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As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain3]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs [Thp1, Ain3] 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P2(1)2(1)2(1), with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) A. The structure has been refined to R = 0.071 for 2301 independent reflections with I greater than 1.5 sigma.

The peptide H-Lys-Val-Leu-Asp-OH (KVLD), derived from antiflammins, and its analog H-Lys-Val-Asp-Leu-OH (KVDL) were prepared by solution synthesis. KVLD does not inhibit platelet aggregation in contrast to the results recently published.

HLA B51 specificity is strongly associated with Behcet's disease (BD) (for references see Baricordi et al., 1986), a multisystem vasculitis of unknown aetiology, for which an immunological pathogenesis has been proposed (O'Duffy et al., 1983; O'Duffy et al., 1990). Neutrophil abnormalities observed in BD patients even during clinical remission suggest prominent involvement of these phagocytic cells in the pathogenesis of the disease (Niwa et al., 1982). In order to clarify how HLA B51 antigen might confer susceptibility to BD, we have investigated neutrophil function in 13 B51-positive and 13 B51-negative healthy subjects. Lymphocyte spontaneous proliferation and circulating immune complexes were also evaluated. Whereas neutrophils from B51-positive subjects showed an increase in the chemotactic response toward casein (P = 0.003) and LPS (P = 0.033) and also in the PMA-induced superoxide production (P = 0.008) no evidence of enhanced lymphocyte activation emerged. These results suggest that the HLA region can exert a regulatory control on PMN functions.

Previous studies have suggested that phorbol ester-activated neutrophils kill both antibody non-coated and antibody-coated K562 target cells. In this report the contribution of the receptors Fc gamma III (CD16) and CR3 (CD11b/CD18) in the lytic process was investigated. In neutrophils CD16 and CR3 are up-regulated by the phorbol ester up to 4 and 10 times, respectively. As expected, lysis of non-immunized K562 targets is not affected by the treatment of neutrophils with anti CD16, AB8.28, whereas lysis of immunized targets is decreased by 50%. In addition, the interaction of CD16 and AB8.28 induces calcium mobilization and increases granule secretion. Surprisingly, the simultaneous binding of AB8.28 and anti-CR3 OKM1 to neutrophils completely abolishes the lysis of antibody-coated targets. Unlike CD16, CR3 does not possess a functional role and binding of OKM1 to CR3 does not affect cytotoxicity of immunized K562 targets, but it blocks lysis of non-coated target almost completely, indicating a function as adhesion protein for CR3. These studies demonstrate a distinct role of CD16 and CR3 in mediating antibody-dependent and antibody-independent cellular cytotoxicity, respectively.

The expression of the complement receptor CR1 has been evaluated using an immunoalkaline phosphatase staining method on peripheral blood neutrophils and granulocyte precursors from 22 patients with chronic myeloid leukaemia (CML) and 15 healthy subjects. The immunocytochemical labelling pattern of CR1 was evaluated semiquantitatively on cell smears using three different anti-CR1 monoclonal antibodies. The scoring method showed that seven patients with CML had a marked reduction in CR1 expression which did not change with in vitro stimulation of neutrophils with phorbol-myristate-acetate (PMA) whereas control cells responded to PMA, increasing the receptor level two-fold. In addition, functional analysis of neutrophils with low CR1 expression from CML patients showed a very low cytolytic activity against K562 tumour target, suggesting a relationship between the cellular content of CR1 and neutrophil tumouricidal activity. The involvement of CR1 in neutrophil-mediated lysis is consistent with complete lack of tumour toxicity following receptor neutralization by anti-CR1 monoclonal antibodies. Interferon therapy improved CR1 expression and the cytolytic response of neutrophils in three out of five CML patients with a moderately low CR1 score. CML patients non-responding to interferon therapy and those with a very low CR1 score, independent of the clinical stage, progressed more rapidly into the advanced clinical stage and blastic crisis.

We have investigated the motility, superoxide anion production and tumor cell lysis of neutrophils from five patients affected by hairy cell leukemia. Random locomotion and chemotaxis towards denaturated casein and activated serum was normal as well as the superoxide production by opsonized zymosan. In contrast, chemotaxis and superoxide generation induced by FMLP and TPA were markedly reduced. The low responsiveness of neutrophils to TPA was also observed by evaluating cell lysis, against either non-immunized K562 target cells or antibody-coated tumor targets. In hairy cell leukemia neutrophils showed a selective reduced response to TPA and FMLP that, directly or indirectly, activate PKC, suggesting an impairment in the system of signal transduction.

Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to a surface glycoprotein CD4 present on T4 helper/inducer lymphocytes, monocytes/macrophages and other cells. A simple octapeptide (H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, peptide T) seems to inhibit HIV infectivity and to activate human monocyte chemotaxis. In order to study in vitro metabolic stability and structure-activity relationships, peptide T and a number of analogues were prepared and tested on human monocytes by chemotactic assay. Peptide T and the shorter fragments T(3-8)-OH and T(4-8)-OH displayed potent bioactivity (maximal chemotactic activity in the range 10(-11)-10(-10) M). The C-terminal heptapeptide showed a reduction of potency, while further truncations at N-terminus of T(4-8)-OH abolished the biological action. In the octapeptide series, whereas the alpha-amino butyric acid (Abu) substitution for Thr4 was well tolerated, the same "slight" structural change at Thr5 or Thr8 was very detrimental. Finally, [D-Asn6]T(1-8)-OH analogue has low chemotactic activity. All these results indicate that i) the C-terminal pentapeptide is the minimum sequence required for bioactivity, ii) residues 5 to 8 appear to play a crucial biological role, iii) peptide T chemotaxis is mediated, at least in part, through the polar properties of Thr side chains at the critical positions 5 and 8, while the Thr4 does not interfere with biological characteristics of peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

When polymorphonuclear leukocytes are treated with soluble and particulate substances the cells produce superoxide anion O2-. which plays a role in the activation of the oxidative metabolism. The time-course of O2-. release and the extent to which the anion is produced are related to the nature of the stimulating agents. Anti-inflammatory non steroid drugs, such as indomethacin and oxamethacin, exert a variable depression of the oxidative burst, which does not seem correlated to the inhibition of prostaglandin synthesis.

At least 14 iodinated proteins can be distinguished by SDS polyacrylamide gel electrophoresis followed by autoradiography in the membrane of intact human neutrophils. Neutrophils from patients suffering from rheumatoid arthritis show a modified expression of 4 outer membrane proteins. Three polypeptide components of the 30, 50 and 130 K were decreased by 95%, while the 120 K component dramatically increased. The possible relationship between the altered cell sensitivity and the defective expression of membrane proteins is discussed.

The antibiotic rifamycin SV (RSV) has been successfully used by others on the local treatment of rheumatoid arthritis (R.A.). Since polymorphonuclear leukocytes (PMNL) are involved in the synovial inflammatory process we tested the 'in vitro' effect of RSV on PMNL functions, such as locomotion and phagocytosis. PMNL locomotion was evaluated by using modified Boyden Chamber and phagocytosis was tested by the number of yeast particles ingested and by NBT reduction. The, the functions of PMNL derived from 7 R.A. patients in therapy only with non steroidal anti-inflammatory drugs were compared with those of PMNL from 14 patients with non inflammatory disease (osteoporosis and osteoarthrosis) in the same therapy and with neutrophils from healthy subjects. It was demonstrated that PMNL derived from patients with both R.A. and non inflammatory disease activated their directional locomotion towards RSV, on the contrary, cells from healthy subjects were unresponsive. Moreover, only patients with R.A. showed a defective phagocytic capacity and a depression in NBT reducing activity, when PMNL were treated with RSV. This phenomenon might be correlated with beneficial effect observed after local treatment of R.A. with RSV.

The aim of our study is to evaluate the eventual activity of the total dialysate of two uremic nephrectomized patients in recirculating dialysis and the chromatographic peak of the dialysate fractionated by Sephadex column G 15 on PMN chemotaxis. Only the total dialysate and the chromatographic peak B showed inhibition of chemotaxis. On the contrary the commercial polyamines in the same concentration range, and the other chromatographic peaks, containing polyamines too, did not revealed inhibition. Our data show, therefore, that the chemotaxis inhibition could be due to the middle-molecules present in the peak B, rather than the polyamines itself. Polyamines were determined by dansylation method, separated by thin layer chromatography and quantified by spectrofluorimeter. Chemotaxis was evaluated using the modified Boyden chamber.

