Directional migration of leukocytes: their pathological roles in inflammation and strategies for development of anti-inflammatory therapies.

Directional migration of leukocytes is indispensable to innate immunity for host defense. However, recruitment of leukocytes to a site of tissue injury also constitutes a leading cause for inflammatory responses. Mechanistically, it involves a cascade of cellular events precisely regulated by temporal and spatial presentation of a repertoire of molecules in the migrating leukocytes and their surroundings (microenvironments). Here I will summarize the emerging evidence that has shed lights on the underlying molecular mechanism for directional migration of leukocytes, which has guided the therapeutical development for innovative anti-inflammatory medicines.

Human leukocyte migration inhibition factor (LIF) increases polymorphonuclear cell endocytosis.

We prepared supernatants of Concanavalin-A activated human lymphocytes containing high titers of leukocyte migration inhibition factor (LIF). A pool of these supernatants was filtered thorough sephadex 6-100 as well as a pool of supernatants from parallel non activated cultures. A migration assay was carried out for each activated fraction, using as control migration the same fraction from non activated supernatants. In this way we found a fraction from activated supernatants with high LIF activity. We assayed the effect of this LIF containing fraction on a yeast endocytosis assay by polymorphonuclear (PMN) cells. We found that the LIF containing fraction increased the number of endocytic PMN in about 80%. This effect was absent from control supernatant and from other fractions from activated supernatant but without LIF activity. The LIF containing fraction did not increase the average number of endocytosed yeast per cell nor the ability to reduce NBT. The endocytosis enhancing effect was blocked by the specific LIF blocker N-acetyl-D-glucosamine. We conclude that LIF can increase the endocytic activity of PMN cells.

Soluble fragments of the low-affinity IgE receptor (CD23) inhibit the spontaneous migration of U937 monocytic cells: neutralization of MIF-activity by a CD23 antibody.

U937 monocytic cells were found to respond by diminished spontaneous migration when confronted with affinity-purified soluble fragments of the low-affinity receptor for IgE (FcER2/CD23). Unlike B lymphoma cells, U937 cells could not be activated to respond with enhanced DNA synthesis through their membrane-bound CD23 antigen by MHM6, a monoclonal antibody within the CD23 cluster. MHM6 did, however, effectively neutralize the U937-directed MIF (migration inhibition factor) activity contained within the soluble CD23 preparations. The findings not only suggest a role for soluble CD23 as a novel cytokine at sites of inflammation but also indicate different functions for the membrane-bound forms expressed on B cells and monocytes.

Regulation of lymphokine response during reinfection by influenza virus. Production of a factor that inhibits lymphokine activity.

Mononuclear cells, obtained from the spleens and lungs of influenza virus-seropositive C57BL/6 mice at 2 to 4 days after re-infection with homologous virus (strain A/Bangkok/1/79), produced a low m.w. factor in vitro that prevents the biologic expression, but not production, of the lymphokine, leukocyte migration inhibition factor (LIF). The low m.w. factor inhibited LIF activity without destroying the LIF molecule inasmuch as simple dialysis restored lymphokine activity to culture supernatants. Production of the low m.w. factor was observed from 2 to 4 days after re-infection, at which time the delayed-type hypersensitivity response to viral Ag was suppressed. In contrast, LIF was produced by splenocytes and lung mononuclear cells obtained at all times tested after re-infection (from 2 to 30 days). Production of the low m.w. factor required re-infection of influenza A virus-seropositive mice with type A virus; re-infection with influenza B virus failed to induce production. Ag specificity was also required in vitro for splenocytes to produce the factor; cells from type A virus-re-infected mice required type A Ag stimulation. Cell depletion studies with mAb plus C revealed that macrophages and T cells along with Ag stimulation were required for factor production by spleen cells. However, mononuclear cells obtained within 4 days from the lungs of re-infected mice did not require in vitro Ag stimulation for production of the low m.w. factor, and factor production was dependent upon the presence of CD4+ (L3T4) cells in the culture. Fractionation of culture supernatants over a Sephadex G-50 column indicated that the factor had a molecular mass of 2 to 3 kDa, and by FPLC chromatofocusing over a Mono P column, the factor eluted at a pH of approximately 8.2. Thus, re-exposure of influenza virus-seropositive mice to homologous virus resulted in the production of a low m.w. factor that prevented the biologic expression of LIF, but not its production. Lymphokines are an important component of the delayed-type hypersensitivity response; the presence of mononuclear cells secreting a low m.w. factor and LIF concomitantly at the site of virus replication (lungs) and the capacity of the factor to block the biologic expression of LIF in vitro suggest that the factor may have a role in the regulation of a delayed-type hypersensitivity response in vivo during re-infection.

Monocyte regulation of lymphokine production in cancer patients.

The effect of monocytes from patients with gastric cancer on lymphokine production was investigated. Patients' monocytes both augmented and reduced production of T-cell migration inhibitory factor (TIF) by normal and patients' T lymphocytes stimulated with PHA, while normal monocytes did not change or decreased the TIF production. This regulatory effect of cancer patients' monocytes on lymphokine production was independent of the initial potential of T cells to produce TIF. These observations suggest that the regulation of lymphokine production by monocytes is altered in some cancer patients.

Prevention of leukocyte migration inhibitory factor activity by glucagon.

Leukocyte migration inhibitory factor (LMIF) activity was tested before and after glucagon administration both in vivo and in vitro. In vivo glucagon 1 mg i.v. vs saline administration inhibited LMIF production by T lymphocytes in 85.21% patients (p less than 0.01). In vitro glucagon in physiologic (125 pg/ml) and pharmacologic (50 ng/ml) doses increased the migration area vs PPD 250 microL (migration index 0.5127 vs 0.3210; p less than 0.05). These results show a significant inhibitory effect of glucagon upon LMIF activity. We suggest that glucagon acts by enhancement of the intralymphocytic cAMP/cGMP ratio (cyclic adenosine monophosphate/cyclic guanosine monophosphate).

