Altered in vitro handling of Mycobacterium avium complex by monocytes and serum from HIV(+) patients.

In patients with acquired immunodeficiency syndrome (AIDS), mycobacterial diseases are leading opportunistic infections. The reasons for the peculiar propensity for disseminated infection with Mycobacterium avium complex (MAC) remain unclear. We have previously examined, in detail, the ability of monocytes from healthy donors to take up and kill MAC under both nonopsonic and opsonic conditions. We have now evaluated the in vitro ability of peripheral blood monocytes from HIV(+) patients to take up and kill MAC organisms, and have discovered a reduced ability under both nonopsonic and opsonic conditions. This reduction is due to: 1) apparent defect(s) in the phagocytes themselves, and 2) substance(s) in the HIV(+) serum which actively suppresses phagocyte activity.

Hyaluronan receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages.

CD44 glycoproteins are present on the surfaces of many hematopoietic cells and in some cases can bind hyaluronan, a major component of the extracellular matrix. In the present study, we have found that newly explanted human peripheral blood monocytes (PBMs) exhibit a major CD44 band of 85 kDa, whereas autologous alveolar macrophages (AM phi) express multiple isoforms ranging from 85 to 200 kDa. Within 4 h in culture, PBMs began expressing new CD44 isoforms of 120, 150, and 180 kDa. Newly explanted AM phi specifically bound [3H]hyaluronan (135 cpm/microgram protein), but newly explanted PBMs did not. However, in vitro cultured PBM progressively acquired the ability to bind [3H]hyaluronan and exhibited specific binding of hyaluronan similar to that of AM phi (113 cpm/microgram protein) after 4 days in culture. In both case, the binding of [3H]hyaluronan was specifically inhibited by the addition of monoclonal antibody directed against CD44. AM phi readily degraded [3H]hyaluronan and reached a plateau after 4 days in culture (115 cpm/microgram protein). Newly explanted PBM exhibit no hyaluronan degradation and only a small degradative activity after 4 days in culture (6 to 11 cpm/microgram protein). Thus, CD44 expression and function appear to change as PBM mature in vitro resembling more that found in AM phi.

Human alveolar macrophage mediated vasodilation and the role of arginine compounds.

To determine whether human alveolar macrophages (AM) generate a compound similar to the endothelium-derived relaxing factor, we studied the effect of AM on the isometric response of the pre-contracted rat aorta preparation in the presence and absence of L-arginine or N-substituted L-arginine compounds. Addition of AM to the pre-contracted aorta preparation was ineffective even in the presence of millimolar concentrations of L-arginine. But, AM in the presence of the substituted L-arginine, N alpha-benzoyl L-arginine ethyl ester, significantly increased vasodilation. The enhanced relaxation was associated with an increase in vascular cyclic guanosine 3,5'-monophosphate formation. Hemoglobin and N omega-nitro L-arginine methyl ester are inhibitors of the endothelium-dependent relaxation, and both attenuated the vasodilation elicited by AM. Human AM were found to metabolize N alpha-benzoyl L-arginine ethyl ester to a citrulline derivative. No such metabolism was observed with L-arginine. A specific, high-pressure liquid chromatographic assay for guanidines revealed that the lack of effect of external L-arginine is not due to the presence of an excess amount of endogenous L-arginine in AM. These results demonstrate that nonactivated human AM, unlike rodent macrophages, possess an enzyme system(s) that metabolize(s) arginine derivatives but not L-arginine to a vasodilator, and this vasodilator has properties similar to that of endothelium-derived relaxing factor. This human AM-derived vasodilator may have an important role in regulating airway smooth muscle function.

Nonopsonic uptake of Mycobacterium avium complex by human monocytes and alveolar macrophages.

The uptake of Mycobacterium avium complex (MAC) microorganisms by human peripheral blood monocytes (PBMs) and alveolar macrophages (AMs) is not well understood. We have previously shown, under opsonic conditions, that humoral factors are important in mediating the uptake of MAC by PBMs. However, the receptor-ligand interactions occurring under nonopsonic conditions remain unclear. We compared the uptake of untreated human PBMs and AMs in a serum-free medium with phagocytes treated to remove surface receptors. Removal of complement receptors CR1 and CR3, the Fc receptor (FcR), and the transferrin receptor (TfR) resulted in significantly lower levels of MAC uptake in serum-free medium by both PBMs and AMs. The addition of barley beta-glucan or mannan from Saccharomyces cerevisiae inhibited MAC uptake by untreated phagocytes in a dose-dependent manner. MAC uptake by PBMs or AMs was never completely abrogated by combining treatments (removal of CR1, CR3, FcR, and TfR and adding mannan or beta-glucan), indicating still-unknown mechanisms of uptake under nonopsonic conditions. We conclude that CR1, CR3, FcR, TfR, the mannose receptor, and possibly a separate beta-glucan-inhibitable receptor all may be involved in nonopsonic uptake of MAC by both PBMs and AMs.

