In this study, exploring mycoplasma-induced inflammation in mast cell–deficient mice unmasked a previously underrecognized contribution of PMNs to local production of histamine. In the context of the hypotheses tested by these studies, some findings were contrary to expectation and others were entirely unanticipated, thereby yielding fresh insights regarding the nature of inflammation in chronic tracheobronchitis and pneumonia. At the start of these studies, the main hypothesis was that M. pulmonis (which is a common respiratory tract pathogen in wild populations of rodents) degranulates mast cells, thereby boosting local inflammation and linking infectious and allergic inflammation in the airway. Although we found no evidence of direct stimulation of mast cells by mycoplasma, our results show that there may indeed be a link between mycoplasma infection and allergic inflammation. However, this link is forged in part by direct, inducible expression and release of the classic allergic inflammatory mediator, histamine, from an unexpected source, PMNs. Some implications of these findings are discussed further below.
Because of the focus on comparing sources of histamine in wild-type and selectively mast cell–deficient mice, these studies establish definitively that mast cells are the chief source of lung and airway histamine at baseline, that is, in mice without respiratory tract inflammation. Although a mast cell origin for respiratory tract histamine will not surprise many investigators, these findings for the first time reveal the extent of the contribution. Levels of free histamine in the airway lumen, as assessed by content in BAL supernatants from +/+ and W-sh
mice, are almost entirely (99%) contributed by mast cells. Given that BAL histamine does not increase in mice after parenchymal lung mast cell populations are augmented by adoptive transfer of BMMCs, it is likely that much or most free airway histamine in healthy mice originates from blood or plasma, like many components of epithelial lining fluid. In turn, most plasma histamine likely was produced in multiple organs. When studied in vitro, mast cells from a variety of sources exhibit a baseline level of histamine release without specific stimulation (20
), as also seen in our BMMC data in . This study's comparisons of serum histamine levels in +/+ and W-sh
mice suggest that the vast majority of serum histamine is from mast cells, a possibility raised previously by studies of IgE-dependent anaphylaxis in mast cell–deficient KitW/KitW-v
). The failure of BMMCs adoptively transferred to the lung to increase BAL histamine levels (as revealed by data in ), despite a large increase in histamine content of whole lung extracts, is consistent with a plasma origin of most BAL histamine in healthy mice.
Our study does not support our original hypothesis that M. pulmonis
degranulates mast cells acutely because live organisms did not affect cultured mast cells and did not increase histamine levels in BAL at early time points after infection in vivo. However, it is possible that the baseline levels of histamine prevented detection of acute, low-level release from mast cells. Unexpectedly, BAL and lung histamine levels rose progressively in W-sh
mice at later time points, especially 1–2 wk after infection when levels came to equal or exceed those in +/+ mice. The histopathological analysis established that these increases were not caused by the appearance of mast cells in W-sh
mice and coincided with progressive worsening of neutrophilic tracheobronchitis and pneumonia. Similarly, we cannot attribute the large increases in W-sh
histamine to recruitment of basophils, which decreased in number as inflammation progressed, as shown by FACS. Indeed, the principal source of histamine appeared to be PMNs, which populated the airway and alveolar lumen in large numbers, and which tracked well with the rise in histamine as revealed by comparing the panels in . As also noted in our prior work with this model (17
), the severity of neutrophilic lung and airway inflammation was greater in W-sh
than in +/+ mice. The difference was especially large by 1 wk, when inflammation in +/+ mice had substantially resolved but in W-sh
mice continued to worsen.
