, counted, and their concentrations adjusted to 2.5 million cells/ ml all using CM in preparation for chromium ( 51 Cr) release assays; LAK-cells were our effectors (E).

Cr release inhibition assay
The procedure for 51 Cr release assay was modified due to mAb additions as previously shown [10,11]. Briefly, to 96-well flat-bottom plates (Corning Glassware) were dispensed 0.25 million LAK-cells/well/100 µl. Identical number of these wells then received 100 µl of one of the following reagents: RH1-38 at 1:100 (E:Ab), RH-138 at 1:400 (E:Ab), NMS-1 at 1:100 (E:Ab), and NMS-1 at 1:400 (E:Ab). Plates were then left at 4°C for 30 minutes to permit Ab to E binding. Subsequently, single types of H were pipetted at 50:1 E:H ratio into equal number of E with Ab containing wells at every four predesigned E:Ab ratio. Maximum E strength was measured by incubation of E and H alone at 50:1 ratio; maximum isotope release was measured by incubation of H with 0.1 N HCl alone; and spontaneous release was measured by incubation of H with CM alone. Plates were then exposed to 5% CO 2 / 37°C for 4 hours. The culture supernatants were harvested using the Skatron Titertek System (Skatron A.S. Libergyn, Norway) and counted in a gamma counter (Packard Auto-Gamma-500; United Technologies Packard, Laguna Hills, CA, USA). The percentage (%) specific lysis was calculated by the formula: All determinations were made in at least triplicate and data calculated as mean ± standard error of mean (SEM). Experiments were repeated at least once.

Authors's Contributions
Author 1 initials performed all laboratory procedures, conducted all experimental research, compiled a thorough literature survey, and drafted the manuscript for submission. Author 2 initials provided the funding, facilities, sources of reagents, experimental direction and manuscript peer review. experimental count cpm -spontaneous count cpm maxim ( ) ( ) u um count cpm -spontaneous count cpm ( ) ( )