Intermediate filament, laminin and integrin expression in lacrimal gland acinar cells: comparison of an immortalized cell line to primary cells, and their response to retinoic acid.

PURPOSE: The goal of this study was to characterize intermediate filament, integrin and laminin expression by rabbit lacrimal gland acinar cells in culture, to determine whether retinoic acid (RA) alters expression of these proteins and to compare primary cells to an immortalized rabbit lacrimal gland acinar cell line using flow cytometric analysis. METHODS: Primary cells, maintained in serum free medium, were exposed to 10(-6) M retinoic acid for 24 hours. Immortalized cells were grown in defined medium with Nu-Serum and exposed to retinoic acid. Cells were labeled with monoclonal antibodies to cytokeratins (AE1, AE2, AE3, AE5, CK10/13, CK18), integrins (alpha(3), alpha(6), alpha(V), beta(1), beta(2), beta(3) and beta(4)), laminin, or vimentin and with FITC-conjugated secondary antibodies. Cells were analyzed for antigen expression by flow cytometry and immunocytochemistry. RESULTS: Primary and immortalized cells expressed type I and type II epithelial cytokeratins (AE1 and AE3), cytokeratin 18, and cytokeratin 3 (AE5) Both cell types were negative to AE2 and CK10/13. Primary and immortalized cells expressed vimentin in culture, with immortalized cells expressing this protein at higher levels. Lacrimal acinar cells appear to synthesize laminin which was detected intracellularly in both cells types. Integrins alpha(6) (CD49f) and alpha(V) (CD51) were expressed by primary and immortalized cells. Expression of integrin alpha(6) was 10-fold higher in immortalized cells compared to primary cells. Retinoic acid increased integrin alpha( V) expression by primary and immortalized cells 1.3-fold and 3-fold, respectively, and caused a slight increase in integrin alpha(6) expression by primary cells. Both cell types also expressed integrins beta( 1), beta(2) and beta(3), but beta(4) was detected only in immortalized cells. Lacrimal acinar cells do not express integrin alpha(3). CONCLUSIONS: Expression of cytokeratins, laminin and integrins by primary and immortalized cells was similar, suggesting that the immortalized cell line is a good model for the study of lacrimal structure and function. Since retinoic acid up-regulated only integrin alpha(V), but not cytokeratins, these cells appear to be highly differentiated. Flow cytometry is a useful method for analysis of protein expression by lacrimal acinar cells.

Chemokines are present in the genital tract of HIV-seropositive and HIV-seronegative women: correlation with other immune mediators.

In this cross-sectional study, 53 cervicovaginal lavage samples (CVL) from 41 women were analyzed for the chemokines interleukin-8 (IL-8), regulated-on-activation normal T-expressed and secreted (RANTES) factor, and macrophage inflammatory protein-1alpha (MIP-1alpha) by enzyme-linked immunosorbent assay (ELISA). IL-8 was detected in 81% of CVL, whereas RANTES was detected in 32%, and MIP-1alpha in 15% of the CVL. The mean levels of IL-8, RANTES, and MIP-1alpha in positive samples were 396 pg/ml, 102 pg/ml, and 34 pg/ml, respectively. IL-8 levels correlated positively with IL-1beta and IgG in a subset of CVL samples. RANTES levels correlated positively with complement protein levels. Additionally, the levels of RANTES, but not MIP-1alpha, reached levels reported in previous studies of the effects of beta chemokines to inhibit HIV replication. These results suggest that measuring chemokines in CVL specimens can provide important information regarding immune responses in the genital tract.

Transfer of host T-cell membrane HLA-DR and CD25 to target cells by human retroviruses.

Many enveloped viruses incorporate host membrane proteins, some of which remain functionally active and significantly affect viral phenotype. We investigated whether human retroviruses can transfer host membrane proteins to target cells. Following incubation with HTLV-I, HLA-DR and CD25 were detected on up to 70% of HPB-ALL cells. Similarly, HLA-DR and CD25 were also detected on cells following incubation with HIV-1. Cyclohexamide or azidothymidine (AZT) had no effect on detection, indicating that binding of virus or infection did not induce expression of these proteins. Detection of host proteins on target cells depended on binding as well as fusion of virus to the cell membrane, indicating that these proteins were inserted into target cell membranes. Virions also transferred host proteins to peripheral blood mononuclear cells (PBMCs). This aberrant transfer of T-cell activation proteins by HIV or HTLV may alter the state of activation or proliferation of target cells and contribute to the immunodeficiencies associated with infection by these viruses.

A potent activator of HIV-1 replication is present in the genital tract of a subset of HIV-1-infected and uninfected women.