Lymphocyte dialysates obtained from healthy volunteers and leukemic subject were chromatographed on Bio-Gel P-4. Partially purified fractions were assayed for neutrophil locomotion: a positive chemokinetic effect and no chemotactic activity was observed in both samples.

Fifteen pentapeptide analogs of C-terminal fragment of peptide T, H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, were prepared and tested for human monocyte chemotaxis. Structure-activity studies suggest that the potent chemotactic activity of H-Thr-Thr-Asn-Tyr-Thr-OH is mediated through the polar properties of the C-terminal carboxyl group and Thr side chains at the critical positions 5 and 8, while the hydroxyl group of N-terminal Thr and its free amino function are not essential requirements for CD4 receptor interactions.

Non steroidal anti-inflammatory drugs, such as oxametacine, are generally used in treatment of rheumatoid disease. In an 'in vitro' experimental model, the drug efficacy was tested on leukocyte functions. Locomotion, both random and directional, phagocytic activity and superoxide production of normal and rheumatoid PMNL were tested in the presence of varying concentrations of oxometacine. Locomotion was evaluated by using modified Boyden chambers; phagocytosis was tested by number of yeast particles injested and by NBT reduction; superoxide production was assayed by reduction of ferricytochrome C. In our conditions the drug exhibited a strong anti-inflammatory effect. In fact, chemotaxis and anion production were specifically depressed in a dose-dependent way.

Human neutrophils stimulated with phorbol myristate acetate were able to damage human erythroleukemic K562 cells, in the absence of specific antibody, as assessed by a two hour 51Cr release assay. Neutrophils treated with formyl-peptide fMet-Leu-Phe did not display tumoricidal response, but the addition of diacylglycerol kinase inhibitor R59022 together with formyl-peptide induced the cytotoxic capacity against tumor target cells. Phorbol ester is a potent activator of certain functions of neutrophils because of its ability to directly and irreversibly stimulate protein kinase C; formyl-peptide, on the contrary, activates protein kinase C by inducing a rapid and transient production of diacylglycerol, that is quickly metabolized. The addition of an inhibitor of diacylglycerol kinase, R59022, however potentiated the action of formyl-peptide. These results indicate that protein kinase C is involved in the tumoricidal activity of neutrophils against K562 cells, and that maximal activation of the enzyme is required to achieve the cytotoxic response.

Treatment of human neutrophils with the two chemoattractants formyl-methyonyl-leucyl-phenylalanine and opsonized zymosan or with phorbol myristate acetate induce the production of superoxide anion (O-.2), which plays a role in the inflammatory reaction and microbicidal activity. The time-course of O-.2 release and the extent to which the anion is produced are related to the nature of the stimulating agents. Anti-inflammatory non steroid drugs, such as indomethacin and oxamethacin, exert a variable depression of the oxidative burst, which does not seem correlated to the inhibition of prostaglandin synthesis, but to the lipophilic character of the drug molecule.

We have studied the effect of the two antibodies, MF 25.1 and GF 26.7.3, on leucocyte function: these antibodies did not mimic the effect of cellular stimuli, but the pre-treatment of neutrophils with MF 25.1 and GF 26.7.3 modulated several cell activities. Both antibodies significantly reduced superoxide anion production in response to various stimuli. Despite similar cellular response MF 25.1 and GF 26.7.3 bound to different antigenic determinants, as shown both by immunoblotting studies and by treating neutrophils first with GF 26.7.3 and subsequently with MF 25.1, and vice-versa; the inhibition of superoxide production corresponded, in fact, to the total of that seen for each antibody. Moreover, in the absence of attractant, measures of locomotion of neutrophils treated with monoclonal antibody showed that 43% of the subjects responded to antibody binding by twofold activation of spontaneous movement. Chemotaxis and lysosomal enzyme release were not affected by antibody treatment.

PGE1 and its 8-aza-analogous were assayed "in vitro" for leukocyte functions; the natural compound was not a chemotactic agent and exhibited positive chemokinesis. On the contrary, the synthetic compound chemically related to PGE1, was a chemotactic factor and inhibited the reduction of NBT during phagocytosis.