O(6)-methylguanine-DNA methyltransferase and uracil DNA glycosylase in human broncho-alveolar lavage cells and peripheral blood mononuclear cells from tobacco smokers and non-smokers.

Because interindividual variations in the activities of DNA repair enzymes may be a risk factor in the pathogenesis of lung diseases, O(6)-methylguanine-DNA methyltransferase (O(6)-MT) and uracil DNA glycosylase (UDG) were measured in broncho-alveolar lavage cell (BALC) and peripheral blood mononuclear cell (PBM) samples from 57 healthy volunteers (25 smokers and 32 non-smokers). According to cotinine determination in 39 cases where serum for this was available, 38% of the self-acclaimed non-smokers had greater than 10 ng/ml of cotinine in their serum. Whether grouped into smokers and non-smokers according to clinical history or by serum cotinine, there were no statistically significant differences between these groups in O(6)-MT or UDG in either of the cell types. However, a tendency towards lower values in smokers was seen. The highest intraindividual variation in O(6)-MT activity was 7-fold, while the highest interindividual variation reached 18-fold. For UDG, the respective values were 24- and 307-fold. Although the distribution of O(6)-MT in BALC was different from that in PBM, the data are consistent with unimodality in both of the cell types. These findings suggest that exposure to cigarette smoke is not entirely responsible for the wide interindividual variation in O(6)-MT and UDG DNA repair activities.

Natural killer cell-mediated lysis of Mycobacterium-avium complex-infected monocytes.

Since the precise mechanism of host responses to infection with Mycobacterium-avium complex (MAC) is unclear and since cytotoxic lymphocytes may be involved in the destruction of cells infected with intracellular pathogens, we investigated the ability of normal peripheral blood lymphocytes to kill MAC-infected monocytes in a short-term isotope release assay. Nylon wool-passed lymphocytes lysed MAC-infected but not uninfected monocytes during a 4-hr assay. Infected monocytes were less sensitive to cell-mediated killing than the standard natural killer (NK) cell-sensitive cell line K562, although the kinetics of lysis were similar. The release of lymphocyte-derived mediators such as tumor necrosis factor, interleukin-2 (IL-2), and interferon-alpha and -gamma could not be implicated as a cause of monocyte death. Through the use of cell-specific monoclonal antibodies plus complement, the phenotype of the effector cell was that of an NK cell (CD3 negative, partially CD8 negative, and CD16 positive). The use of highly purified, negatively selected NK cells confirmed these results. NK cell-mediated lysis of infected monocytes decreased MAC viability, indicating that this cytotoxic activity would not favor dissemination of the organism. The killing of MAC-infected monocytes was reduced by K562 cells, suggesting that these targets shared common recognition/binding structures. These results suggest that NK-cell function may be important in the prevention of or response to MAC infection and may help explain the predilection of AIDS patients to develop widespread disease.

Therapeutic evaluation of free and liposome-encapsulated amphotericin B in the treatment of systemic candidiasis in mice.

Various doses of amphotericin B encapsulated into unilamellar vesicles of 0.1 micron diameter (lip-AMB) (1.0 to 20.0 mg/kg of body weight) were compared with free amphotericin B (AMB) (0.5 to 2.0 mg/kg of body weight) in a murine model of disseminated candidiasis. CD2F1 mice injected intravenously with 3 x 10(5) Candida albicans cells were treated with either single- or multiple-dose regimens. Untreated infected mice had a median survival of 7 days, with all mice dead by 12 days. Single doses of AMB resulted in a median survival range from 18 to 23.5 days, with less than or equal to 38% survival by day 42. Single doses of lip-AMB resulted in 88 to 100% survival by day 42. The multiple-dose AMB regimen provided median survival of only 30 to 33 days, with less than or equal to 38% survival by day 42. The multiple-dose lip-AMB regimen resulted in greater than 90% survival by day 42. With single-dose regimens, lip-AMB levels in plasma were severalfold higher than AMB levels in plasma. By 10 h, at equivalent doses, lip-AMB levels in plasma were much higher, whereas AMB levels in plasma were not detectable. Compared with normal values, the blood urea nitrogen, serum glutamic pyruvic transaminase, serum glutamic oxaloacetate transaminase, and serum lactate dehydrogenase levels were not significantly altered by high doses of lip-AMB treatment. Viable C. albicans was recoverable from the kidneys of some of the lip-AMB-treated mice at day 42. Thus, encapsulation into unilamellar liposomes enhances the antifungal efficacy of amphotericin B while reducing the toxicity normally associated with administration of free amphotericin B.

Differences in uptake of mycobacteria by human monocytes: a role for complement.

