Biophysical characterization of macrophage migration enhancement factor (MEF)

Macrophage migration enhancement factor (MEF), a lymphokine produced in the spleen by suppressor-like lymphoid cells, may be an important immunoregulatory molecule of macrophage function. MEF appears to be a potent positive chemokinetic factor and is unusual in that it lacks chemotactic activity. To aid in the development of a purification scheme for MEF we have employed biophysical characterization techniques to define its physical properties. Using the technique of velocity sedimentation in isokinetic sucrose gradients, the S_20w for MEF was determined to be 2.25. The Stoke's radius for MEF was determined by Sephadex G-100 gel filtration to be 28.9 Å. From these measurements the D_20w was calculated to be 7.55 × 10⁻⁷cm²/sec, the mol. wt was calculated to be 28,000, the frictional ratio (|/|₀) was calculated to be 1.45, the axial ratio was calculated to be 1:8, and the dimensions of the molecule were estimated to be 2 × 160 Å. Using the technique of isoelectric focusing in liquid density gradients, the isoelectric point for MEF was estimated to be 8.8. We have also determined by enzyme treatment that MEF is resistant to DNase and RNase and susceptible to proteinase K and L-fucosidase. In addition, MEF partitioned to the aqueous phase during methanol-chloroform extraction procedures. MEF was inactivated at pH 12; at 100°C MEF was stable for 10 min but was inactive after 1 hr. Collectively, these data will facilitate the development of a purification scheme for MEF which will ultimately permit the analysis of the molecule and its function.     

Macrophage-Agglutinating Factor Produced In Vitro by BCG-Sensitized Lymphocytes

Supernatant fluids from cultures of BCG-sensitized rabbit lymph node and spleen cells contained a factor that strongly agglutinated normal rabbit alveolar macrophages within 3 min at room temperature. In contrast, fluids from nonsensitized cell cultures did not agglutinate normal rabbit alveolar macrophages. This factor was designated macrophage-agglutinating factor (MAgF) because it is similar to the previously described factor found in lung lavages of rabbits exhibiting a BCG-induced pulmonary delayed hypersensitivity reaction. The kinetics of MAgF production in vitro by sensitized lymph node cells and its inhibition by puromycin and actinomycin D suggest active synthesis; sensitized spleen cells exhibited kinetics resembling release rather than synthesis. Studies on purified lymphocyte and macrophage populations from sensitized spleen and lymph nodes indicated that lymphocytes are responsible for MAgF production. However, MAgF production was not induced in normal cells incubated in vitro with concanavalin A or phytohemagglutinin. Fractionation of cell culture supernatant fluids in Sephadex G-100 or Ultrogel AcA-34 clearly separated MAgF from migration inhibition factor; MAgF was present in the void volume of the eluates, suggesting a molecular weight of over 400,000, whereas migration inhibition factor was recovered in the same peak as albumin. The role of MAgF in vivo is unknown, but it is postulated that it may cause the adherence of macrophages during granuloma formation.     

Musculoskeletal infection, microbial adhesion, and antibiotic resistance

Osteomyelitis and intra-articular infection are septic diseases that present pathogenic features characteristic of molecular mechanisms involving adhesion to substrata. In this review, mechanisms of microbial adhesion to bone and cartilage as substrata are presented and related to host tissue response and to antibiotic treatment.     

Priming of rabbit alveolar macrophages for enhanced oxidative responses by herpes simplex virus type 2 infection.

The effect of herpes simplex virus type 2 (HSV-2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 microgram PMA/ml) or latex (250 micrograms/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex-elicited but not PMA-elicited CL response. HSV-2 infection (moi = 1.0) of infant AM for 2 hr at 37 degrees C did not alter the PMA or latex-elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV-2 infection (moi = 1) of adult AM for 2 hr further increased both PMA- and latex-elicited CL responses. Increasing the moi to 10 inhibited both PMA- and latex-elicited CL responses. Incubation of uninfected control and HSV-2 infected adult AM for 18 hr at 37 degrees C resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV-2 alone failed to elicit a CL response in adult AM. Infection with heat-inactivated HSV-2 (moi = 1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV-2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV-2 infection for an increased CL response elicited by either PMA or latex.     

Macrophage migration inhibition induced by MDP, LPS, PMA, and MIF/MAF: reversal by macrophage migration enhancement factor (MEF), L-fucose, L-fucosyl BSA, D-mannose, and D-mannosyl BSA

Our data establish that migration inhibition factor (MIF) and migration enhancement factor (MEF) mutually neutralize the effect of each other in a concentration-dependent manner. The monosaccharides L-fucose and D-mannose were also shown to reverse MIF and additionally to stimulate alveolar macrophage (AM) migration in the absence of MIF. The specific activity of these sugars was increased 200-fold when conjugated to bovine serum albumin (BSA). Macrophage activation is usually observed concurrently with migration inhibition when macrophages are incubated with MIF preparations. Migration inhibition occurred also when AM were incubated in the presence of known metabolic activators (MDP, PMA, and LPS). It was found that L-fucose, D-mannose, L-fucosyl BSA, and D-mannosyl BSA could reverse migration inhibition caused by MIF as well as by these metabolic activators. These observations suggest that reversal of MIF by L-fucose is unexplained solely on the basis that L-fucose is functioning as a competitive inhibitor; instead, they suggest that MEF and the above sugars and their conjugates stimulate AM through a receptor system different from the MIF receptor. These observations support the concept that MEF is an important macrophage modulator in CMI responses.     

Selective suppression of granuloma formation and delayed hypersensitivity in rabbits

The relationship between dermal delayed hypersensitivity (DH) and granulomatous hypersensitivity was studied in rabbits sensitized with killed mycobacteria. Specific antigen challenge of sensitized animals resulted in extensive pulmonary granulomatous inflammation and induced suppression of both dermal DH and dermal granuloma formation. Whereas suppression of DH was concomitant with pulmonary granuloma formation, as is the case in a number of granulomatous diseases, a causal relationship between the two did not exist. Both DH and dermal granulomatous hypersensitivity were significantly suppressed whether or not the antigen challenge was of a granulomagenic (particulate) or nongranulomagenic (soluble) form. The data presented indicate that granulomatous hypersensitivity and DH are selectively suppressed with regard to different anatomical sites.