The data presented here demonstrate that the EC50s in response to LPS for peripheral blood monocytes from subjects either heterozygous or homozygous for mutations in CFTR were 100-fold lower compared to those observed for monocytes from healthy control subjects. The increased levels of basal IL-8 secretion seen only from monocytes from CF patients and not those from obligate heterozygotes or healthy controls may be due to the chronic infectious state of the CF patients. Although this could also explain the lower EC50 for monocytes from CF patients in response to LPS, the fact that monocytes from healthy obligate heterozygotes, who had no recent history of infections, show similar results would imply that the enhanced LPS response is a result of CFTR dysfunction. Although the healthy controls did not undergo CFTR genotyping, only 1 in 29 would be expected to be a carrier, and the presence of a carrier among the healthy controls would have minimized the differences that were observed.
It is interesting that this response of monocytes from heterozygotes was of a magnitude similar to that observed for monocytes from CF patients, even though the amount of functional CFTR is decreased by ≤50% in heterozygotes. This is in contrast to the finding that cholera toxin-induced intestinal secretion is 50% less in mice heterozygous for CFTR mutations than in mice homozygous for CFTR mutations (7
) and suggests that CFTR may have more of a threshold effect and not a graded response on IL-8-induced secretion in human monocytes.
Since the difference in EC50
s was seen only with LPS and was not observed in response to IL-1β, the priming of monocyte function would have to be due to either increased numbers of LPS receptors or, alternatively, upregulation of intracellular signaling pathways activated by LPS. LPS binds to serum binding protein and is transferred to CD14 on the cell surface (for a review, see reference 8
). This complex interacts with Toll-like receptor 4 and the accessory protein MD-2, which leads to activation of the MAPKs ERK, p38, and JNK. In addition, LPS activates the I kappa B kinase pathway. The result is activation of multiple transcription factors, including NF-κB. Our data demonstrate that the expression of both CD14 and Toll-like receptor 4 on monocytes did not differ between healthy controls and CF patients. Hence, the lack of difference between CF patients and healthy control subjects in response to IL-1β implies that in the setting of CFTR alleles there is an alteration either in MD-2 or in downstream factors that lead to the activation of NF-κB-inducing kinase. It is at this point that the LPS and IL-1β signaling pathways converge (8
The phosphorylation of ERK in monocytes from patients either homozygous or heterozygous for CFTR mutations occurred with lower doses of LPS than that seen in healthy control monocytes. It should be noted that 1 ng/ml was the dose found to cause 50% of maximal IL-8 secretion (EC50
), as shown in Fig. . Hence, the changes in MAPK activity seen at this dose and higher doses parallel the alterations in IL-8 secretion. Although similar trends were observed for p38, the differences were not statistically significant. How CFTR may regulate MAPKs in monocytes to modulate IL-8 levels is unknown. Whether MD-2 or other components of cell membrane signaling are altered in the setting of CFTR dysfunction remains to be determined. It has been shown in epithelial cells expressing defective CFTR that, following stimulation with P. aeruginosa
, IL-8 expression is increased whether one examines cell lines (5
), naïve human CF airway grafts (23
), or CF knockout mice (24
). In addition, constitutive activation of NF-κB leading to IL-8 synthesis occurs in the setting of either an impairment in CFTR chloride channel function or the accumulation of ΔF508 CFTR protein in the endoplasmic reticulum, which leads to cell stress (27
). In addition, the levels of cytosolic IκBα, which inhibits the activation of NF-κB, are decreased in ΔF508 CF bronchial tissues as well as cultured CF bronchial gland cells (20
). Taken together these data indicate that CFTR dysfunction is associated with activation of NF-κB in epithelial cells, although the precise mechanism remains unknown. Although a similar effect on NF-κB activation may be expected in monocytes/macrophages, this remains to be tested.
The fact that peripheral blood monocytes from healthy obligate heterozygotes demonstrate enhanced LPS-induced production of IL-8 has two implications. First, these data would suggest that a single allelic mutation is sufficient to modulate LPS-induced cytokine secretion. CFTR dysfunction has been shown to lead to other abnormalities in obligate heterozygotes, as evidenced by elevated sweat chloride concentrations (6
) and diminished levels of pancreatic juice bicarbonate and chloride secretion as well as diminished water flow in infants (12
); resistance to cholera toxin-induced intestinal fluid secretion in CF knockout mice (7
); and the association of the CFTR dysfunction with the development of CF-related diseases in humans, such as sinusitis (25
), the congenital bilateral absence of the vas deferens (3
), and chronic pancreatitis (4
). The second implication of these data may relate to the high frequency of single allelic mutations in the general population. The enhanced response to LPS may be advantageous in priming the innate immune response, which leads to the rapid eradication of bacteria. In the setting of overt CF, persistent bacterial infections due to viscous secretions and poor clearance would result in continued excessive secretion of cytokines by these primed monocytes and would explain, at least in part, the excessive host inflammatory response in patients with CF.