We investigated the influence of serum factors on the uptake of various species of mycobacteria by human peripheral blood monocytes (PBM). On the basis of the percentage of PBM involved during in vitro uptake, the mycobacteria were of two distinct groups. The mycobacteria of one group, which consisted of Mycobacterium avium complex and M. chelonae, were taken up by many PBM; the other group, consisting of M. tuberculosis, M. kansasii, M. fortuitum, and M. gordonae, were taken up by fewer PBM. M. scrofulaceum was intermediate to these two groups on the basis of its uptake by PBM. Serum depleted of complement by heating or treatment with cobra venom factor significantly reduced the extent of PBM involvement with M. avium complex, indicating that complement is an important serum component mediating the uptake of M. avium complex organisms. Preincubation of mycobacteria with serum containing 10 mM EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and 10 mM MgCl2 resulted in uptake by a high percentage of PBM, while preincubation in heated serum or serum containing 10 mM EDTA resulted in a significantly reduced percentage of PBM involved in uptake of M. avium complex organisms, indicating that these organisms are activators of the alternative pathway of complement. Incubation of M. avium complex organisms in human serum consumed 51% of the hemolytic complement activity. Parallel experiments indicated that serum had a lesser effect on the uptake of M. tuberculosis. Thus, serum is important in in vitro M. avium complex uptake by PBM; complement has a major role in the effect of serum, but this role is less important with M. tuberculosis.

Inheritance of susceptibility to phototumorigenesis and persistent hyperplasia in F1 hybrids between SENCAR mice and BALB/c or C57BL/6 mice.

SENCAR mice are selectively bred for hypersusceptibility to two-stage chemical skin carcinogenesis. They are also hypersusceptible to UV radiation tumorigenesis with single high-dose, but not chronic low-dose, exposures. In addition, SENCAR mice exhibit an exaggerated and persistent epidermal hyperplasia (due to sustained proliferation of the basal cells) in response to UV-induced tissue damage. In the present study, we have examined the inheritance of susceptibility to both phototumorigenesis and persistent hyperplasia in the F1 offspring of SENCAR mice crossed with either of two inbred strains (BALB/c or C57BL/6) which are relatively resistant to phototumorigenesis. A total of 428 mice from the parental strains and reciprocal F1 crosses were given a single high dose (8.64 x 10(4) J/m2) of UV radiation (FS40 sunlamps) which causes persistent hyperplasia and tumorigenesis in many SENCAR, but no BALB/c or C57BL/6, mice. F1 hybrids between SENCAR and C57BL/6 mice did not develop persistent hyperplasia or skin tumors, which indicates that susceptibility to both traits is completely recessive to the C57BL/6 genotype. In contrast, F1 hybrids between SENCAR and BALB/c mice developed both persistent hyperplasia and skin tumors, although at a much lower incidence than the SENCAR mice, indicating that susceptibility to both traits is only partially (incompletely) recessive to the BALB/c genotype. Thus, in either F1 cross, susceptibility to phototumorigenesis decreased in parallel with persistent hyperplasia. These results are consistent with the hypothesis that the two characteristics are mechanistically related.

Suppression of delayed-type hypersensitivity to radiation [UV, 280-320 nm (UVB)]-induced tumor cells with serum factors from UVB-irradiated mice.

Irradiation--UV, 280-320 nm (UVB)--of the shaved dorsal skin of mice induces a systemic immunosuppression that interferes with the normal rejection of highly antigenic UVB radiation-induced skin cancers. Sera from C3H/HeNCr- female mice, exposed either to a single dose of UVB radiation (8.6 X 10(4) J/m2) or who received a 5-second thermal injury, were evaluated for ability to induce suppression of delayed-type hypersensitivity (DTH) to a syngeneic UVB-induced fibrosarcoma cell line, UV-2240. Sera from both UVB-irradiated and thermally injured donors could suppress the DTH responses of recipients, but only sera from UVB-irradiated donors could induce suppressor spleen cells to UV-2240 in recipient mice. The molecular-weight range of the UVB-induced serum factors is between 1,000 and 10,000.

Role of UVB-induced serum factor(s) in suppression of contact hypersensitivity in mice.

Ultraviolet, 280-320 nm (UVB), irradiation of the shaved dorsal skin of mice results in suppression of the development of contact hypersensitivity (CHS) to antigens applied subsequently to a distant nonirradiated skin site. Serum from BALB/cAnNCr mice exposed to a single dose of UVB radiation (8.6 X 10(4) J/m2) was evaluated for its ability to induce suppression of CHS to 2-chloro-1,3,5-trinitrobenzene (TNCB), a contact allergen, after transfer to normal recipients. Serum from UVB-irradiated donors was capable of inducing immunosuppression only when collected and transferred within a restricted time period, i.e., approximately 2-6 h post irradiation, and at least 400 microliters of serum per recipient was required. Serum from UVB-irradiated donors was sufficient to induce splenic suppressor cells in recipient mice.