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Species differences in NO formation by rat and hamster alveolar macrophages in vitro
Abstract:
Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.
Insights
Hamster alveolar macrophages (AM) do not produce nitric oxide (NO) or express inducible NO synthase (iNOS) protein, unlike rat AM, despite both species expressing iNOS mRNA. This highlights species-specific differences in NO regulation in rodents.
Area of Science:
- Immunology
- Cell Biology
- Toxicology
Background:
- Nitric oxide (NO) is a key regulator of biological functions, with alveolar macrophages (AM) implicated in pulmonary injury.
- While NO production and inducible NO synthase (iNOS) expression are known in rat macrophages, this pathway's existence in other species is debated.
- Rodent models, like rats and hamsters, exhibit differing pulmonary responses to inhaled substances, necessitating investigation into their cellular mechanisms.
Purpose of the Study:
- To investigate and compare nitric oxide (NO) production and inducible NO synthase (iNOS) expression in alveolar macrophages (AM) from rats and hamsters.
- To determine if hamster AM possess the capacity for NO generation and iNOS protein expression, contrasting with established rat AM responses.
- To elucidate species-specific differences in iNOS regulation and NO generation relevant to pulmonary toxicology research.
Main Methods:
- Alveolar macrophages (AM) from rats and hamsters were stimulated in vitro with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha).
- Nitrite levels, a stable marker of NO, were measured using the Griess reaction.
- iNOS expression was assessed via immunocytochemistry, Western blot analysis, and reverse transcriptase-polymerase chain reaction (RT-PCR).
Main Results:
- Rat AM demonstrated dose- and time-dependent NO production upon stimulation with LPS and/or IFN-gamma, confirmed by iNOS protein expression.
- Hamster AM failed to produce detectable NO or express iNOS protein under identical stimulation conditions.
- Both rat and hamster AM exhibited inducible iNOS mRNA expression following LPS and IFN-gamma treatment, indicating a post-transcriptional regulatory difference.
Conclusions:
- Hamster AM, unlike rat AM, lack the ability to translate iNOS mRNA into functional protein, thus inhibiting NO generation in response to common stimuli.
- Significant species-specific differences exist in the regulation of iNOS and NO production between rats and hamsters.
- These findings underscore the importance of considering rodent species-specific responses in the interpretation of pulmonary toxicology and immunology studies.