OBJECTIVE AND DESIGN: To determine whether the female genital tract contains factors that affect HIV-1 replication. Cervicovaginal lavage (CVL) samples were collected from HIV-1-seropositive and seronegative women and added to cell cultures. METHODS: HIV p24 production was used to measure the effects of CVL on replication of HIVMN in a T-cell line, of a primary isolate in peripheral blood mononuclear cells, or on HIV expression by the latently-infected monocytic U1 cell line. The effects of CVL on the HIV long terminal repeat (LTR) were determined in 1G5 T cells by measuring luciferase activity. RESULTS: Increased replication of HIVMN and a primary isolate were observed in T cells cultured with CVL samples from three out of 38 HIV-infected women, one out of four uninfected high-risk women, and none of 12 low-risk women. The CVL factor increased replication by enhancing virus expression via activation of the HIV LTR. The HIV-inducing activity was highly stable to heat but was sensitive to proteases, indicating that the activity was distinct from heat-labile cytokines including tumour necrosis factor-alpha. CONCLUSIONS: This is the first study to show that a factor which can stimulate HIV-1 replication is present at biologically active levels in the reproductive tract of women. This factor could potentially affect sexual or vertical transmission of HIV-1 by altering genital tract virus load or virus expression.

Detection of HLA-DR associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type 1.

This study determined whether HLA-DR was incorporated into human immunodeficiency virus type 1 produced in vivo or by primary cultured cells. HLA-DR was associated with virions from primary isolates, macrophage cultures, and blood plasma. These results represent the first demonstration of major histocompatibility complex molecules associated with an in vivo source of virus.

Susceptibility of HIV-1 plasma virus to complement-mediated lysis. Evidence for a role in clearance of virus in vivo.

This study was undertaken to directly assess the susceptibility of HIV-1 plasma virus to C-mediated lysis. Plasma from HIV-infected individuals was collected and ultracentrifuged over 20% sucrose to isolate virions from plasma components including anticoagulants, which inhibit C activity. Treatment with C alone in the absence of exogenously added Ab caused lysis of virus from all patients (n = 18) (range 14 to 86%). This lysis occurred via the classical C pathway and was not due to cross-reactive Abs in the C source. Protein A bound a fraction of isolated plasma virus and this binding was blocked by purified human Ig suggesting that anti-HIV Abs bound to plasma virus could be responsible for inducing C activation. A portion of virus bound to CR2 on cells in the absence of exogenously added C indicating that virus activated C in vivo. C levels from six of six patients were determined to be sufficient to lead to lysis of virus in vivo. Since plasma virus appeared more sensitive to C than primary isolates, isolated virus was evaluated for the presence of C control proteins. While primary isolate virions contained CD46, CD55, and CD59, only CD59 was detected on plasma virus. The results of this study strongly suggest that C is activated by a portion of plasma virus in vivo due to the binding of Ab. The resultant opsonization plus subsequent lysis may be important routes of clearance and destruction of plasma virus in infected persons.

Elevated levels of iC3b and C4d, but not Bb, complement fragments from plasma of persons infected with human T cell leukemia virus (HTLV) with HTLV-I-associated myelopathy/tropical spastic paraparesis.

Plasma levels of complement (C) fragments iC3b, C4d, and Bb from human T cell leukemia virus (HTLV)-positive subjects with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) were analyzed by EIA. Both iC3b and C4d levels were significantly elevated in persons with HAM/TSP. These levels were similar to those in patients with human immunodeficiency virus (HIV) infection or rheumatoid arthritis (RA), who are known to have increased C fragments. Bb levels in persons with HAM/TSP wer unaffected, suggesting that C activation occurred only via the classical pathway. This differed from findings in HIV-infected or RA patients, who had elevated levels of Bb. The results showed an increase in C activation in persons with HAM/TSP and activation via the classical pathway, likely mediated by virus or immune complexes. It is possible that the C activation observed in these subjects contributed to the inflammatory pathogenesis of HAM/TSP.

Efficacy of HIV-specific and 'antibody-independent' mechanisms for complement activation by HIV-infected cells.

Previous studies in this laboratory have shown that efficient activation of complement (C) on HIV isolates and HIV-infected cells requires the binding of specific anti-HIV antibodies, while other investigators have observed 'antibody-independent' C activation. In an attempt to clarify these disparate findings, we investigated the effect of several variables on C activation by HIV-infected cells using flow cytometric analysis of C3 deposition. Antibody-mediated C activation using pooled sera from infected persons or human MoAbs directed against the V3 region of gp120 was always substantially higher than activation without antibody. Normal human serum (NHS) from a subset of HIV antibody-negative donors did, however, induce low levels of C3 deposition. Differences in C3 activation between the various NHS did not correlate with total haemolytic C levels or mannose-binding protein (MBP) levels. IgM isolated from NHS that induced high levels of C activation was at least partly responsible for the 'antibody-independent' C activation. Although there appeared to be a correlation between NHS that induced C activation and the presence of anti-blood type B IgM, absorption of anti-B did not abrogate the C3 deposition. Additionally, MoAb to the B antigen did not induce C3 deposition. These studies show that IgM in sera from HIV-uninfected donors can induce C3 deposition on HIV-infected cells, but that specific antibody-dependent C activation is substantially more efficient. Therefore, 'antibody-independent' C activation on HIV-infected cells may, in some cases, be more accurately described as HIV-cross-reactive antibody-dependent C activation.

Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis.

Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 micrograms/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 micrograms/ml (Ran-lo) or 143 micrograms/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 micrograms/ml, or (c) cimetidine (Cim) at 107 micrograms/ml, all in the drinking water on days 5-24; (3) IL-2 (1.5 x 10(3) Cetus U i.p. every 8 h on days 10-14 and 20-24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (51Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no difference observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy.

Effects of cancer immunotherapy with indomethacin and interleukin-2 on murine hemopoietic stem cells.

We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumor-bearing mice subjected to interleukin-2 (IL-2) therapy alone or in combination with chronic indomethacin therapy have any detrimental effects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols on CFU-S numbers in host hemopoietic organs. Effects of in vitro-generated LAK cells (normal C3H/HeN mouse splenocytes cultured with 1000 units IL-2/10(6) cells for 72 h) on BM CFU-S were examined by incubating macrophage-depleted BM cells with LAK cells at 1:2.5 and 1:5 BM:LAK cell ratios or with LAK cell supernatant for 4 h. The cells were washed and subsequently injected into irradiated mice. Irradiated mice were also reconstituted with BM cells or LAK cells incubated alone. Spleen colonies were scored macroscopically and microscopically on day 7 after reconstitution of lethally irradiated mice with the various cell combinations. A comparison of colony numbers produced by LAK and BM cell mixture revealed that LAK cells at either dose had no suppressive effect on the colony-forming ability of BM at the macroscopic and microscopic levels of analysis. The supernatant of cultured LAK cells had a minor suppressive effect on colony formation at the macroscopic but not the microscopic level of analysis, indicating the presence of one or more suppressive factors capable of mediating a short-term inhibitory effect. In the immunotherapy experiment, C3H/HeN mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells received either vehicle alone (controls), IL-2 (1.5 x 10(4) Cetus units i.p. every 8 h on days 10-14 and days 20-25), or chronic indomethacin therapy (10 micrograms/ml in drinking water from day 5 onwards) plus IL-2 as above. Animals were killed 24-25 days after tumor transplantation to examine: (a) the number of metastatic lung nodules; (b) the effects of co-incubating therapy-generated splenic effector cells with normal BM cells for 4 h on BM CFU-S, and (c) the CFU-S content of host BM and spleen. Results revealed a drop in spontaneous lung metastases from a mean of 50 in control mice to 18 with IL-2 therapy alone, and to 5 with chronic indomethacin therapy plus IL-2 therapy. Splenocytes from normal and tumor-bearing control or treated mice, when incubated with normal BM, had no effect on spleen colony formation at the macroscopic level.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunotherapy of mammary adenocarcinoma metastases in C3H/HeN mice with chronic administration of cyclo-oxygenase inhibitors alone or in combination with IL-2.

In this study the efficacy of treatment of two cyclo-oxygenase inhibitors, ibuprofen (Ibu) and indomethacin (Indo), are compared in the immunotherapy of metastasis designed to reverse prostaglandin E2 (PGE2)-mediated inactivation of interleukin-2 (IL-2)-dependent host killer cell lineages. These agents were tested either alone for the prevention of metastasis or in combination with IL-2 for the eradication of established metastasis. C3H/HeN mice were placed on chronic oral Ibu (CIbT; 200 and 600 micrograms/ml of water) or Indo (CIT; 10 micrograms/ml) 5 days after s.c. transplantation of 5 x 10(5) metastatic C3L5 mammary carcinoma for the prevention of spontaneous lung metastases. They showed intolerance to Indo at a dosage of 14 micrograms/ml, which was well tolerated by other mouse strains in previous studies, but tolerated the Ibu dosages used. Control and treated mice were killed on day 30 to score metastatic lung colonies, to evaluate killer activity in splenocytes against natural killer (NK)-sensitive YAC-1 lymphoma or NK-resistant C3L5 adenocarcinoma and 8911 lymphoma targets, and to phenotype the surface markers of killer cells. CIbT and CIT alone at the above dosage significantly reduced the number of lung colonies, retarded local tumor growth and restored NK activity of splenic killer cells expressing AGM-1+, Thy-1-, Lyt-2- phenotype. To treat established lung metastasis, mice bearing 15-day C3L5 transplants were given CIbT or CIT alone or in combination with two 4-day rounds (days 20-23, 31-34) of IL-2 (15,000 Cetus units, i.p. every 8 h) and were killed on day 35 to score lung colonies and characterize splenic killer cells. CIbT or CIT alone reduced the number of spontaneous lung metastases and restored anti-YAC-1 killer function of splenocytes with NK-like phenotype (AGM-1+, Thy-1-, Lyt-2-); some anti-C3L5 killer function was also generated in the high dose Ibu group and the killer cell showed AGM-1+, Thy-1+ and Lyt-2+ phenotype. Combined therapies with CIbT or CIT plus IL-2 were more effective in reducing metastases and promoting killer cell function, the best results being achieved with high dose Ibu+IL-2. All killer cells expressed AGM-1 and Thy-1. In addition, C3L5 killer cells also expressed Lyt-2, suggesting T-cell stimulation. PGE2 synthesis in the host was inhibited by at least 50% in mice subjected to CIbT or CIT.(ABSTRACT TRUNCATED AT 400 WORDS)