Prostaglandin (PG) E1 and chemically related compounds 15 alpha-11-deoxy-8-azaPGE1 and 15 epi-11-deoxy-8-azaPGE1 were examined in vitro for their capacity to modulate some physiological functions of human polymorphonuclear leukocytes (PMN). PGE1 was completely devoid of chemotactic activity and did not affect leukocyte phagocytosis. On the contrary, 15 alpha-11-deoxy-8-azaPGE1 showed a potent leukotactic activity. In addition, this synthetic substance significantly depressed the phagocytic activity and the concomitant NBT reduction by leukocytes. The analogue 15 epi-11-deoxy-8-azaPGE1 did not exert any of these effects. From these results it appeared that the substitution of the carbon-8 by the nitrogen and the lack of hydroxyl group at the position 11 into the PGE1 structure, make the compound active towards human neutrophils.

Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

The conformational behavior of the chemotactic peptide analogs formylmethionylleucylphenylalanine methyl ester (CHO-Met-Leu-Phe-OMe) and formylmethionylleucylcyclohexylalanine methyl ester (CHO-Met-Leu-Cha-OMe) has been studied in solvents of different polarity by circular dichroism and infrared absorption. Both analogs and very probably the chemotactic peptide formylmethionylleucylphenylalanine (CHO-Met-Leu-Phe-OH) preferably adopt in solution a folded "active" conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.

Neutrophils derived from peripheral blood of patients with rheumatoid arthritis (RA) exhibited a defective responsiveness to natural mediators of inflammation, namely histamine and serotonin, and to the anti-inflammatory drugs ibuprofen and naproxen, in spite of the fact that the basic status of motility was normal. Not even pretreatment of granulocytes with substances restored the capacity to modulate the random and directional locomotion. This neutrophil functional defect was correlated with an anomalous response to rifamycin SV, previously observed in rheumatic states.

36 unrelated individuals with neutrophil MPO deficiency, (10 totally MPO deficient) were found on screening a population of 148,000 subjects. A further 2 subjects with total and 22 with partial MPO deficiency were identified through family studies. The assessment of neutrophil function, i.e., peroxidase activity, superoxide anion generation, microbicidal activity towards fungi and bacteria, and locomotor behaviour, was carried out in 10 subjects with total and 4 with partial MPO deficiency. We found that the enzyme defect is associated with a marked impairment in the killing of both S. aureus and C. albicans, without affecting microbicidal activity against S. faecalis. There appears to be a high incidence of malignancy in patients with complete MPO deficiency, suggesting a relationship between a defective MPO system and neutrophil-mediated tumor cell cytotoxicity.

Two tripeptides, related to the chemotactic formyl-peptide, are tested for its ability to affect random and directional locomotion and to induce chemotaxis of polymorphonuclear leukocytes. The results indicate that the methyl ester of formyl-methyonyl-leucyl-phenylalanine possesses a biological activity towards human phagocytes, including a chemotactic potency, comparable to that of unmodified peptide. Therefore, the free carboxyl group does not seem essential to generate active leukoattractant. On the contrary, the replacement of the formyl group by the butyloxycarbonyl results in a drastic loss of biological activity. Our data may indicate that the two formyl-peptides interact with the same binding site on the cell membrane.

Eighteen patients with established hereditary myeloperoxidase deficiency underwent morphological, ultrastructural, cytochemical and functional analysis in order to correlate the lack of peroxidase from phagocytes with other leucocyte activities. Cytochemical and ultrastructural findings only confirmed the peroxidase defect in neutrophil and monocyte population, whereas normal peroxidase activity was detected in eosinophil granulocytes ("Alius-Grignaschi anomaly"). Morphological analysis, as determined by both ligh and electron microscopy, showed in two patients with total MPO-deficiency a large number of neutrophils (50-60%) with nuclear abnormalities very similar to Pelger-Huet's heterozygous form (two lobed neutrophils having a typical pince-nez appearance and a nuclear chromatin coarser than that of normal PMNL). Other 2 cases displayed a 50-60% five-lobed neutrophils, as occur in congenital nuclear hypersegmentation of PMNL. These findings suggest that Alius-Grignaschi anomaly and Pelger-Huet syndrome can be found associated in the same individuals, since both these abnormalities have a genetic origin. Finally, since an impaired bactericidal and fungicidal activity was observed, no patients displayed particular susceptibility to persistent or severe infections, thus confirming the presence of MPO-independent enzymatic systems.

