The HL-60hca cell line has several notable features. The NOS2 gene is expressed, and high levels of nitric oxide are generated following stimulation with LPS or 1,25-D3. This unusual degree of NO inducibility is of particular interest since the cells have a macrophage-like phenotype. Our data indicate that 1,25-D3 acts to inhibit the growth or viability of M. tuberculosis in HL-60hca cells through an NO-dependent mechanism.
It is puzzling that human macrophages generally fail to produce NO in response to stimuli such as LPS plus gamma interferon in vitro, considering the critical role of NO in defending mouse macrophages against a variety of intracellular pathogens. Since NOS2 expression has been demonstrated in pulmonary alveolar macrophages from humans with tuberculosis, it is conceivable that inducible NO synthesis in humans depends on specific processes of macrophage differentiation in vivo (27
). HL-60 is a human promyelocytic cell line which can be induced to differentiate in various ways: for example, a monocyte/macrophage-like phenotype can be induced by vitamin D or gamma interferon, whereas neutrophil- or eosinophil-like phenotypes are induced by DMSO or alkaline media, respectively (11
). The undifferentiated parent cell line is nonadherent, and the present investigation stems from our chance observation that a significant proportion of the cells spontaneously became adherent after several weeks in culture at 7.5% CO2
, allowing us to select an adherent line that we have termed HL-60hca
. The adherent properties of HL-60hca
cells appear to depend on continual high CO2
concentration, but we cannot exclude the possibility that unknown factors have contributed to differentiation of this phenotype. Importantly, adherence is preserved after storage in liquid nitrogen. HL-60hca
cells express CD14 and readily phagocytose latex particles, whereas they do not reduce NBT. They also ingest M. tuberculosis
. Taking these findings together with the published literature on HL-60 differentiation pathways (2
), we conclude that the HL-60hca
line is essentially macrophage-like.
A striking feature of HL-60hca
cells is that LPS alone is sufficient to induce NO production. This contrasts with the case for undifferentiated HL-60 cells, which require more complex combinations of stimuli to produce NO (20
). Our data confirm that the parent HL-60 cell line can produce NO after stimulation with PMA and LPS in combination but not with either agent alone. Levels of RNI in the culture supernatant of LPS-stimulated HL-60hca
cells can reach almost half the levels obtained from murine macrophages optimally stimulated with LPS plus gamma interferon (34
Several lines of evidence indicate that the LPS-induced NO response in HL-60hca
cells is due to activity of an NOS, which we identify as NOS2. Diaphorase activity is present in LPS-stimulated but not in unstimulated cells, and both this and the inducible rise in RNI are inhibited by l
-NMMA. Following LPS stimulation, there is a marked rise in the rate of formation of [14
C]citrulline from [14
C]arginine, whose time course is consistent with the rate of accumulation of RNI in the culture medium. RT-PCR of LPS-stimulated cells yields a product, confirmed as human NOS2 by sequencing, which is absent in the unstimulated state. Northern analysis confirms that levels of NOS2 mRNA rise appreciably after LPS stimulation. Taking these data together, we can exclude the possibility that the accumulation of RNI is simply due to alternate mechanisms, as has been recently proposed for other experimental systems involving cultured human macrophages (38
Our time course experiments with HL-60hca
cells suggest that the relationship between NOS2 mRNA expression and the rate of NO production is complex. Both Northern and RT-PCR analyses show that levels of NOS2 mRNA rise markedly within 12 h and are maintained for at least 14 days after stimulation with LPS. The daily rate of conversion of arginine to citrulline rises significantly during the 2nd day, reaches a peak during the 5th and 6th days, and declines to resting levels on the 10th day after stimulation. Consistent with the latter observation, the amount of RNI in the culture medium rises progressively for 7 or 8 days and thereafter remains more or less constant. That is, high levels of NOS2 mRNA are accompanied by increasing rates of NO generation in the first few days, but after day 6 the rate of NO generation declines while NOS2 mRNA levels remain high. The decline does not appear to be due to nutrient depletion, since we replaced 10% of the culture supernatant with fresh medium every 24 h, and a similar time course was observed in experiments where the medium was not replaced (data not shown). In other experimental contexts it has been found that NO generation can remain low despite high levels of NOS2 mRNA and NOS2 protein (31
), and there is growing evidence of mechanisms that can modulate the functional activity of NO synthases (17
). Thus, it is interesting to speculate that the eventual decline in rates of NO generation in LPS-induced HL-60hca
cells might be caused by autoregulatory processes on NOS2 gene expression (1
), and further investigation of this phenomenon is warranted.