The basis of the difference in neutrophilic response between +/+ and W-sh
mice is not clear. In part it may be because of the higher mycoplasma burden developing in W-sh
mice in the first few days of infection. Later, when mycoplasma burden becomes similar in both types of mice, greater neutrophilia in W-sh
mice may be caused by loss of a suppressive effect of histamine on PMN influx, as suggested by exaggerated PMN flux into a skin pouch model of inflammation in mice lacking HDC (23
). If this is the case, it is conceivable that if not for the production of histamine by PMN, the neutrophilia would be even more exaggerated in W-sh
mice. Despite differences between +/+ and W-sh
mice in the magnitude of the neutrophilic response to a given dose or burden of M. pulmonis
—and notwithstanding higher baseline BAL histamine levels in +/+ mice tending to mask the PMN contribution to histamine at lower levels of neutrophilic inflammation—the present data () establish that airway histamine levels rise markedly in +/+ mice when neutrophil numbers comparable to those seen in mice are provoked by larger initial inocula of mycoplasma. Thus, a major neutrophil contribution to airway histamine is not a phenomenon limited to mast cell–deficient mice. In this regard, the lack of an intrinsic difference between +/+ and W-sh
mice in vivo is underscored by the absence of major differences between naive PMN from W-sh
and +/+ mice in mycoplasma-stimulated histamine synthesis and release in vitro. Greater histamine content in W-sh
mice at the lower mycoplasma dose (0.5 × 106
CFU) was more likely caused by higher numbers of mycoplasma-stimulated PMNs than to intrinsic differences in W-sh
and +/+ neutrophil responsiveness to mycoplasma. Furthermore, the increase in local production of histamine in W-sh
mice infected for 2 wk was sufficiently great to increase histamine levels in serum. This independently supports the validity and importance of the increases in lung and airway histamine. The elevations of serum histamine in W-sh
mice, however, remained well below levels in +/+ animals, with or without infection, providing further evidence that extra-pulmonary sources (perhaps most notably mast cells in the skin) make the largest contribution to circulating levels of histamine in +/+ mice.
The marked increase in airway epithelial remodeling in infected W-sh
mice versus infected +/+ mice, as reflected by goblet cell hyperplasia, also probably relates to more severe and persistent infection in W-sh
). Unlike effects of live mycoplasma on PMN production of HDC mRNA and histamine, effects on epithelial remodeling probably were not caused by live mycoplasma and its products interacting alone with respiratory epithelium and goblet cell precursors. This is because epithelial remodeling, and mycoplasma-stimulated tracheal angiogenesis and lymphangiogenesis, occur only in the context of an inflammatory response and development of mycoplasma-specific antibodies and immune complexes (16
). Indeed, transfer of immune serum to B cell–deficient mice reconstitutes mycoplasma-induced remodeling (16
). The higher burdens of bacterial antigen and higher titers of mycoplasma-specific antibodies in W-sh
) presumably increase the formation of antigen–antibody complexes and inflammation in the airway, thereby augmenting antibody-dependent goblet cell hyperplasia.
These results provide the first evidence that mycoplasma stimulates PMN production of histamine. The significance of this novel finding is not so much that PMNs and other leukocytes can be sources of histamine, because past studies suggested that BM-derived cells other than mast cells and basophils can produce small amounts of this mediator (3
). Rather, the significance lies in direct stimulation of PMNs by a common respiratory pathogen strongly inducing PMN production of histamine, and also in the unexpected magnitude of the effect on histamine levels in airway lumen, lung tissues, and serum. The finding of mycoplasma-induced increases in histamine content of PMNs purified from BM further argues that internalization of histamine from other sources is not a likely explanation of our findings.