Partial purification and physicochemical properties of human T cell migration inhibitory factor (TIF).

Supernatants from 24 hr cultures of PHA-pulsed human T lymphocytes inhibit the migration of human peripheral blood T lymphocytes and guinea pig macrophages in vitro. The factor responsible for the inhibition of T lymphocytes provisionally called TIF (T cell migration inhibitory factor) was separated from MIF by preparative PAGE, had apparent molecular weight (m.w.) of 1,000-10,000 daltons and isoelectric point of 3.1. TIF activity was resistant to treatment with trypsin, chymotrypsin and neuraminidase but sensitive to PMSF (phenyl-methyl-sulfonyl-fluoride). This suggests that TIF is presumably different from human MIF and may represent a novel lymphokine which preferentially affects T cell migration in vitro.

Counter inhibitor: a low molecular weight cytokine derived from human leukocyte dialysates reverses antigen dependent PMN and macrophage migration inhibition.

A low molecular weight component, termed counter inhibitor (CI), has been partially purified from human dialyzable leukocyte extracts. Addition of CI to either a direct leukocyte or macrophage migration inhibition system results in reversal of antigen-induced migration inhibition. CI activity requires the presence of antigen for expression, but does not require that the donor of the CI be immune to the antigen used in the migration inhibition assay. Reversal of migration inhibition by CI appears to be a consequence of its ability to prevent PMNs or macrophages from responding to lymphokines which induce migration inhibition.

Effect of histamine on leukocyte migration tests in vertebrates. II. Study of the lack of species specificity of leukocyte inhibiting factor--production inhibitor (LIF-PI).

Immune regulatory effects of histamine are now well established. We have previously demonstrated that human T lymphocytes having histamine receptors, produce a soluble factor inhibiting the production of LIF. In this present paper we have shown that production and function of this factor is not species specific. Indeed the leukocyte inhibiting factor-production inhibitor (LIF-PI) produced by human, guinea pig or dog lymphocytes inhibits the production of LIF by human, guinea pig and murine lymphocytes to a similar extent. This property allows for easier testing of LIF-PI particularly from human subjects.

Spontaneous production of lymphocyte migration inhibitory factor (sLyMIF) by T and B lymphocytes from peripheral rabbit blood.

Nonstimulated 24 h cultures of lymphocytes from rabbit peripheral blood, as well as isolated rabbit T and B lymphocytes release into the culture medium a factor inhibiting migration of T and B lymphocytes (LyMIF). Synthesis of the factor can be blocked by puromycin. The stimulatory factor for B lymphocyte migration (LyMSF) can also be produced in T lymphocyte cultures. The synthesis of both factors occurs in suspensions containing monocytes, as well as in those lacking monocytes.

Inactivation of bronchial mucous proteinase inhibitor by cigarette smoke and phagocyte-derived oxidants.

Freshly prepared aqueous solutions of cigarette smoke suppressed the elastase inhibitory capacity (EIC) of the acid-stable proteinase inhibitor present in bronchial mucus (BMPi) and human seminal plasma (HUSI-I). Thin-layer gel-immunofiltration analysis of mixtures of smoke-treated BMPi and human leukocyte elastase showed decreased elastase: BMPi complexes, increased uncomplexed BMPi and increased free elastase. Phenolic antioxidants prevented the suppression of the EIC of BMPi or HUSI-I by cigarette smoke. In addition, treatment of BMPi or HUSI-I with chemical oxidants caused a similar suppression of EIC. Furthermore, treatment of BMPi or HUSI-I with the phagocyte-derived oxidizing system, myeloperoxidase + H2O2 + Cl-, suppressed EIC. Finally, the functional activity of BMPi was significantly reduced in tracheal aspirates of human smokers compared to that of nonsmokers. These results support the hypothesis that local inactivation of BMPi in the conducting airways of the lung by inhaled cigarette smoke or by phagocyte-derived oxidants may play a role in the pathogenesis of obstructive lung disease in smokers.

Determination of the human lymphokine leukocyte migration inhibitory factor (LIF) by a sensitive radioenzymatic assay. Inhibitory effect of cGMP on the esterolytic activity of highly purified LIF.

Indirect experiments using irreversible enzyme inhibitors have shown that the human lymphokine leukocyte migration inhibitory factor (LIF) is a serine esterase and protease exhibiting specific affinity towards arginine esters and amides. A sensitive assay for direct measurement of esterase activity using p-tosyl-L-arginine (3H) methyl ester (3H-TAME) as substrate is described. Esterolytic activities are demonstrated in crude supernatants of human lymphocytes stimulated with concanavalin A (LIF-rich) and, more pronounced, in supernatants of unstimulated cells (control). To follow the effects of purification procedures, the serine esterases of LIF-rich and control preparations were specifically labeled with the irreversible, active site directed agent (1,3-3H)di-isopropylphosphorofluoridate. Most of these enzymes, visualized by Sephadex chromatography, were removed by a gentle three-step procedure, allowing at least 50% of the initial LIF activity to be recovered. The resulting LIF-rich preparation, purified to contain serine esterases at a concentration corresponding to less than 1 ng per ml original supernatant, still showed estrolytic activity towards 3H-TAME. The optimal conditions for the radioenzymatic assay of purified LIF and the inhibitory effect of 10(-4) M cGMP, which on the basis of indirect experiments has been implicated as a specific regulator of LIF activity, are described.