It is well known that vitamin D has potent immunomodulatory properties (6
), but little attention has been paid to the possibility that one of its important immunological functions may be to enhance expression of the NOS2 gene, despite a previous report that undifferentiated HL-60 cells produce NO when stimulated with a combination of 1,25-D3
plus prostaglandin E1
). We find that 1,25-D3
cells to express NOS2 mRNA and generate NO, whereas cholecalciferol and ergocalciferol do not. The ability of 1,25-D3
to stimulate a detectable NO output at concentrations as low as 1 nM suggests that the observed effects are physiologically plausible. The concentration of 1,25-D3
in normal human serum is around 0.1 nM, but that of its immediate precursor 25-hydoxyvitamin D3
is around 100 nM (12
). A critical point is that 25-hydroxyvitamin D3
is converted to 1,25-D3
within the macrophage, and the rate of conversion increases markedly within pulmonary alveolar macrophages and human monocytes following stimulation with gamma interferon (21
The last observation may be highly relevant to the role of NO in the host defense against human tuberculosis. Although gene knockout experiments with mice have formally identified the NOS2 gene as a potentially important component of the host defense against tuberculosis (24
), whether human macrophages can produce NO at antimicrobial concentrations remains controversial (15
). NOS2 protein is expressed in pulmonary alveolar macrophages from tuberculous patients (27
), raising the possibility that the natural NO response in human tuberculosis depends on factors that have not been adequately explored in vitro. Several lines of evidence point to vitamin D as one of the missing factors. There is a substantial body of circumstantial clinical evidence that vitamin D is protective against human tuberculosis (4
), and we have recently found that polymorphisms in the vitamin D receptor gene are associated with susceptibility to pulmonary tuberculosis in an African population (1a
suppresses the growth of M. tuberculosis
in human monocyte-derived macrophages (13
). Taking these observations together with the present data, we can speculate that 1,25-D3
acts to control tuberculosis in human macrophages by an NO-dependent mechanism.
Our observations on the growth of M. tuberculosis in HL-60hca cells support this proposition. The number of CFU recovered from HL-60hca cells 6 days after infection was significantly reduced as a result of adding 20 nM 1,25-D3 to the culture medium, and the level of RNI generation was increased. Both of these effects of 1,25-D3 were reversed by the addition of l-NMMA. However, l-NMMA had no effect on the recovery of M. tuberculosis or RNI generation compared to control cultures without 1,25-D3. Taken together, these results indicate that ingestion of M. tuberculosis does not directly stimulate HL-60hca cells to produce NO but that cells stimulated with 1,25-D3 inhibit M. tuberculosis via a mechanism that involves NO production.
Several important questions arise from these observations. Are there specific differentiation processes and costimulatory factors that permit 1,25-D3 to induce NOS2 gene expression in natural human macrophages? Is gamma interferon the major stimulus for intracellular generation of 1,25-D3, and thereby NO, in the pulmonary alveolar macrophages of humans with tuberculosis, or are there other important immune stimuli? How does NO suppress the intracellular growth of M. tuberculosis; i.e., does it act directly on the microbe, affecting its survival or its ability to replicate, or is the suppression an indirect consequence of metabolic changes in the host cell that are caused by NO? The HL-60hca cell line provides a tool for exploring these issues and the general question of how the human NOS2 gene is regulated.