Casein, a phosphoprotein forming aggregates in solution, exerts chemotactic activity for human neutrophils. The protein was filtered on Sephadex G 100 to obtain fractions of homogeneous mol. weight and the column fractions were tested for chemotactic activity. The chemotactic activity was found only in the lighter peak, which was then digested by trypsin. The digested material was purified by filtration through AcA-54 and the 6.000 M.W. peptide containing the most phosphate groups and tyrosines resulted chemotactic for PMN. Most likely the phosphate groups are important in chemotactic recognition.

We studied possible effects of uremic toxins and polyamines (PAs) on organ cultures of chick embryo. We added on culture media the lyophilized of total dialysate and its chromatographic peak II obtained with chromatography with Sephadex G 15. The total dialysate and peak II showed toxicity with degeneration of the culture, whereas the free PAs, we added, did not effects.

The effect exerted by rifamycin SV, used intra-articularly in the treatment of rheumatoid arthritis, on polymorph function was studied. Random and directional locomotion of synovial fluid neutrophils was compared with that of peripheral blood cells in 10 patients followed up during 5 drug applications. PMNs from the two sources were characterized by different responsiveness to this pharmacological agent: blood cells activated chemotaxis in a dose-response way during rifamycin therapy, whereas synovial polymorphs did not modify their locomotor behaviour. It is proposed that the presence of immune complexes and/or factors produced by cell-cell interactions in the articular space may change the synovial neutrophil response to stimuli.

In this study we have demonstrated that acidic non-steroidal anti-inflammatory drugs in low concentrations inhibited human PMN locomotion in vitro. A speculative mechanism of action is proposed.

Polymorphonuclear leucocytes play an important role in the inflammatory process, and their functions are likely to be regulated by pharmacological agents. In this paper we report the "in vitro" effect of non steroidal anti-inflammatory drugs on random and chemotactic motility of human leucocytes, and demonstrate that the two forms of movement, spontaneous and directional, are differently inhibited by low concentrations of these agents. Leucocytes of patients with recurrent inflammation are unaffected by these drugs.

Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction.

Lymphocytes stimulated with alfa-interferon released a chemotactic factor for neutrophils. The process was not inhibited by cycloheximide, whereas mepacrine completely inhibited release of the chemotactic activity. The chemotactic factor was resistant to storage, heat treatment and proteolysis. Recombinant alfa-interferon did not stimulate lymphocytes to release a neutrophil chemotactic factor.

The tetrapeptides CHO-Met-Leu-Phe-CO-NH-(CH2)n-COOMe (n = 1-5) have been synthesized. These peptides containing an omega-amino acid residue in position 4 exhibit a very high chemotactic activity. Like the chemotactic peptide CHO-Met-Leu-Phe-OMe, these tetrapeptides in solution undoubtedly adopt an "active" conformation which allows a strong interaction with the receptor on the human polymorphonuclear leukocyte surface.

We have isolated peak II, which is the peak of the middle molecules containing polyamines, from dialysate of uremic patients in recirculating dialysis. We investigated the effect of total dialysate, chromatographic peaks, I, II, III, IV and commercial polyamines on polymorphonuclear leukocyte chemotaxis and superoxide production (i.e. phagocytic activity) in healthy and uremic patients. Polymorphonuclear chemotaxis was inhibited by total dialysate and peak II but not by polyamines; polyamines, total dialysate and peak II had no activity on polymorphonuclear phagocytosis.

Granulocyte superoxide production by different stimuli was studied in 14 patients suffering from rheumatoid arthritis, and in four cases defective O(2) generation was shown. The effect of two chemically related drugs, such as indomethacin and oxamethacin, was also evaluated, since we have previously investigated the action of antiinflammatory agents on cell locomotion. Indomethacin did not affect O(2) production, whereas oxamethacin reduced significantly superoxide generation in PMNs from all subjects tested. Moreover, the extent of the effect was dependent on the stimulant used, being larger when the activation of O(2) generating system was induced by opsonized zymosan.

Myelin basic protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation. Pre-treatment of human neutrophils with myelin basic protein selectively inhibits the formyl-peptide-induced chemotaxis, without affecting chemotaxis evoked by casein and activated serum. Furthermore, both the degranulation and superoxide anion production stimulated by the chemotactic peptide are not modified by the prior treatment of the neutrophils with myelin basic protein.