Grover C. Bagby

Interaction of lactoferrin, monocytes, and T lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro.

Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrins inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrins inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.

Glucocorticoid Therapy in the Preleukemic Syndrome (Hemopoietic Dysplasia): Identification of Responsive Patients Using In-Vitro Techniques

Bone marrow cells from 54 patients with the preleukemic syndrome were cultured in agar (granulocyte colony forming units) in the presence and absence of cortisol. Twenty-four patients were given trials of prednisone therapy after the initial culture was performed. Cortisol (in vitro) failed to enhance colony growth in 29 of these 34 cases, and none of the 29 patients responded to prednisone therapy. Cortisol enhanced colony growth in five patients and three of these responded favorably to prednisone therapy. The correlation of in-vivo with in-vitro events is significant (P less than 0.005). Glucocorticoid therapy is of value in the management of a small number of patients with the preleukemic syndrome but is hazardous in those who fail to respond. These preliminary observations suggest that bone marrow cell culture techniques may aid in the identification of those patients who will and those who will not respond favorably to such therapy.

T lymphocytes involved in inhibition of granulopoiesis in two neutropenic patients are of the cytotoxic/suppressor (T3+T8+) subset.

T lymphocyte-mediated bone marrow failure is a recently recognized clinical disorder, but the T lymphocyte subsets responsible for mediating the inhibitory effect have not been identified. We obtained T lymphocytes from the bone marrow of two patients with T lymphocyte-mediated granulopoietic failure, exposed them to monoclonal antibodies (OKT3, OKT4, and OKT8) in cytotoxicity assays, then recombined the treated T cells with autologous T-depleted marrow cells in clonal assays for granulocyte/macrophage progenitors (CFU-GM). Treatment of T cells with OKT3 and OKT8 abrogated their granulopoietic inhibitory effect but treatment with OKT4 did not. Therefore, in these two patients, the lymphocytes that played a role in the inhibition of granulopoiesis were of the cytotoxic/suppressor subset.

Calcium-dependent polymerization of lactoferrin

Summary Lactoferrin is known to have a molecular weight of approximately 77,000. In this study, we show that it undergoes polymerization in calcium-containing fluids and that the predominant species is a tetramer. Preliminary evidence is presented to indicate that the tetramer is also found in human serum, tears and breast milk. When the lactoferrin monomer and the tetramer were tested for biological activity in a system that is known to be highly sensitive to an inhibitory effect of lactoferrin — the production of granulocyte monocyte colony-stimulating activity — it was found that only the monomeric form of lactoferrin was inhibitory.

Influence of volume expansion on proximal tubular sodium reabsorption in congestive heart failure

Abstract Proximal tubular sodium reabsorption was studied in ten patients with severe decompensated congestive heart failure when marked extracellular fluid volume expansion was present and again after diuresis to dry weight. The technique used was pharmacologic blockade of distal nephron sites by diuretic agents. The percentage of the filtered load of sodium reabsorbed by the proximal tubule increased from 68 ± 5 per cent to 85 ± 2 per cent and of chloride from 61 ± 6 per cent to 78 ± 4 per cent following reduction of the expanded extracellular volume. This increase could not solely be accounted for by change in filtered sodium load or enhanced aldosterone activity. It is concluded that although the proximal tubule responds in an appropriate manner qualitatively to volume stimuli, an enhanced fractional proximal sodium reabsorption at any given level of extracellular fluid volume may characterize the patient with congestive heart failure. The exact mechanism of sodium retention in heart failure must remain speculative.

Suppression of Granulopoiesis by T-Lymphocytes in Two Patients with Disseminated Mycobacterial Infection

Two patients with disseminated mycobacterial infection presented with severe neutropenia and hematopoietic failure. Marrow cells were obtained from each patient and were cultured in methylcellulose before and after the removal of mononuclear phagocytes, T-lymphocytes, or both from the marrow cell suspension. Glucocorticosteroid-resistant T-lymphocytes markedly inhibited granulopoiesis, but mononuclear phagocytes did not. Indomethacin inhibition of prostaglandin synthesis did not influence suppression of colony growth by the inhibitory lymphocytes. In the one patient who responded favorably to antituberculous therapy, the in-vitro T-lymphocyte inhibition of granulopoiesis disappeared as the neutropenia resolved. Thus, in some patients with disseminated mycobacterial infection, clinical bone marrow failure may be mediated, at least partly, by T-lymphocytes that suppress hematopoiesis.