The present paper shows that unstimulated B cells from old mice make more TNF-α than young B cells. This can be accounted for primarily by increased TNF-α in old FO B cells, while ABC and MZ B cells do not have a significant increase in TNF-α. Importantly, we demonstrate here that there is an inverse relationship between the amount of TNF-α made by B cells and the ability of these cells to be stimulated in vitro by this cytokine and/or other mitogenic stimuli, e.g. LPS. Moreover, the results herein suggest a connection between the defective B cell response and the increased inflammatory conditions observed in aging. Our hypothesis that the increased inflammatory response in old mice negatively impacts B cell function has been directly demonstrated here in vitro and we have shown that old B cell response can be restored, at least as measured in vitro, by anti-TNF-α given in vivo.
Our results have been obtained in BALB/c mice which is a strain widely used in aging studies. Because we do not perform experiments with transgenic or KO mice, we do not have the necessity to use C57BL/6 mice. As the cells studied here are not antigen-experienced cells, which appear to be expanded in old C57BL/10 (25
), the data we report here should be applicable to both BALB/c and C57BL/10.
TNF-α can positively or negatively modulate immune responses. It is a potent enhancer of both T-dependent antibody responses and T cell responses against pathogens (11
), arguing that the ability of B cells to produce pro-inflammatory cytokines is positive, especially in younger individuals, as in response to antigenic challenge/stimulus. TNF-α has been discovered as a cytokine that could kill tumor cells, but it can also contribute to tumorigenesis by mediating the proliferation, invasion and metastasis of tumor cells, and it is an autocrine growth factor for a wide variety of tumors (34
). In general, inflammation is a protective response of the body to infection. However, increased plasma levels of TNF-α, which contribute to the chronic, low-grade inflammation typical of old age, can have deleterious effects as are implicated in the pathogenesis of several disabling diseases of the elderly, such as type II diabetes mellitus (35
), osteoporosis (36
), Alzheimer’s disease (37
), rheumatoid arthritis (38
), and coronary heart disease (39
We here show that TNF-α stimulation of B cells induces TTP, which is a negative regulator of the stability of mRNA for cytokines and transcription factors (18
). We have previously shown that TTP mRNA and protein levels are higher in old as compared to young stimulated B cells (18
), suggesting that TTP induction in B cells may help to down-regulate the production of inflammatory cytokines, and may contribute to an autocrine negative feedback loop to keep levels of TNF-α and perhaps other pro-inflammatory cytokines below toxic/cell death amounts. As a side effect, TTP reduces optimal B cell immune responses, down-regulating E47, AID and class switch. Because TTP levels are also higher in old as compared to young unstimulated B cells, the higher levels of TNF-α mRNA in old versus
young unstimulated B cells can be due to increased transcription. Our model, shown in , emphasizes that the increased autocrine TNF-α released by aged B cells impairs their function.
Model for differential activation of CSR in B cells from young versus old mice
Healthy aging results not only from the ability to control/make less distructive inflammatory responses, but also from the ability to mount effective anti-inflammatory responses. If chronic inflammation prevails, frailty and common age-related pathologies may occur (20
). Therefore, it is important to understand the regulation of inflammatory signaling pathways in order to develop effective therapeutic strategies to fight age-related immune and inflammatory diseases and in particular, as we have shown here, for optimal B cell functional responses.
In conclusion, results herein may indicate that B cells make TNF-α and therefore contribute to the systemic modification of the cellular microenvironment typical of old age. We show that unstimulated B cells from old mice make more TNF-α mRNA and protein than B cells from young mice, but after stimulation the old make less than the young, i.e. old B cells are pre-activated and refractory to further stimulation. Unstimulated follicular B cells, which represent the major splenic B cell subset, make more TNF-α in old than in young mice, but after stimulation they make less, thus recapitulating the results obtained with the whole population of B cells. If B cells are pre-incubated with TNF-α before stimulation with LPS, both young and old B cell responses are inhibited. B cells can in fact be induced by TNF-α to secrete IgA but not IgG, and this response is down-regulated in old B cells, emphasizing the importance of unique stimuli for a complete evaluation of the aged B cell response. The inhibiting effect of pre-incubation with TNF-α is more pronounced with longer TNF-α incubation times. This inhibitory effect correlates with the induction of TTP, a physiological regulator of mRNA stability of the transcription factor E47, crucial for CSR, down-regulated in old B cells. Finally, anti-TNF-α antibody increases the LPS response in young and more significantly in old cultured B cells. Moreover, anti-TNF-α antibody given in vivo increases B cell function in old but not in young FO B cells stimulated in vitro. Taken together, our results show that inflammation and B cell function are inversely related in old mice and these studies should help further understanding of the mechanisms leading to reduced antibody responses in aging. These will also help to design new possibilities for novel therapeutic approaches for age-related immune diseases.