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Mast cells are preferentially found in large numbers at the interface of the host and environment and are primarily known for their capacity to secrete significant amounts of pharmacologically active mediators (4-6, 8, 9, 12, 14). They are also an extremely heterogeneous collection of cells exhibiting striking differences in morphology and responsiveness to agonists (13, 17, 18, 32). These distinct traits are seen among mast cells from different species and even among mast cells from different sites in the same animal (17, 33). Recently, several in vitro and in vivo studies involving mucosal-type and connective tissue-type rodent mast cells have suggested that mast cells play a critical and hitherto unrecognized role in host defense against infectious agents (3, 7, 15, 23, 24). There exist two subpopulations of human mast cells, some containing measurable amounts of the neutral proteases tryptase and chymase, and others containing tryptase but no chymase (18, 34). In the present study, we sought to investigate if cultured CBHMC expressing tryptase but not chymase (2, 29, 31) were capable of recognizing and responding to different gram-negative and gram-positive bacteria.

Binding of CBHMC to gram-negative and gram-positive bacteria. CBHMC were obtained following 10 weeks of culture of cord blood progenitor cells in the presence of stem cell factor and interleukin 6 (11). The cells were determined to be >97% CBHMC from their morphological appearance following May-Grünwald-Giemsa staining and by their positivity for tryptase (10, 28, 29). To examine the binding of CBHMC to various bacteria, we exposed monolayers of CBHMC grown on eight-chamber tissue culture slides (Nalge Nunc, Naperville, Ill.) to suspensions of various bacteria at a ratio of 100 bacteria to 1 mast cell (22). After 1 or 3 h of incubation, nonadherent bacteria were removed by gentle rinsing, and the CBHMC monolayers were fixed and stained. The number of adherent bacteria on at least 400 CBHMC was determined by light microscopy. Adherence to the following clinical bacterial isolates was investigated: Citrobacter freundii CI125, Streptococcus faecium CI126, Staphylococcus aureus CI127, and Klebsiella pneumoniae CI128. Also included in this study was the laboratory Escherichia coli strain ORN103(pSH2), which expresses recombinant type 1 fimbriae and its FimH-negative derivative, ORN103(pUT2002) (21, 22).

CBHMC-mediated phagocytosis and killing of bacteria. The morphological aspects of the interaction between CBHMC and selected bacteria were examined by scanning and transmission electron microscopy (SEM and TEM, respectively) (22). These techniques showed that mast cells employ protoplasmic protrusions on their surfaces to entrap bacteria. The formation of these mast cell protrusions around E. coli ORN103(pSH2) is shown in Fig. 1. Examination of cross-sections of mast cells after exposure to bacteria revealed a significant number of bacteria encased in vacuoles. A cross-section of mast cells containing several intracellular S. faecium CI126 cells is shown in Fig. 2. It is interesting that whereas most of the S. faecium CI126 associated with the CBHMC appeared to be intracellular, a significant number of the gram-negative bacteria still remained attached on the outside of the cell (data not shown), implying that the rate of internalization of the gram-positive bacteria was markedly higher than that of the gram-negative bacteria. Conceivably, the uptake of S. faecium CI126 is a rapid process triggered spontaneously upon contact with the mast cell membrane, whereas uptake of the gram-negative bacteria could be a more gradual process which is initiated only after the adherence of a critical number of bacteria.

View larger version (99K): [in this window] [in a new window]   FIG. 1.   SEM examination of CBHMC surface following exposure to E. coli ORN103(pSH2). Notice the formation of filopod-like structures employed by the mast cell to grip bacteria.

View larger version (129K): [in this window] [in a new window]   FIG. 2.   TEM examination of cross-sections of CBHMC showing internalized bacteria following exposure to S. faecium CI126. Notice the degraded bacteria (arrowheads) within vacuoles.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Viability of S. aureus CI127 (A and C) and E. coli ORN103(pSH2) (B and D) incubated with CBHMC or HUVEC. Bacteria were added to a monolayer of CBHMC or HUVEC in antibiotic-free and serum-containing medium in wells of a 96-well tray at a ratio of 100:1. The mixture was incubated for 30 min, after which all unbound bacteria were removed and fresh medium was added. At different time points thereafter, the viability of residual bacteria was assessed by solubilizing the cells in each well with Triton X-100, and the number of viable bacteria was assessed by plating onto agar.

CBHMC release of TNF-. Mast cells are renown for their capacity to release a large array of pharmacologically active mediators. Among mast cell mediators, tumor necrosis factor alpha (TNF-) is of particular interest because mast cells are the only cell type in the body known to prestore TNF- and spontaneously to release the cytokine upon activation (16). We examined the capacity of CBHMC to release TNF- following exposure to various bacteria. CBHMC at a concentration of 10⁶ per ml were incubated with 10⁷ bacteria for 0.5, 1, 3, or 6 h at 37°C. After centrifugation (1,000 × g for 10 min at 4°C), cell-free supernatants were collected and TNF- was measured by an enzyme-linked immunosorbent assay (ELISA) method (human TNF- ELISA kit; Genzyme, Cambridge, Mass.). The results were expressed in picograms per milliliter, with the detection limit of the kit being 15 pg/ml. As shown in Table 2, CBHMC elaborated a substantial TNF- response even after 0.5 h of incubation with various bacteria. However, it is not known at this time if this TNF- is actually derived from presynthesized stores or from the rapid translation of untranslated TNF- mRNA. Further incubation with bacteria (1, 3, and 6 h) revealed even greater amounts of TNF- release, reflecting substantial de novo synthesis and release of TNF-. The greatest amounts of TNF- were elicited from CBHMC by S. faecium CI126 and S. aureus CI127, and the least amounts were elicited by K. pneumoniae CI128 and the FimH-negative mutant E. coli ORN103(pUT2002) (Table 2). It is interesting that at 6 h, the amounts of mast cell TNF- elicited by S. faecium CI126 and S. aureus CI127 were comparable to the amount elicited by the combined action of the well-known mast cell agonists phorbol myristate acetate and calcium ionophore. Although on the whole there appeared to be a correlation between CBHMC binding of bacteria and TNF- release (Tables 1 and 2), there were some notable exceptions. For example, the laboratory E. coli strain ORN103(pUT2002) exhibited a low level of binding but elicited appreciable TNF- release from mast cells, especially after 6 h. It is conceivable that during the lengthy incubation period, bacteria could have released agents such as proteases and toxins which can potentially elicit a TNF- response. Indeed, mast cell activation by such bacterial products as cholera toxin and lipopolysaccharide has been demonstrated (19, 20, 25, 26). Nevertheless, the capacity of CBHMC to release large amounts of TNF- following contact with bacteria is relevant to host defense, considering the recent findings for mice showing that the early burst of mast cell-derived TNF- following bacterial challenge was critical to the survival of mice (7, 23, 27).

View this table: [in this window] [in a new window]   TABLE 2.   Time-dependent release of TNF- by CBHMC following exposure to various bacteria and mast cell agonists