Our data also allow a quantitative comparison of histamine levels in PMNs versus mast cells. As shown in , highest expression of PMN histamine (~10 pmol/106 cells = ~1.1 ng/106 cells) was seen in PMNs purified from W-sh BM. Similar levels (~6.4 pmol/106 cells = ~0.70 ng/106 cells) were seen in PMNs from infected lung, as shown in . On the other hand, unstimulated PMNs contained only ~0.04 ng/106 cells. This compares to ~0.64 μg of histamine per 106 BMMCs. Based on these data, there is a 580–910-fold difference between the histamine content of BMMCs and mycoplasma-stimulated PMNs, compared with a 16,000-fold difference between BMMCs and unstimulated PMNs. It is possible that the histamine content of BMMCs does not accurately reflect that of lung and airway mast cells. Nonetheless, the estimates of histamine content obtained by assaying purified PMNs from infected lung fit well with the hypothesis that PMNs are the major source of BAL histamine in the independent measurements shown in . Total histamine content of the 2.4 ml of BAL fluid from infected W-sh mice 7 d after infection was ~16 ng compared with the ~9 ng expected to lie within the observed ~13 × 106 PMNs (based on ~0.7 ng/106 cells observed in PMNs purified from infected lungs), with the difference being accounted for mainly by extracellular, secreted histamine. Thus, in healthy uninflamed lung, which contains resident mast cells but very few PMNs or basophils, mast cells are by far the major source of extracellular and stored histamine. This conclusion receives strong support from , which shows data obtained very early after infection, before major neutrophilic inflammation has had an opportunity to develop. However, in the setting of established mycoplasma-induced tracheobronchitis and pneumonia, the number of PMNs relative to mast cells becomes so great (easily a several thousandfold difference in the airway lumen) that PMN production of histamine, augmented by exposure to bacteria, is substantial, even surpassing the mast cell contribution. This previously unsuspected capability of PMNs was uncovered fortuitously by examining the phenomenon in mast cell–deficient mice. Thus, PMNs become a major source of airway histamine by two principal mechanisms: (a) ramping up histamine production in each cell in response to bacteria, and (b) increasing their numbers to achieve overwhelming numerical superiority over other histamine-producing cells.
The finding that indices of tracheobronchitis and pneumonia in mast cell–deficient mice are diminished by treatment with pyrilamine or cimetidine suggests that neutrophil-derived histamine is proinflammatory in this model. The magnitude of this effect may be underestimated, because more mice died in the untreated control group before lung harvest than in antihistamine-treated groups, thereby precluding assessment of inflammation in the sickest mice. This effect may have prevented the observed reduction in indices of lung inflammation from achieving statistical significance in the group treated with the combination of pyrilamine and cimetidine. In any case, further investigations are needed to determine whether these observations in mycoplasma-infected mice apply to other microbes and other mammals. If human PMNs respond similarly, the histamine produced could contribute to worsening of allergic symptoms in acute respiratory infections, which are associated with PMN influx into the airways and with most exacerbations of asthma severe enough to require emergency treatment or hospitalization (26
). These findings also suggest a mechanism for some of the inflammation and bronchoconstriction in chronic purulent respiratory tract infections and noninfectious neutrophilic inflammation, as in cystic fibrosis (30
) and chronic, cigarette-associated bronchitis (32
Finally, our studies suggest that the mechanism of the increase in histamine production by naive PMNs in vitro involves induction of transcription of the gene encoding the rate-limiting enzyme in endogenous synthesis, HDC. Similar elevations of HDC transcript levels were found in PMNs purified from airways of infected mice. Thus, this mechanism for stimulated histamine production appears to apply in vivo and in vitro. Furthermore, the magnitude of the increase in HDC mRNA parallels that of the increase in histamine production. Thus, the mechanism of the mycoplasma effect on PMNs is likely to involve increased production of HDC, rather than an alternative mechanism, such as slower rates of histamine degradation. This conclusion is consistent with studies suggesting that leukocytes infiltrating foci of allergic inflam mation express HDC transcripts by in situ mRNA hybridization and also contribute to increased HDC activity (25
). Further studies will be required to establish the nature of the signals passed between mycoplasma and PMN, and to dissect intracellular pathways leading to an increase in HDC transcription.
In conclusion, this study shows that PMNs can become a major source of histamine in airway mycoplasma infection, which increases histamine production by up-regulating transcripts encoding the rate-limiting enzyme in histamine production. Thus, histamine is a mediator shared by allergic and infectious varieties of airway inflammation.